Super Critical Fluid Extraction......

SupercriticalFluid Extraction

of Nutraceuticalsand BioactiveCompounds

7089_C000.indd 1 10/22/07 12:02:14 PM

7089_C000.indd 2 10/22/07 12:02:14 PM

SupercriticalFluid Extraction

of Nutraceuticalsand BioactiveCompounds

Edited by

Jose L. Martínez

CRC Press is an imprint of theTaylor & Francis Group, an informa business

Boca Raton London New York

7089_C000.indd 3 10/22/07 12:02:14 PM

CRC PressTaylor & Francis Group6000 Broken Sound Parkway NW, Suite 300Boca Raton, FL 33487-2742

© 2008 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government worksPrinted in the United States of America on acid-free paper10 9 8 7 6 5 4 3 2 1

International Standard Book Number-13: 978-0-8493-7089-2 (Hardcover)

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the conse-quences of their use.

No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers.

For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged.

Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data

Supercritical fluid extraction of nutraceuticals and bioactive compounds / [edited by] Jose L. Martinez.

p. cm.Includes bibliographical references and index.ISBN 978-0-8493-7089-2 (alk. paper)1. Supercritical fluid extraction. 2. Functional foods. 3. Bioactive compounds. I.

Martinez, José L. (José Luis), 1966-

TP156.E8S835 2007660.6’3--dc22 2007025441

Visit the Taylor & Francis Web site athttp://www.taylorandfrancis.com

and the CRC Press Web site athttp://www.crcpress.com

7089_C000.indd 4 10/22/07 12:02:18 PM

Dedication

To Marlene and Alejandro

7089_C000.indd 5 10/22/07 12:02:18 PM

7089_C000.indd 6 10/22/07 12:02:18 PM

vii

ContentsPreface.......................................................................................................................ixAcknowledgments......................................................................................................xiContributors............................................................................................................ xiiiEditor........................................................................................................................xv

Chapter 1 Fundamentals.of.Supercritical.Fluid.Technology.................................1

Selva Pereda, Susana B. Bottini, and Esteban A. Brignole

Chapter 2 Supercritical.Extraction.Plants:.Equipment,.Process,.and.Costs........25

Jose L. Martínez and Samuel W. Vance

Chapter 3 Supercritical.Fluid.Extraction.of.Specialty.Oils................................. 51

Feral Temelli, Marleny D. A. Saldaña, Paul H. L. Moquin, and Mei Sun

Chapter 4 Extraction.and.Purification.of.Natural.Tocopherols.by.Supercritical.CO2............................................................................... 103

Tao Fang, Motonobu Goto, Mitsuru Sasaki, and Dalang Yang

Chapter 5 Processing.of.Fish.Oils.by.Supercritical.Fluids................................ 141

Wayne Eltringham and Owen Catchpole

Chapter 6 Supercritical.Fluid.Extraction.of.Active.Compounds.from.Algae..... 189

Rui L. Mendes

Chapter 7 Application.of.Supercritical.Fluids.in.Traditional.Chinese.Medicines.and.Natural.Products....................................................... 215

Shufen Li

Chapter 8 Extraction.of.Bioactive.Compounds.from.Latin.American.Plants.... 243

M. Angela A. Meireles

Chapter 9 Antioxidant.Extraction.by.Supercritical.Fluids................................ 275

Beatriz Díaz-Reinoso, Andrés Moure, Herminia Domínguez, and Juan Carlos Parajó

7089_C000.indd 7 10/22/07 12:02:18 PM

viii Contents

Chapter 10 Essential.Oils.Extraction.and.Fractionation.Using.Supercritical.Fluids...........................................................................305

Ernesto Reverchon and Iolanda De Marco

Chapter 11 Processing.of.Spices.Using.Supercritical.Fluids............................... 337

Mamata Mukhopadhyay

Chapter 12 Preparation.and.Processing.of.Micro-.and.Nano-Scale.Materials.by.Supercritical.Fluid.Technology.................................................... 367

Eckhard Weidner and Marcus Petermann

Index....................................................................................................................... 391

7089_C000.indd 8 10/22/07 12:02:19 PM

ix

PrefaceIn.the.last.decade.new.trends.in.the.food.industry.have.emerged,.enhanced.concern.over. the. quality. and. safety. of. food. products,. increased. preference. for. natural..products,. and. stricter. regulations. related. to. the. residual. levels.of. solvents..Addi-tionally,.the.nutraceutical.and.functional.food.sector.represents.one.of.the.fastest.growing.areas.in.a.consumer-driven.trend.market..These.trends.have.driven.super-critical.fluid.(SCF).technology.to.be.a.primary.alternative.to.traditional.solvents.for.extraction,.fractionation,.and.isolation.of.active.ingredients..The.aim.of.this.book.is. to.present. the.current. state.of. the.art. in.extracting.and. fractionating.bioactive.ingredients.by.SCFs.

This.book.contains.twelve.chapters.that.primarily.focus.on.implemented.indus-trial.processes.and.trends.of.the.technology..The.content.of.the.chapters.includes.a.review.of.the.major.active.components.in.the.target.material,.including.chemical,.physical,. nutritional,. and. pharmaceutical. properties;. an. analysis. of. the. specific.SCF. process. used;. a. comparison. of. traditional. processing. methods. versus. SCF.technology;.and.a.set.of.conclusions.with.supporting.data.and.insight..A.review.of.the.fundamentals.of.the.technology.and.an.examination.of.SCF.extraction.systems.and.process.economics.are.also.included.

The. contributing. authors. are. international. experts. on. the. topics. covered,. and.I.would.like.to.thank.them.for.their.thoughtful.and.well-written.contributions..This.book.is.addressed.to.food.scientists,. technologists,.and.engineers.as.well.as.other.professionals.interested.in.the.nutraceutical.and.functional.food.sector..Additionally,.I.hope.that.this.book.will.serve.to.stimulate.academia.and.industry.to.search.for.new.process.and.product.developments.as.well.as.their.industrial.implementation.

7089_C000.indd 9 10/22/07 12:02:19 PM

7089_C000.indd 10 10/22/07 12:02:19 PM

xi

AcknowledgmentsThe.authors.of.the.chapters.of Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds wish.to.acknowledge.the.following.funding.agencies.for.their.support.and.assistance.

Dr.. Feral. Temelli. would. like. to. acknowledge. the. financial. support. from. the.Natural.Sciences.and.Engineering.Research.Council.of.Canada.(NSERC).

Dr..Fang.et.al..gratefully.acknowledge.the.21st.COE.program.“Pulsed.Power.Sicence”.and.Wuhan.Kaidi.Fine.Chemical.Industries.Co.,.Ltd.,.for.their.support.

Dr..Shufen.Li.would.like.to.thank.Dr..Can.Quan,.Dr..Shaokun.Tang,.Dr..Wenqiang.Guan,.Dr..Yongyue.Sun,.Ms..Luan.Xiao,.and.Ms..Ying.Zhang.for.their.contributions.to.the.research.work.as.well.as.their.assistance.in.the.preparation.of.Chapter.7.

Dr..Maria.Angela.Meireles.thanks.CNPq,.CAPES,.and.FAPESP.for.supporting.the.research.done.at.LASEFI.–.DEA/.FEA.–.UNICAMP.

Dr.. Eckhard. Weidner. and. Dr.. Marcus. Petermann. would. like. to. thank. their.coworkers.and.students.from.the.University.Bochum.as.well.as.Prof..Knez.and.his.coworkers.from.the.University.of.Maribor.and.Adalbert-Raps.Research.Center..They.would.also. like. to. thank.Adalbert-Raps.Stiftung,. the.European.Union,. the.Ewald.Doerken.AG,.and.Yara.Industrial.GmbH.for.their.support.

7089_C000.indd 11 10/22/07 12:02:19 PM

7089_C000.indd 12 10/22/07 12:02:19 PM

xiii

Contributors

Susana B. Bottini, Ph.D.Planta.Piloto.de.Ingeniería.QuímicaUniversidad.Nacional.del.SurBahía.Blanca,.Argentina

Esteban A. Brignole, Ph.D.Planta.Piloto.de.Ingeniería.QuímicaUniversidad.Nacional.del.SurBahía.Blanca,.Argentina

Owen J. Catchpole, Ph.D.Industrial.Research.LimitedLower.Hutt,.New.Zealand

Iolanda De Marco, Ph.D.Dipartimento.di.Ingegneria.

Chimica.ed.AlimentareUniversita.di.SalernoSalerno,.Italy

Beatriz Díaz-Reinoso, M.Sc.Department.of.Chemical.EngineeringFacultade.de.Ciencias.de.OurenseUniversidade.de.VigoOurense,.Spain

Herminia Domínguez, Ph.D.Department.of.Chemical.EngineeringFacultade.de.Ciencias.de.OurenseUniversidade.de.VigoOurense,.Spain

Wayne Eltringham, Ph.D.Industrial.Research.LimitedLower.Hutt,.New.Zealand

Tao Fang, Ph.D.Department.of.Applied.Chemistry.and.

BiochemistryKumamoto.UniversityKumamoto,.Japan

Motonobu Goto, Ph.D.Department.of.Applied.Chemistry.and.


Shufen Li, Ph.D.School.of.Chemical.Engineering.&.

TechnologyTianjin.UniversityTianjin,.China

Jose L. Martínez, Ph.D.Thar.Technologies,.Inc.Pittsburgh,.Pennsylvania

M. Angela A. Meireles, Ph.D.LASEFI-DEAFEA.–.UNICAMPSao.Paulo,.Brazil

Rui L. Mendes, Ph.D.Departamento.de.Energias.RenovaveisINETILisboa,.Portugal

Paul H.L. Moquin, B.Sc.Department.of.Agricultural,.Food,.and.

Nutritional.ScienceUniversity.of.AlbertaEdmonton,.Canada

Andrés Moure, Ph.D.Department.of.Chemical.EngineeringFacultade.de.Ciencias.de.OurenseUniversidade.de.VigoOurense,.Spain

Mamata Mukhopadhyay, Ph.D.Chemical.Engineering.DepartmentIndian.Institute.of.TechnologyBombay,.India

7089_C000.indd 13 10/22/07 12:02:20 PM

xiv Contributors

Juan Carlos Parajó, Ph.D.Department.of.Chemical.EngineeringFacultade.de.Ciencias.de.OurenseUniversidade.de.VigoOurense,.Spain

Selva Pereda, Ph.D.Planta.Piloto.de.Ingeniería.QuímicaUniversidad.Nacional.del.SurBahía.Blanca,.Argentina

Marcus Petermann, Ph.D.University.BochumParticle.TechnologyBochum,.Germany

Ernesto Reverchon, Ph.D.Dipartimento.di.Ingegneria.

Chimica.ed.AlimentareUniversita.di.SalernoSalerno,.Italy

Marleny D.A. Saldana, Ph.D.Department.of.Agricultural,.Food,.and.


Mitsuru Sasaki, Ph.D.Department.of.Applied.Chemistry.and.


Mei Sun, M.Sc.Department.of.Agricultural,.Food,.and.


Feral Temelli, Ph.D.Department.of.Agricultural,.Food,.and.


Samuel W. Vance, P.E.Thar.Technologies,.Inc.Pittsburgh,.Pennsylvania

Eckhard Weidner, Ph.D.University.BochumProcess.TechnologyBochum,.Germany

Dalang Yang, M.Sc.Wuhan.Kaidi.Fine.Chemical.Industries.

Co..Ltd.Wuhan,.Hubei,.China

7089_C000.indd 14 10/22/07 12:02:20 PM

xv

EditorDr. Jose L. Martínez,.a.native.of.León,.Spain,.received.his.B.S..and.Ph.D..degrees.from.the.University.of.Oviedo.(Spain)..He.is.currently.General.Manager.of.Thar.Technologies,. Inc.,. Process. Division. (Pittsburgh,. USA),. a. company. dedicated.exclusively.to.supercritical.fluid.technology..He.has.nearly.two.decades.of.experi-ence. in.conducting.R&D.and. implementing. industrial.processes. in.supercritical.fluid.technology,.including.applications.in.extraction,.fractionation,.chromatogra-phy,.particle.formation,.coating,.and.impregnation.for.the.food,.nutraceutical,.and.pharmaceutical.industries.

7089_C000.indd 15 10/22/07 12:02:20 PM

7089_C000.indd 16 10/22/07 12:02:20 PM

�

1 Fundamentals of Supercritical Fluid Technology

Selva Pereda, Susana B. Bottini, and Esteban A. Brignole

Contents

1.1 Introduction.....................................................................................................11.2 SupercriticalFluids.........................................................................................2

1.2.1 PhysicalPropertiesofSupercriticalFluids..........................................41.3 PhaseEquilibriumwithSupercriticalFluids..................................................4

1.3.1 SolidSolubilities..................................................................................41.3.2 MultipleFluidPhaseEquilibrium.......................................................6

1.4 PhaseEquilibriumEngineeringofSupercriticalProcesses...........................81.4.1 UnderstandingPhaseBehavior............................................................9

1.5 ConceptualSupercriticalProcessDesign..................................................... 111.5.1 OxychemicalExtractionandDehydration......................................... 111.5.2 ParticleMicronizationwithSupercriticalFluids............................... 151.5.3 Extraction,Purification,orFractionationofNaturalProductswith

SupercriticalFluids............................................................................ 171.5.3.1 FractionationofOils............................................................. 171.5.3.2 ExtractionfromVegetableMatrices.................................... 18

1.5.4 SupercriticalReactions...................................................................... 19References................................................................................................................ 21

�.� IntroduCtIon

Solventsareusedinlargeamountsinthechemical,pharmaceutical,food,andnatural-product industries. In thesearch forenvironmentally friendlysolvents, increasingattentionisbeingpaidtosupercriticalfluids(SCFs)forawidevarietyofapplica-tions.Forinstance,supercriticalsolventsareusedinextractions,materialprocessing,micronization,chemicalreactions,cleaning,anddrying,amongotherapplications.SCFsandnear-criticalfluidsaddanewdimensiontoconventional(liquid)solvents: their density-dependent solvent power.ThedensityofSCFscanbeeasilytunedtotheprocessneeds,withchangesintemperature,pressure,and/orcomposition.OtherimportantpropertiesofSCFsaretheirverylowsurfacetensions,lowviscosities,andmoderatelyhighdiffusioncoefficients.

7089_C001.indd 1 10/8/07 11:10:40 AM

� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds

Thedesignofprocessesusingsupercriticalsolventsisstronglydependentonthephaseequilibriumscenario,whichishighlysensitivetochangesinoperatingcondi-tions.Therefore,phaseequilibriumengineeringplaysakeyroleinthesynthesisanddesignoftheseprocesses.

�.� superCrItICal FluIds

The different physical states of a pure substance can be visualized in a three-dimensionalpressure–volume–temperature(PVT)diagram,asshowninFigure1.1.Thesurfacesrepresentthedifferentstates—solid,liquid,orvapor—thatcorrespondtoparticularvaluesofpressureandtemperature.Accordingtothephaserule, thetwo-phase(solid–liquid,solid–vapor,andliquid–vapor)regionsofapuresubstancehaveonlyonedegreeoffreedom.Therefore,theequilibriumpressureineachcaseisafunctionoftemperature.ThePTprojectionsofthesolid–liquid,solid–vapor,andliquid–vaporequilibriumlinesareshownontheleftofFigure1.1.Inparticular,thevapor–liquidlinerepresentsthevaporpressurecurvethatstartsatthetriplepoint(TP)ofsolid–liquid–vaporcoexistenceandendsatthecriticalpoint(CP).ThenatureoftheCPcanbeunderstoodfollowingthechangesofthefluidpropertiesalongthevaporpressurecurve.Withincreasingvaluesoftemperature,thedensityoftheliquidphasediminishesandthevapordensityincreasesduetothehighervaporpressure.Eventually,bothdensitiesconvergeat theCPanddifferentiating the liquidor thevaporstateisnolongerpossibleabovethecriticaltemperature.Whenbothtempera-tureandpressureareabovethecriticalvalues(Figure1.1),thesystemisconsideredtobeinthesupercriticalregion.

Withinaregionclosetothecriticalconditions,thesystempropertiesarehighlysensitivetopressureandtemperature;thisregionisconsiderednear-critical.Usually,theSCFsolventisappliedatatemperatureclosetoitscriticalvalueandatapres-surehighenoughforitsdensitytobecomegreaterthanthefluidcriticaldensity.A

Volume

Pc

Tc

Vc

Vapor

Solid

Liquid

sv

lv sl

slv TP

sl

lv

sv

Pres

sure

Tempera

ture

Supercritical Region

CP CP

FIgure �.� PVTdiagramofapuresubstanceanditsprojectiononthePTplane.

7089_C001.indd 2 10/8/07 11:10:55 AM

Fundamentals of Supercritical Fluid Technology �

listoffluidsthathavebeenproposedasSCFsolventsisshowninTable1.1.Thesefluidscanbeclassifiedasa) low-critical temperature(low-Tc)andb)high-criticaltemperature(high-Tc)solvents.Somecondensablegases,likecarbondioxide(CO2),ethane, andpropane,areconsidered low-Tcsolvents,whereas thehigheralkanes,methanol, andwater can be considered high-Tc solvents. Strong differences insolventpowerandselectivitycharacterizethelow-Tcandhigh-Tcsolvents.

Francis[1]madeasignificantcontributiononthesubjectofCO2solventproper-tiesbystudyingitsbehaviorwithalargenumberofsolutes.LiquidCO2ismisciblewithalkanesuptoapproximatelycarbonnumber10,while therangeofmiscibil-ity increasesforethaneup to20,andpropaneup to35.Therefore, thesesolventsshowselectivityforrelativelylow-molecular-weightmaterial.StahlandQuirin[2]havereportedtheextractabilityofawiderangeofnaturalproductsusingCO2;theyshowedthat:“1)hydrocarbonsandotherlipophilicorganiccompoundsofrelativelylowmolecularmassandpolarityareeasilyextractable;2)theintroductionofpolarfunctionalgroups,hydroxylorcarboxylgroupsrendertheextractionmoredifficultorimpossible;3)sugarsandaminoacidscannotbeextracted;4)fractionationeffectsarepossibleiftherearemarkeddifferencesinmass,vaporpressureorpolarityoftheconstituentsofamixture.”

Regardingtheuseofhigh-Tcsolvents,suchastolueneorwater,theextractioniscarriedoutat temperaturesfrom500to700K,whereevenamildpyrolysisofhigh-molecular-weightmaterialtakesplace.Thesolventpowerofhigh-Tcfluidsismuchhigherthanthatoflow-Tcsolvents,andhigh-Tcsolventsarepropersolventsfor high molecular weight materials. However, they have low selectivity and thesevereoperatingconditions,ontheotherhand,degradethermallylabilematerials.Agoodfeatureoflow-Tcsolvents,ascomparedwithconventionalliquidsolvents,is that theyoperate atmoderate temperature andhave low solvent power.There-fore, by carefully choosing the pressure and temperature of operation, selectivefractionscanbeextractedfromvegetablematrices,suchasessentialoils,alkaloids,lipids, or oleoresins. These are the preferred solvents for the pharmaceutical andnatural-productindustries.Akeyadvantageoflow-Tcsolventsisthattheyareeasilyseparatedfromtheextract.

table �.�Critical properties of Fluids of Interest in supercritical processes

FluidCritical temperature

tc/KCritical pressure

pc/barCritical Volume Vc/cm�·mol–�

CO2 304.12 73.7 94.07

Ethane 305.3 48.7 145.5

Propane 369.8 42.5 200.0

Water 647.1 220.6 55.95

Ammonia 405.4 113.5 72.47

n-Hexane 507.5 30.2 368.0

Methanol 512.6 80.9 118.0

7089_C001.indd 3 10/8/07 11:10:57 AM


SCF-soluteinteractionsintheliquidphasemayoriginateasecondliquidphase(gas salting out effect), improving process selectivity and making it possible, forinstance, to separate chemical reaction products in situ [3]. A better understand-ingofsupercriticalsolventpropertieswillbeobtainedafterconsideringthephaseequilibriumbehaviorofbinarysystemsthatshowadifferentdegreeofasymmetryinsizeorintermolecularinteractions.

1.2.1 Physical ProPerties of suPercritical fluids

ThephysicalpropertiesofSCFsarein-betweenthoseofagaseousandliquidstates.Typical values of different physical properties for each fluid state are listed inTable1.2.

DensityandviscosityofSCFsarelowerthanthoseofliquids;however,diffusivi-tiesarehigher.ThermalconductivitiesarerelativelyhighinthesupercriticalstateandhaveverylargevaluesneartheCPbecause,inprinciple,theheatcapacityofafluidtendstoinfinityattheCP.Interfacialtensionisclosetozerointhecriticalregion.Ingeneral,thephysicalpropertiesinthecriticalregionenhancemassandheattransferprocesses.

�.� phase equIlIbrIum wIth superCrItICal FluIds

1.3.1 solid solubilities

TheconditionsofphaseequilibriumbetweenaSCF(1)andasolidcomponent(2)areformulatedonthebasisoftheisofugacitycriterion.Ifthesolidphaseisassumedtobeapurecomponent(2),thesolubilityinthegasphasecanbedirectlyobtainedas:

y E

p

P

s

22= (1.1)

table �.�Comparison of the physical properties of gas, liquid, and supercritical Fluidsphysical property gas (tambient) sCF (tc, pc) liquid (tambient)

Densityr(kgm–3) 0.6–2 200–500 600–1600

Dynamicviscositym(mPa.s) 0.01–0.3 0.01–0.03 0.2–3

Kinematicviscosityha(106m2s–1) 5–500 0.2–0.1 0.1–5

Thermalconductivityλ(W/mK) 0.01–0.025 Maximumb 0.1–0.2

DiffusioncoefficientD(106m2s–1) 10–40 0.07 0.0002–0.002

Surfacetensionσ(dyn/cm2) — — 20–40

a Kinematicviscositydefinedasη = µ/ρb Thermalconductivitypresentsmaximumvaluesinthenear-criticalregion,highlydependent

ontemperature

7089_C001.indd 4 10/8/07 11:10:58 AM


whereEistheenhancementfactorovertheidealsolubilityand ps2 isthesublima-

tionpressureofthesolute(2).Foralow-volatility,incompressiblesolidsolute,theenhancementfactorcanbecalculatedasfollows:

E

P p vRT

S sol

=

−

exp

( )2 2

2Φ (1.2)

whereΦ2isthefugacitycoefficientofthesolidsoluteinthegasphaseandv2

solisthesolidmolarvolume.Φ2isstronglydependentontheSCFdensity.Figure1.2showstheregionofSCFextraction.Thisregionischaracterizedbyastrongvariationoffluiddensitywithpressure,attemperaturesclosetotheSCFcriticaltemperature.Foragivenisotherm,theincreaseinsolubilitycloselyfollowstheincreaseindensity,as indicated inFigure1.2.Thedrastic increase insolubility in thevicinityof thecriticalregioncanbeofseveralordersofmagnitudeandismainlyduetoasharpdecreaseofthesolutefugacitycoefficientΦ2inthefluidphase.Thisistheclassicalenhancementeffectatthenear-criticalregion.

Theinfluenceoftemperatureonthesolidsolubilityistheresultoftwocompet-ingeffects:theincreaseofsolidvolatilityandthedecreaseofsolventdensitywithtemperaturerise.Nearthecriticalpressure,theeffectoffluiddensityispredominant.Therefore, a moderate increase in temperature leads to a large decrease in fluiddensityandaconsequentreductioninsolutesolubility.However,athigherpressures,the increase of solid sublimation pressure with temperature exceeds the densityreductioneffect,andthesolubilityincreaseswithtemperature.Thisbehaviorleadstoaregionofretrogradebehaviorofthesolidsolubility,asillustratedinFigure1.3.AtpressureswellabovetheSCFcriticalpressure,theisothermsexhibitamaximumin solubility.Thismaximum is usuallyobserved in the rangeof30 to100MPa.

Pressure

Den

sity

(δ)

Near Critical Region

Solu

bilit

y (y 2

)

T1

T1 < Tc1 < T2

T2

Pc1

FIgure �.� Density(δ)andsolidsolubilityinfluidphase(y2)asafunctionofpressure.

7089_C001.indd 5 10/8/07 11:11:01 AM


KurnikandReid [4]have shown that themaximum is achievedwhen thepartialmolarvolumeofthesoluteinthefluidphaseisequaltoitssolidmolarvolume.

Aquantitativecorrelationandpredictionof thesolubilityofapuresolid inaSCFispossibleifthefugacitycoefficientofthesolidinthefluidphaseiscomputedusinganequationofstate.Cubicequationsofstate,withconventionalmixingrulesandadjustablebinaryinteractionparameters,havebeenwidelyusedsincetheearlyworks of Deiters and Swaid [5] and Kurnik and Reid [4]. However, equations ofstate thatuseclassicalmixingrules,evenwithenergyandsizebinaryinteractionparameters, may fail to predict or correlate the solubility of solids with polar orhydrogen-bonding interactions.For instance,Kurnik andReid [4] found that thisapproachisnotabletomodelthesolubilityofstearicacidorn-octanolinCO2.Thelimitationsofcubicequationsofstatetomodelthesolubilityofpolarsolidscanbetackledbyusingcubicequationsofstatewithlocalcompositionmixingrules[6].

Whenanonpolarsupercriticalsolvent isused, theseparationprocessdoesnotpresent specific selectivities; in this case, the addition of a proper cosolvent canenhancesolubilityandselectivity.Nonpolarcosolventsincreasethesolubilityofsolidaromaticsseveraltimes,whereaspolarcosolventsenhancethesolubilityofsolutesthat present specific interactions with the cosolvent. For example, Brenecke andEckert[7]showedadramaticeffectofthecosolventtributilphosphateonthesolubil-ityofhydroquinoneinCO2.Thecosolventselectionfollowsthegeneralrulesappliedforclassicsolventselectioninsolidorliquid-liquidextraction.Brunner[8]studiedtheeffectsofcosolventsontheextractionoflow-volatilityliquidsandshowedthattheuseofacetoneormethanol,forinstance,improvesselectivityandsolventpowerintheextractionofhexadecanolfromoctadecane.

1.3.2 MultiPle fluid Phase equilibriuM

Equilibriumpredictionsinsystemshavingtwoormorefluidphasesaremorecom-plexthanthoseincasesofsolidsolubilitiesduetotheneedtocomputefugacities

T2

T1

Pressure

T1 > T2 > Tc1

y2

Pc1

Supercritical Region

FIgure �.� TypicalisothermsofsolidsolubilityinSCF.

7089_C001.indd 6 10/8/07 11:11:02 AM


inseveralphasesofdifferentcompositions.Theuseofthesameequationofstatetocomputefugacitycoefficientsinallphasesgivestherequiredcontinuityinthepre-dictionofphaseequilibriumatthecriticalregion.CubicequationsofstateofthevanderWaalsfamilyhavebeensuccessfullyappliedinthecorrelationandpredictionofphaseequilibria inmixturesofsubcriticalandsupercriticalnonpolarcomponentsinthenaturalgasandpetrochemicalindustries.However,theirapplicationtosizeand energy asymmetric systems, typical of the supercritical extraction of naturalsubtracts, has found little success. De la Fuente et al. [9] tried to correlate bothvapor-liquidequilibrium(VLE)andliquid-liquidequilibrium(LLE)ofthesystemsunfloweroil+propaneusingtheSoave[10]equationofstatewithquadraticmixingrulesandbinaryinteractionparametersforboth,theattractiveenergyparameterandthecovolume.ItwasnotpossibletoquantitativelydescribebothVLEorLLEusingonlyonesetofparametersfortheattractiveenergyparameterandthecovolume.ThisindicatesthelimitationsofthevanderWaalsrepulsivetermtodescribetheseasym-metricmixtures.Thefailureofcubicequationsofstatetomodelphaseequilibriainsizeasymmetricmixturescanbeattributedtothelargedifferencesinthepure-componentcovolumes[11].

Espinosaetal.[12]andFerreiraetal.[13]extensivelydiscussedtheapplicationofequationsofstatetomodelthesupercriticalprocessingofnaturalproducts.Agroupcontribution approach is particularly useful when dealing with natural productsbecausealargenumberofcompounds,suchastriglycerides,fattyacids,esters,andalcohols,canberepresentedwithasmallnumberoffunctionalgroups.Groupcon-tributionequationsofstate,suchasModifiedHuron-Vidal2(MHV2)[14,15]andgroupcontributionequationofstate (GC-EOS)[16,17],areparticularlyuseful tomodelthecomplexphasebehaviorobservedinasymmetricmixturesatnear-criticalconditions.Bottinietal.[18]extendedtheGC-EOSmodeltodescribebothVLEandLLEinmixturesofsupercriticalgases+vegetableoilmixturesusingthesamesetofparameters.Grosetal.[19]andFerreiraetal.[20]extendedthismodeltorepresentassociatingmixtures(GCA-EOS),usingagroupcontributionapproachfordealingwith self- andcross-associations.TheGCA-EOSequationcanbederived fromathree-term(repulsive,attractive,andassociating)Helmholtzresidualenergy:

A=Arep+Aatt+Aassoc (1.3)

The repulsive (rep) term is givenby theCarnahan-Starling equation for hardspheres, the attractive (att) term is a group contribution version of a density-dependent local composition Non-Random Two Liquids (NRTL) model, and theassociation (assoc) term is a group contribution expression based on Wertheim’sstatisticalassociationfluidtheory[21].ThehardspheretermperformsbetterthanthevanderWaalsrepulsivetermwhendealingwithhighlysize-asymmetricsystemsandtheothertwotermsareabletohandlestrongnonidealspecificinteractions.TheGC-EOSmodelwascompared toMHV2andPSRK[22] byEspinosaetal. [23].Allthreemodelsperformsimilarlyformoderatelypolarsystemsoflowmolecularweightcompounds.However,theMHV2andPSRKmodelspresentsomelimitationswhentheyareappliedtoveryasymmetricsystems.

7089_C001.indd 7 10/8/07 11:11:02 AM


�.� phase equIlIbrIum engIneerIng oF superCrItICal proCesses

Phase equilibrium engineering is the systematic application of phase equilibriumknowledgetoprocessdevelopment.Thisknowledgecomprisesdatabanks,experi-mentaldata,phenomenologicalphasebehavior,thermodynamicanalysis,andmath-ematical modeling procedures for phase equilibrium process calculations. EachSCFapplicationhasasetofspecificationsandphysicalrestrictions.Insupercriticalreactions,forinstance,homogeneousphaseconditionsmayberequiredatthereactiontemperature.Thesolutiontothisproblemisgivenbytheselectionofthepropersolventandthedeterminationofafeasibleoperatingpressurerangeandfeedcompositiontoachieve homogeneity in the reaction mixture. On the other hand, aheterogeneoustwo-phasesystemmayberequiredtodevelopsupercriticalextractionorfractionationprocesses.Additionalphaseequilibriumrestrictionsmayincludenosolidphasepre-cipitation,azeotropeformation,specificsolventsolubilities,orsaturationconditions.

Amulticomponentfluidcanbeasupercriticalmixture,asubcooledliquid,asuper-heated vapor, or a heterogeneous liquid-liquid, liquid-vapor, or liquid-liquid-vapormixture.Ausefulplottoidentifyeachregionisapressurevs.temperaturediagramshowingthebubbleanddewpointphasetransitionscurves,aswellastheCPofagivenglobalcomposition.Theselinesdeterminethemixturephaseenvelope.Differentphasescenarioscanbeselectedfromthisphaseenvelope(Figure1.4):a)homogeneouscon-ditionsforasupercriticalreaction,b)homogenousandheterogeneousconditionsforatunablephasesplitreactor,orc)phasesplitforaseparationprocess.Certainly,differentphaseenvelopesareobtainedduringthecourseofthereactionorseparationprocess.However,theprocesstrajectoryshouldalwaysremainattherequiredphasescenario.Generalconditionscanalsobesetfromthisplot;forinstance,abovethemaximumpressureofthephaseenvelopetherewillbeasinglephaseatanytemperature.

Rigoroussimulationsofequilibriumstageseparationsatnear-criticalconditionsare needed for the design and optimization of supercritical processes. However,equilibriumcalculationsinthenear-criticalregioncanpresentseriousconvergence

Sometimes We Look for Bothb) Tunable Reactors

Sometimes We Look for Phase Splitc) Separation Processes

Temperature

Pres

sure

Sometimes We Look for Homogeneitya) Supercritical Reactions

Heterogenous RegionBubble P

oint C

urve

Liquid

Vapor

CriticalPoint

Dew Point Curve

FIgure �.� Possibleprocessphasescenarios.

7089_C001.indd 8 10/8/07 11:11:04 AM


difficulties.Inthatrespect,Michelsen’s[24]phasestabilitycriterion,multiple-phaseflashalgorithms,andglobalphasecomputationsareofparticularinterestforsuper-criticalextractionapplications.

Solventrecycleisamajorissueintheeconomicoptimizationoftheseprocesses,becauseitisthemainfactorindeterminingcapitalandoperatingcosts.Designandsynthesis problems have been increasingly solved by formulating mathematicalmodels,whichinvolvecontinuousandintegervariablestorepresentoperatingcon-ditionsandalternativeprocesstopologies[25].Withregardtosupercriticalextrac-tions,Grosetal. [26]haveaddressed thesynthesisofoptimumprocesses for theextractionanddehydrationofoxychemicalsasamixedintegernonlinearprogram-mingproblem.Espinosaetal.[23]andDiazetal.[27]haveappliedtheseproceduresforthesynthesisandoptimizationofcitrusoildeterpenationprocesses.

1.4.1 understanding Phase behavior

VanKonynenburgandScott[28]haveshownthatthefluidphasebehaviorobservedinbinarymixturescanbeclassifiedintofivemaintypes.IntypeIphasebehavior,com-pleteliquidmiscibilityisobservedatalltemperatures.Whenpartialliquidmiscibilityoccursatlowtemperatures,thesystemisoftypeII.TypeIphasebehaviorisusuallyfoundinsystemswithcomponentsofsimilarchemicalnatureandmolecularsize,likemixturesofhydrocarbons,noblegases,orsystemsthatdonotdeviategreatlyfromidealbehavior.TypeII is typicalofnonidealmixturesofsimilarsizecompounds,inwhichnonidealityleadstoliquidphasesplitatsubcriticalconditions.Whentheliquidimmiscibilitypersistsevenathighpressuresandtemperatures, thesystemsareoftypeIII.Thisbehaviorischaracteristic,forexample,ofmixturesofCO2withhigh-molecular-weight alkanes or vegetable oils. When the difference in molecu-larsizebecomessignificant,inalmostidealsystems,liquid-liquidimmiscibilityisobservednearthelight-componentcriticaltemperature(solventTcinsupercriticalprocesses).However,completemiscibilityisrecoveredatlowertemperatures; thiscorrespondstotypeVphasebehavior.TypeIV,ontheotherhand,showsdiscon-tinuedliquid-liquidimmiscibility(i.e.,liquidimmiscibilityoccursatlowandhightemperaturesbutnotatintermediatetemperatures).Figure1.5isamasterchartofthedifferent typesofbinaryfluidphasediagrams [29].Thearrows inFigure1.5qualitativelyindicatethetypeoffluidphasebehaviorthatcanbeexpectedwhenthesystemcomponentsexhibitgreatermolecularinteractions,sizedifferences,orboth.

Figure1.6illustrates,inmoredetail,aTypeVphasediagram.Thelinesinthisdiagram indicate the boundaries of phase transitions and the critical locus. Thethree-phaseequilibriumline(l1l2g)startsatthelowercriticalendpoint(LCEP)andfinishesattheuppercriticalendpoint(UCEP).Thisbehavioristypicalofmixturesofpropanewith triglycerides,suchassunfloweroilor tripalmitin [30].When theprocess operating temperatures are above the critical temperature of the solvent,pressuresshouldbehigherthanthecriticalpressureofthemixtureinordertoensurecompletemiscibility(i.e.,thepressureshouldbeabovethel1=l2line).

In the search for an adequate supercritical solvent to achievehomogenous orheterogeneousconditions,twodifferentapproachescanbefollowed:1)tocomparethephasebehaviorofagivensubstratewithdifferentsolventsor2) to followthe

7089_C001.indd 9 10/8/07 11:11:04 AM

�0 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds

change in thephasebehaviorofagivensolventwithdifferent familiesofchemi-calcompounds.Inthemoregeneralcase,whenthecomponentsofthemixtureareofdifferentchemicalnature, thesecondapproachshouldbefollowedtotakeintoaccountanypossiblechangeinthephasebehaviorduringprocessevolution.

Theliquid-liquidimmiscibilityoftypeVphasebehaviorappearsinmanybinarymixtures between supercritical solvents and organic substrates beyond a certaincarbon number. Figure1.7 shows the regions of liquid-liquid immiscibility forbinarymixturesofsupercriticalsolvents(ethaneandpropane)withhydrocarbonsofdifferentchainlength[31].Peters[31]alsopresentedsimilardataontheliquid-liquid

T

P

lg(2)

lg(1)

l1 = l2

l1 = g

l1l2g

l2 = g

UCEP

LCEP

FIgure �.� TypeVphasebehavioraccordingtoVanKonynenburgandScottclassification.

CL

CL

CL

CL

CL

CH

CH

CH

CH

CH

T

T T

P

P P

P Type II

Type IV Type I

T

Type V

Type III

Molecular Interaction



+ Size

Size

Size

Pure Component Vapor Pressure Critical Locus �ree Phase Region (LLV)

P T


+ Size

FIgure �.� Modificationsofbinaryphasebehaviorwith sizeandenergyasymmetries.CLandCHarethecriticalpointsofthelightandheavycompounds,respectively.

7089_C001.indd 10 10/8/07 11:11:07 AM

Fundamentals of Supercritical Fluid Technology ��

immiscibilitydomainsofthesystemsethane+alcohols,ethane+aromatichydro-carbons,andethane+alkanes.Itbecomesclearfromthesedatathatethaneisnotanadequatesupercriticalsolventfornormalalcoholsbecauseitpresentsliquid-liquidimmiscibilityevenwithmethanol.However,ethaneseemstobeabettersolventforaromatichydrocarbonsorparaffinsbecausetheliquid-liquidimmiscibilityappearsatcarbonnumbersgreaterthan15or18,respectively.

CO2 has been the most studied solvent for supercritical processes. However, itexhibitsstrongliquid-liquidandgas-liquidimmiscibilityforhydrocarbonswithcarbonnumbersgreaterthan13.Inaddition,CO2presentsaratherlowcriticaltemperaturetobeusedas a solvent for reactionscarriedout atmoderateorhigh temperatures.Figure1.8showsdataonthetypeofphasetransitionforthefamiliesofCO2+alkanescompiledbyPeters[32],whoalsoshowedthebehaviorofCO2+alkanolsystems.

Unfortunately,thetypeofdatashowninFigures1.7and1.8isonlyknownforalimitednumberoffamiliesoforganiccompoundswithsomesupercriticalsolvents.Therefore,reliablethermodynamicmodelsareneededtoexplorethepossiblephasescenariosfoundinmixturesbetweenprocesscomponentsandsupercriticalsolvents.

The phase equilibrium engineering approach will be illustrated with severalexamples,where thermodynamicandmodeling toolsareapplied for supercriticalprocessdevelopment.Theexamplestobecoveredarealcoholextractionanddehy-dration,gasantisolventcrystallization,purificationofvegetableoils, supercriticalfractionation,extractionwithnearcriticalfluids,andsupercriticalreactions.

�.� ConCeptual superCrItICal proCess desIgn

1.5.1 oxycheMical extraction and dehydration

Thesupercriticalextractionoforganicoxygenatedcompoundsfromaqueoussolu-tionsisofgreatinterestinbiotechnologicalprocesses.Oxygenatedcompoundsand

280

300

320

340

360

380

400

10 15 20 25 30 35 40 45 50 55 60 65 70

UCEP

UCEP

LCEP

LCEP

Ethane

Propane

SL1L2V

SL1L2V

Number of Carbon Atoms

Tem

pera

ture

, (K)

FIgure �.� Phasetransitionsforthebinariesofethaneandpropanewithparaffinsofdif-ferentchainlength.UCEPandLCEPpointsareupperandlowercriticalendpoints,respec-tively.SL1L2Vstandsforsolid–liquid–liquid–vaporequilibria.

7089_C001.indd 11 10/8/07 11:11:08 AM

�� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds

water have strong hydrogen bonding interactions that complicate their separationwithconventionalsolvents.Moreover,anoxygenatedcompounddissolvedinanon-

polarnear-criticalsolventwillhavearatherhighactivitycoefficient( γ oxySCF),leading

toalowvalueofthedistributioncoefficient:

moxyoxyH O

oxySCF

=γγ

2

(1.4)

This is even more pronounced in the case of alcohols or acids that exhibit self-association.AstrategytoovercomethisproblemmaybebasedontheKoenenandGaube [33] diagram that classifies binary mixtures in an excess Gibbs function(GE)versusexcessenthalpy(HE)diagram(Figure1.9).Wecanderivetheeffectoftemperature on the activity coefficients directly from this diagram. The aqueoussolutionsoforganicoxygenatedcompoundsarelocatedonthesecondquadrantofthediagramwithnegativeHEvalues,whereasthesupercriticalsolutionsthatcorrespondtopositiveHEvaluesarelocatedonthefirstquadrant.Inbothcases,therearepositivedeviationstononideality(positiveGE).Fromthisdiagram,wecanseethattheactiv-itycoefficientsintheaqueousphaseincreasewithtemperature;however,thereverseoccurswiththeactivitycoefficientsintheSCFphase.Therefore,extractingathightemperatures leads to more attractive values of the distribution coefficients. ThisbehaviorisfoundintheextractionofisopropanolorethanolfromaqueoussolutionsusingCO2,ethane,orpropaneasnear-criticalsolvents.However,weshouldconsideranotherfacttomakeapropersolventselection:Atoptimumextractiontemperatures(around380 to400K), the solvent powerofCO2or ethane is drastically reducedduetofluiddensitydecreaseattemperatureswellabovethecriticaltemperatureofbothfluids(around304K).Torecoverthesolventpower,relativelyhighpressuresshouldbeusedfortheextractionprocess.Thismakespropaneabettercandidateas

200

220

240

260

280

300

320

340

5 7 9 11 13 15 17 19 21 23 25

Type II Type VType IV

UCEP

LCEP

Number of Carbon Atoms

Tem

pera

ture

, (K)

FIgure �.� PhasetransitionsforthebinariesofCO2withparaffinsofdifferentchainlength.UCEPandLCEPpointsareupperandlowercriticalendpoints,respectively.Dashedline(opensquares):SL1L2Vequilibria.

7089_C001.indd 12 10/8/07 11:11:11 AM


anextractionsolventbecauseitscriticaltemperatureiscloseto370KandithasalowercriticalpressurethanCO2orethane.Horizoeetal.[34]andBrignoleetal.[35]

verifiedthepotentialofpropaneasanextractingsupercriticalsolvent.Dehydrationbynear-critical solventsfinds importantapplications, forexample,

intheextractionofsolutesfromaqueoussolutionsandinthedryingofsolidparticlesaftermicronization.Wewillconsiderfirstthedehydrationofextractedsolutes.Inlow-pressure separations, entrainment agents like cyclohexane or solvents like ethyleneglycolhavebeenusedtoseparatewaterbyazeotropicorextractivedistillations.Incon-nectionwithsupercriticalprocesses,itisofinteresttostudytheequilibriumbetweenwaterandanear-criticalfluidasafunctionoftemperatureandpressure.InthecaseofCO2,thedataofWiebbe[36]andCoanetal.[37]showthesolubilityofwaterinCO2asafunctionofpressureatsubcriticalandsupercriticaltemperatures.Thesedataindicatethatwaterfollowstheclassicalsupercriticaleffect:theconcentrationofwaterintheCO2phaseincreasesoncethesupercriticalpressureisexceeded(Figure1.10).

AttheCO2saturationpressure,atsubcriticalconditions,wewouldhaveathree-phase VLL equilibrium condition, where the concentration of water in the con-densedCO2phaseexceedstheconcentrationofwaterinthevaporphase.Hence,inaCO2–waterseparationprocess,therelativevolatilityofwaterwithrespecttoCO2islowerthanone.ThisbehaviorhasimportantconsequencesfortheseparationofwaterfromCO2extracts.Water,asexpected,islessvolatilethanCO2;therefore,theextractcannotbeobtainedfreefromwaterinthesolventrecoveryoperation.

Whenthesamephaseequilibriaanalysisismadeforwaterandlightalkanes,suchasethaneandpropane,adifferentpictureisobtained.ThedataofKobayashiandKatz[38]forthesolubilityofwaterinpropaneareplottedagainstpressureatdifferenttemperatures(Figure1.11).Fornear-criticalpropane,thesolubilityofwaterdecreaseswhenthecriticalpressureisexceeded(seeTable1.1forthecriticalproper-tiesofpropane).Thisphenomenoncanbecalledanonclassical supercritical effect.

< 0

GE

HE

γ > 1

Regular SolutionSE= 0

> 0δγδT

γ < 1

> 0δγδT

γ < 1

< 0δγδT

γ > 1

< 0δγδT

FIgure �.� Value and temperature derivative of activity coefficients, according to therelativevaluesofGEandHE.

7089_C001.indd 13 10/8/07 11:11:12 AM


Averyattractivepropertycanbederivedfromthiseffect.Whenworkingatsubcriti-caltemperatures,atthepropanesaturationpressure,weagainhaveVLLequilibria.Inthiscase,thecompositionofwaterinthevaporphaseisgreaterthanthatinthecondensedpropanephase,leadingtoawater-propanerelativevolatilitygreaterthanone.Thismakesitpossibletoobtaindehydratedorganicoxygenatedproductsduringtheprocessofsolventrecoveryfromtheextract[39].

0

0.002

0.004

0.006

0.008

0.01

0.012

0 200 400 600 800Pressure, (bar)

Gra

ms o

f Wat

er P

er L

iter o

f Exp

ande

d G

as at

s.t.p

.

298 K323 K348 K

FIgure �.�0 CompositionoftheCO2-richphaseasafunctionofpressureandtempera-ture.ExperimentaldatafromWiebbe[34].

0.1

1

10

100

0 20 40 60 80 100P(bar)

Mol

e Fra

ctio

n %

of W

ater

327.6 K

377.6 K

369.6 K

FIgure �.�� Experimental water composition in liquid and vapor propane. Data fromKobayashiandKatz[38].

7089_C001.indd 14 10/8/07 11:11:13 AM


On thebasisof thephase equilibriumengineering concepts presented above,a process for the production of bioethanol or for the dehydration of isopropanolwithanear-criticalsolvent(propane)canbedeveloped.Thekeyfeaturesoftheseprocessesare:

a) Hightemperaturesandpressuresofextractionfavorthesolubilityofalcoholinpropane.

b) Liquid-liquidequilibriumatlowtemperaturesisbeneficialforreducingthewatercontentintheextract.

c) Thealcoholproductisobtaineddehydratedbecausetherelativevolatilityofwaterwithrespecttopropaneisgreaterthanoneoveracertainconcen-trationrangeofethanolintheextractmixture.

All these properties were first predicted by group-contribution thermodynamicmodelingandthereafterverifiedbyexperimentalandpilotplantinformation.

1.5.2 Particle Micronization with suPercritical fluids

Supercriticalmicronizationprocessesarebasedoncreatingahighdegreeofsolutionsupersaturationthatleadstotheformationofagreatnumberofnucleationsitesandverysmallcrystals.Theseprocesseshavefoundmanyapplicationsinthelastdecade[40,41],mainlyinthemicronizationofpharmaceuticalsolidcompounds.Usually,several components may participate in the process: the solute to be crystallized,thesolvent,a supercriticalfluid,andacosolvent.Thephaseequilibriumbetweenthesecomponentsplaysakeyroleintheselectionofthepropertechnologyforthemicronizationprocesses.Abetterunderstandingofprocessselectioncanbemadeon thebasis of thebinaries behavior.First,we shall consider the solute+ super-criticalfluidbinary. If the solute solubilityunder supercriticalconditions ishigh,thenonlythesecomponentsparticipateintheprocessandmicronizationisobtaineddirectlybyadrasticreductioninthesolutesolubilitybytherapidexpansionofthesupercriticalsolution(RESSprocess)throughanozzleorotherconvenientdevice.ThemainlimitationofthisRESSprocessisthatitcanonlybeappliedtosoluteswithhighsolubilitiesinthesupercriticalfluid.ThelowsolventpowerofsupercriticalCO2forpolarormedium-tohigh-molecular-weightmaterialmakesthisapproachuneconomicalforthesemixtures.

Whenthesolutecannotbedissolvedinsignificantamountsinthesupercriticalfluid,wecanlookforagoodliquidsolventforboththesoluteandthesupercriticalgas.Inthiscase,aconcentratedsolutionofthesoluteinthesolventispreparedandahighdegreeof supersaturation isobtainedbydissolving the supercriticalfluidintheliquidphaseathighpressure.Thistechnologyiscalledthegasantisolvent(GAS)processanditcanbecarriedoutinabatchorsemicontinuousprocess.Theseprocessescanbeappliedtoavarietyofsolutes,butinthiscase,theternaryphaseequilibria shouldalsobeevaluated toassureahighdegreeof supersaturationattheoperatingpressureand temperature. In the semicontinuousprocess,both thesolutionandthesupercriticalfluidentertogetherintheprecipitationvesselthrough

7089_C001.indd 15 10/8/07 11:11:13 AM


amixingdevice.Verygoodprecipitationconditionsareachievediftheoperatingconditions are above the CP of the solvent + supercritical fluid mixture. Undertheseconditions,bothfeedsarecompletelymiscibleandnointerfacialresistanceisofferedtomasstransfer[42].

AnotherpossiblephasescenarioappearswhenthesolidsoluteisnotsolubleintheSCF,butthesolubilityoftheSCFinthemeltedsolidishighatelevatedpres-sures.Therefore,ifthesolutionisexpandedtoatmosphericpressure,alargecoolingeffectoccursthatgivesrisetotheprecipitationofmicronizedsoluteparticles.

Adifferentsituationariseswithsolutesthatareonlysolubleinwater,suchassomeorganicsaltsandproteins[41].TypicalnonpolarsupercriticalfluidslikeCO2andethanearenotsolubleinaqueoussolutions,evenathighpressures.Therefore,noantisolventeffectcanbeobtainedinatypicalGASprocess[43].Inthiscase,acosolventthatshowscompletemiscibilitywithboththeSCFandwatercanbeintro-duced.Forexample,ethanolwasusedasapropercosolventfortheprecipitationofanorganicsaltfromaqueoussolution[43].Inthisapplication,theaqueoussolutionisfedasasprayormistintoaprecipitationvesselalreadyfilledupwithamixtureofethanol+CO2attherequiredcomposition.Toobtainafeasibleprocess,theoperat-ingconditionsoftheprecipitationchambershouldlieinsidethehomogeneousregionofthetriangularphasediagramforwater+CO2+ethanolatagivenpressureandtemperature,asshowninFigure1.12.Inthisway,thefinewaterdropletsbecomequicklysupersaturatedbytheethanol+CO2dissolutioninthedropsandthesimulta-neousfastevaporationofwater.Asaresultofthisprocess,highlymicronizeddriedsaltparticlesareobtained[43].Alltheseexamplesillustratethataphaseequilibriumengineeringanalysisisaprerequisiteforpropertechnologyselectionandsuccessfuladequatechoiceofmicronizationoperatingconditions.

CO2

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Ethanol

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Water + Lobenzarit

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Feasible Region

A

B

FIgure �.�� Feasible operating region for Lobenzarit precipitation using supercriticalCO2andethanolascosolvents.

7089_C001.indd 16 10/8/07 11:11:15 AM


1.5.3 extraction, Purification, or fractionation of natural Products with suPercritical fluids

�.�.�.� Fractionation of oils

Intheprocessingofvegetableoils,itispossibletotakeadvantageofthelowsolu-bilityof triglycerides inCO2.For instance,bothpalmoilandsunfloweroilgiveliquid-liquidorliquid-SCFimmiscibilitywithCO2,evenathighpressures,typicaloftypeIIIsystems.Inthesecases,eithersupercriticalorliquidCO2canbeusedasasolventtoremoveundesirablecomponentsfromtheoil—forinstance,removalofoleicacidfromoliveoil[44].Likewise,liquidornear-criticalCO2canbeappliedtorecovervaluablecomponentsliketocopherolsorsqualenefromfishoil[45].Whendealingwiththeseseparationprocesses,itispossibletofindoptimumextractionoperatingconditions thatminimize the solvent-feed ratio and, at the same time,keep the coextraction of oil at a low value. Other solvents that have regions ofliquid-liquidimmiscibilitywithfattyoils,suchasethaneandpropane,maybeusedasalternativesolvents.

SCFsolventscanalsobeusedasfractionatingagents.Thisisofinterestintheseparationof low-volatilesubstancesofcloserelativevolatility.Forinstance,CO2andethanehavebeenproposedasdensegasextractantstoremovetheterpenefrac-tionfromcitrusessentialoils[27]andalsoforthefractionationofhighlyunsaturatedfishoilmethylesterstoobtainricheicosapentaenoicacidanddocosahexaenoicacidfractions[46].ThebinarysystemsbetweenCO2andthesefamiliesofcompoundsare generally of type II, so complete miscibility for all compositions is obtainedabovethemaximumpressureofthevapor-liquidcriticallocus.

Asingledense-gasfractionationcolumnschemeisshowninFigure1.13.Themixturetobefractionatedisfedatanintermediatepointinthecolumn.Adensegas

N=40 N=40 N=40

Feed

Separator

CO2

Heat Exchanger

Extractor

Fresh CO2

Raffinate

N=40

Extract

FIgure �.�� Dense-gasfractionationschemeprocess.

7089_C001.indd 17 10/8/07 11:11:17 AM


(CO2,forexample)isintroducedatthebottomofthecolumnanditflowscountercur-rentlytotheliquidmixturetobeseparatedorenriched.Atthetopofthecolumn,theextractphaseisheatedandexpandedtoalowerpressuretorecoverthelightfractionandCO2isrecycledtothebottomofthecolumn.Acompressororcondenser-pumpcyclecanbeselectedforthispurpose.Thesetypesofseparationprocessesfollowtheprinciplesofastrippingoperation.Oneofthemaindifferenceswithordinarygasstrippingisthatthedensegasisverysolubleinthefeed.Therefore,theliquidphaseflowrateinthecolumnismuchlargerthanthefeedflowrate.Ontheotherhand,thelowvolatilityofthesubstratesbeingfractionatedleadstoarelativelyhighgas-feedstrippingratio.Botheffectscontributetogiveafairlyconstantmolaroverflowforbothphasesinasimplecounter-currentcolumn.Thedesignoftheseseparationprocessesishighlydependentontherelativevolatilitybetweenthekeycomponentsof theoils ineachseparationstage. Itcanbeshown thatasimplecountercurrentseparationislimitedbytherecoveryofeachkeycomponentinthebottomandtopproducts.Inthiscase,thelimitingrecoveriesofthekeycomponents(φ1, φ2)inthetopandbottomproductsaredeterminedbytherelativevolatility(α12)betweenbothcomponentsunderprocessconditions:

α φ φ12 1 21= −/ ( ) (1.5)

In most simple countercurrent extraction columns, this constraint limits therecoveryandpurityoftheproductsintheseparationofcomponentsofcloserelativevolatility.Therefore,theuseofrecycle(reflux)ofthetopproductisrequired:1)toincreaserecoveryandpurityand2) toassure that the trajectoryof theseparationprocessliesinsidethetwo-phaseregion.Thus,thecolumnandseparatoroperatingconditions(pressure,temperature,andcompositions)shouldalwaysbecheckedinordertoverifyaheterogeneousoperation.

�.�.�.� extraction from Vegetable matrices

The extraction of lipids and oils from vegetable matrices has been extensivelycoveredinthemonographeditedbyKingandList[47].Intheextractionoffattyoilsfromgroundedseeds,itisadvantageoustoselectasolventthatpresentscompletemiscibilitywiththeoil.CO2isacheap,nontoxicsolvent;however,theoilsolubilityinthisSCFisverylowevenatpressuresoftheorderof30MPa(typeIIIbinary).Ontheotherhand,liquidpropaneiscompletelymisciblewithvegetableoilsbelowtheLCEPofthisbinary.PropanehastypeIIortypeIVglobalphasediagramswithvegetableoils.Themaindrawbackofusingpropaneasasolventfortheextractionofoilsfromgroundedseedsisthatitisflammable.Recently,Hegeletal.[48]studiedtheuseofpropane+CO2solventmixturesforoilextraction, lookingforefficientandsafesolventmixtures.Peter[45]hasstudiedthesetypesofmixturestoimproveselectivitiesintheseparationoflecithinfromvegetableoils.IntheworkofHegeletal.[48],theselectedphasescenariowastooperateinaregionofcompleteliquidmiscibilityoftheoil+solventmixture,withanonflammablevaporphase.Theselec-tionofoperatingconditionswasbasedonexperimentaldataontheLLVregionatconstant temperature, for the systemsunfloweroil+propane+CO2.Atconstant

7089_C001.indd 18 10/8/07 11:11:18 AM


temperature,forathree-componentsystem,theLLVequilibriumisonlyafunctionofpressure.Therefore,abinodalcurvecanbedrawnonatriangulardiagram,withtielineslinkingthetwoliquidphasecompositionsatspecifiedpressures(seetrian-gulardiagramonFigure1.14).Thebinodalcurvegives theboundaryof theLLVregion.ThediagramalsoshowstheminimumpressureforwhichLLimmiscibilityarisesatagiventemperature.Atlowerpressures(i.e.,lowerCO2composition),thesolvent has complete miscibility with the oil. However, there is also a minimumoperatingpressure toavoidvaporphaseflammabilitybecause, atpressures lowerthanthis,thepropanecontentofthevaporphaseistoohigh.ThefeasibleoperatingregioncanbeeasilydeterminedwiththehelpofFigure1.14.

1.5.4 suPercritical reactions

In general, gas-liquid catalyzed reactions are diffusion controlled. The use of anadequate supercritical SCF can bring the reactive mixture into homogeneous

0

10

20

30

40

50

60

70

80

0 0.2 0.4 0.6 0.8 1.0

0 0.2 0.4 0.6 0.8 1.0

CO2 Weight Fraction

CO2 Weight Fraction

Pres

sure

, (ba

r)

00.10.20.30.40.50.60.70.80.91.0

Prop

ane W

eigh

t Fra

ctio

n

Minimum CO2Content in theGaseous Phase

Liquid-Liquid-Vapor Region

Oil

CO2 + Propane at 308 K

CO2 + Oil + Propane at 308 K

MaximumOperating Pressure

MinimumOperating Pressure

Safe Operating Region

FIgure �.�� Safe operating extraction region at 308 K. Experimental data from Hegeletal.[48].

7089_C001.indd 19 10/8/07 11:11:19 AM

�0 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds

conditions,withtheconsequentreductionofthemasstransferresistancebyelimi-natingthegas-liquidinterfaceandbyincreasingthediffusivityofreactants.There-fore,thereactionrateandselectivitycanbegreatlyincreased.Härrödetal.[49]havestudiedexperimentallythehydrogenationofheavysubstratessuchasvegetableoilsandfattyestersundersupercriticalconditions.

The use of batch reactors is a common practice in bench scale experimentalstudiesonsupercriticalreactions.However,thecontrolofhomogeneousconditionsinthesereactorsisquitedifficult.Baikerandcoworkers[50]recommendtheuseofwindowsinthereactionvesselsinordertocontrolthephaseconditions.Eventhoughitispossibletohaveanindependentcontrolofprocessvariablesincontinuousreac-tors, the selection of pressure, temperature, and composition should be carefullydonetoobtainthedesiredhomogeneousstate.Knowledgeofthephasebehaviorofareactionprocesscanhelptounderstandtheresultsofexperimentalstudiesandtoplananddesignexperimentalruns.

Thesolvent tobeused ina supercritical reactionshouldbenonreactiveunderprocess conditions. The critical temperature of the solvent should be lower thanthe reaction temperature toassurecompletemiscibilityofallgaseous reactants inthesupercriticalsolvent.However,thecriticaltemperatureshouldnotbefarfromthereactiontemperaturetomaintainthefavorablepropertiesofthenear-criticalstate.

Toshowtheimportanceofmakinganadequatephaseequilibriumengineeringanalysis,weselectasupercritical reactioncarriedoutbyChouchietal. [51]asanexample.Chouchietal.havestudiedthehydrogenationofα-pineneundersupercriticalCO2 in abatch reactoroperatingat323Kand14MPawithaPd/Ccatalyst.Theauthorsshowedthatthereactionrateandconversionarelowwhenthereactoroper-atesunderhomogenousconditions.Onthecontrary,betterconversionswereachievedwhentheCO2pressurewasreduced,althoughthesystembecameheterogeneous.Aphaseequilibriumengineeringanalysisofthereactoroperatingconditionscangiveanexplanationtotheseseeminglycontradictoryresults.Thebatchreactorwasfirstfedwiththecatalyst,togetherwithaknownamountofα-pinene.Then,thesystemwaspressurizedwithCO2uptothedesiredpressure(8,9,10,or12MPa),and,finally,H2wasfeduntilatotalpressureof14MPawasreached.Theactualmolarcompositionofthereactingmixturewasunknown.Thiscompositionmaybeobtainedbyusingan equationof state suitable for densitypredictionsunder the reaction conditions.OnepossibilityistousetheMHV2[15,48]equationofstate.Thecomputationoftheactualmixturecompositionsrequiresaniterativeprocedureforestimatingthesystemcompressibilityfactor,theamountsofeachcomponentchargedintothecell,andtheevolutionofthereactorcompositionwithconversion.Thisanalysisindicatesthat,atthehigherCO2partialpressure,an important reduction inhydrogenconcentrationoccurs,whichislikelythereasonfortheobserveddecreaseinthereactionrates.

Phaseequilibriumengineeringanalysisofsupercriticalprocessesisoftheutmostimportance in developing new technologies that replace conventional solvents byhigh-pressuregasestoobtainenvironmentallyfriendlychemicalprocesses.Severalexamplesofprocessdevelopmentclearlydemonstratethatagoodunderstandingofphasebehaviorandapplicationofrigorousmodelingtoolsareessentialtoprocesssynthesesinwhichthefluidpropertiesareextremelydependentonpressure,tem-perature,andcomposition.

7089_C001.indd 20 10/8/07 11:11:19 AM


reFerenCes

1. Francis,A.W.,Ternarysystemsofliquidcarbondioxide,J. Phys. Chem,58,1099,1954. 2. Stahl,E.andQuirin,K.W.,Densegasextractionona laboratoryscale:Asurveyof

recentresults,Fluid Phase Equilibria,8,93–105,1983. 3. Eckert,C.A.andChandler,K.,Tuningfluidsolventsforchemicalreaction,J. Supercrit.

Fluids,13,187–195,1998. 4. Kurnik,R.T.andReid,R.C.,Solubilityextremeinsolid-fluidequilibria,AIChE J.,27,

861–863,1981. 5. Deiters,U.K.andSwaid,I.,Calculationoffluid-fluidandsolid-fluidphaseequilibriain

binarymixturesathighpressures,Ber. Bunsenges. Phys. Chem.,88,791–796,1984. 6. Vidal,J.,Phaseequilibriaanddensitycalculationsformixtureinthecriticalrangewith

simpleequationofstates,Ber. Bunsenges. Phys. Chem.,88,784–791,1984. 7. Brennecke, J.andEckert,C.,Phaseequilibria for supercriticalfluidprocessdesign,

AIChE J.,35,1409–1427,1989. 8. Brunner, G., Selectivity of supercritical compounds and entrainers with respect to

modelsubstances,Fluid Phase Equilibria,10,289–298,1983. 9. delaFuente,J.C.B.,Mabe,G.D.,Brignole,E.A.andBottini,S.B.,Phaseequilibriain

binarymixturesofethaneandpropanewithsunfloweroil,Fluid Phase Equilibria,101,247–257,1994.

10. Soave,G.,Equilibriumconstants fromamodifiedRedlich-Kwongequationofstate,Chem. Eng. Sci.,27,1197–1203,1972.

11. Heidemann,R.A.andKokal,S.L.,Combinedexcessfreeenergymodelsandequationsofstate,Fluid Phase Equilibria, 56,17–37,1990.

12. Espinosa,S.,Fornari,T.,Bottini,S.andBrignole,E.,PhaseequilibriainmixturesoffattyoilsandderivativeswithnearcriticalfluidsusingtheGC-EOSmodel,J. Supercrit. Fluids,23,91–102,2002.

13. Ferreira,O.,Modelling of association effects by group contribution: Application to natural products,Ph.D.Thesis,Univ.dePorto,Portugal,2003.

14. Michelsen,M.L.,Amodified Huron-Vidalmixing rule for cubic equations of state,Fluid Phase Equilibria,60,213–219,1990.

15. Dahl,S.andMichelsen,M.L.,High-pressurevapor-liquidequilibriumwithaUNIFAC-basedequationofstate,AIChE J.,36,1829–1836,1990.

16. Skjold-Jørgensen, S., Gas solubility calculations II. Application of a new group-contributionequationofstate,Fluid Phase Equilibria,16,317–351,1984.

17. Skjold-Jørgensen, S., Group contribution equation of state (GC-EOS): A predictivemethod for phase equilibrium computations over wide ranges of temperature andpressuresupto30MPa,Ind. Eng. Chem. Res.,27,110–118,1988.

18. Bottini,S.B.,Fornari,T.andBrignole,E.,Phaseequilibriummodelingoftriglycerideswithnearcriticalsolvents,Fluid Phase Equilibria,158–160,211–218,1999.

19. Gros,H.P.,Bottini,S.B.andBrignole,E.,Agroupcontributionequationofstateforassociatingmixtures,Fluid Phase Equilibria,116,537–544,1996.

20. Ferreira,O.,Brignole,E.A.andMacedo,E.A.,Modelingofphaseequilibriaforasso-ciatingmixturesusinganequationofstate,J. Chem. Thermodynamics,36,1105–1117,2004.

21. Chapman,W.G.,Gubbins,K.E.,Jackson,G.andRadosz,M.,Newreferenceequationofstateforassociatingliquids,Ind. Eng. Chem. Res.,29,1709–1721,1990.

22. Holderbaum, T. and Gmehling, J., PSRK: A Group Contribution Equation of StateBasedonUNIFAC,Fluid Phase Equilibria,70,251–270,1991.

23. Espinosa,S.,Foco,G.,Bermudez,A.andFornari,T.,Revisionandextensionof thegroupcontributionequationofstatetonewsolventgroupsandhighermolecularweightalkanes,Fluid Phase Equilibria,172,129–143,2000.

7089_C001.indd 21 10/8/07 11:11:20 AM


24. Michelsen,M.L.,Calculationofphaseenvelopesandcriticalpointsformulticompo-nentmixtures,Fluid Phase Equilibria,4,1–10,1980.

25. Kravanja,Z.andGrossmann,I.E.,Multilevel-hierarchicalMINLPsynthesisofprocessflowsheets,Comput. & Chem. Eng.,21,S421–S426,1997.

26. Gros,H.P.,Díaz,S.andBrignole,E.A.,Near-criticalseparationofaqueousazeotropicmixtures:Processsynthesisandoptimization,J. Supercrit. Fluids,12,69–84,1998.

27. Diaz,S.,Espinosa,S.andBrignole,E.A.,Citruspeeloildeterpenationwithsupercriticalfluids: Optimal process and solvent cycle design, J. Supercrit. Fluids, 35, 49–61,2005.

28. vanKonynenburg,P.H.andScott,R.L.,CriticallinesandphaseequilibriainbinaryvanderWaalsmixtures,Phil. Trans.,298,495–540,1980.

29. Lucks,K.D.,Theoccurrenceandmeasurementofmultiphaseequilibriabehavior,Fluid Phase Equilibria,29,209–224,1986.

30. Coorens, H.G.A., Peters, C.J. and De Swaan Arons, J., Phase equilibria in binarymixturesofpropaneandtripalmitin,Fluid Phase Equilibria,40,135–151,1988.

31. Peters,C.J.,Supercritical fluids: Fundamentals for application. Multiphase equilibria in near-critical solvents,KluwerAcademicPublisher.Editors:Kiran,E.,andLeveltSengers,M.H.,1994.

32. Peters,C.J.andGauter,K.,Occurrenceofholesinternaryfluidmultiphasesystemsofnear-criticalcarbondioxideandcertainsolutes,Chem. Rev.,99,419–431,1999.

33. Koenen,H-E.andGaube,J.,Temperaturedependenceofexcessthermodynamicproper-tiesofbinarymixturesoforganiccompounds,Ber. Bunsenges. Phys. Chem.,86,31–36,1982.

34. Horizoe,H.,Tanimoto,T.,Yamamoto,I.andKano,Y.,Phaseequilibriumstudyfortheseparation of ethanol-water solution using subcritical and supercritical hydrocarbonsolventextraction,Fluid Phase Equilibria,84,297–320,1993.

35. Brignole,E.A.,Andersen,P.M.andFredenslund,A.,Supercriticalfluidextractionofalcoholsfromwater,Ind. Eng. Chem. Res.,26,254–261,1987.

36. Wiebe,R.,Thebinarysystemcarbondioxide-waterunderpressure,Chem. Rev.,29,475–481,1941.

37. Coan, C.R. and King, A.D., Jr., Solubility of water in compressed carbon dioxide,nitrousoxide,andethane.,J. Am. Chem. Soc.,93,1857–1862,1971.

38. Kobayashi, R. and Katz, D., Vapor-liquid equilibria for binary hydrocarbon-watersystems,Ind. and Eng. Chem.,45,440–446,1953.

39. Zabaloy, M., Mabe, G., Bottini, S.B. and Brignole, E.A., The application of highwater-volatilitiesoversomeliquefiednear-criticalsolventsasameansofdehydratingoxychemicals,Fluid Phase Equilibria,5,186–191,1992.

40. Reverchon, E. and Adami, R., Nanomaterials and supercritical fluids, J. Supercrit. Fluids,37,1–22,2005.

41. Martin, A., Precipitation processes with supercritical carbon dioxide: mathematical modeling and experimental validation,Ph.D.Thesis,UniversidaddeValladolid,Spain,2005.

42. Martin,A.andCocero,M.J.,Numericalmodelingofjethydrodynamics,masstransfer,and crystallization kinetics in the SAS process, J. Supercrit. Fluids, 32, 203–219,2004.

43. Amaro-González, D., Mabe, G., Zabaloy, M. and Brignole, E.A., Gas antisolventcrystallizationoforganicsaltsfromaqueoussolutions,J. Supercrit. Fluids,17,249–258,2000.

44. Simoes,P.C.andBrunner,G.,Multicomponentphaseequilibriaofanextra-virginoliveoilinsupercriticalcarbondioxide,J. Supercrit. Fluids, 9,75–81,1996.

7089_C001.indd 22 10/8/07 11:11:20 AM


45. Peter, S., Supercritical Fluid Technology in Oil and Lipid Chemistry. Chapter VI: Supercritical fractionation of lipids,Editors:King,J.W.andList,G.R.,AOCSPress,Illinois,65–100,1996.

46. Espinosa, S., Díaz, S. and Brignole, E.A., Thermodynamic modeling and processoptimizationofsupercriticalfluidfractionationoffishoilfattyacidethylesters.Ind. Eng. Chem. Res.,41,1516–1527,2002.

47. King, J.W. andList,G.R.,Supercritical fluid technology in oil and lipid chemistry,Editors:King,J.W.andList,G.R.,AOCSPress,Illinois,1996.

48. Hegel, P.E., Mabe, G.D.B., Pereda, S., Zabaloy, M.S. and Brignole, E.A., PhaseequilibriaofnearcriticalCO2+propanemixtureswithfixedoilsintheLV,LL,andLLVregion,J. Supercrit. Fluids,37,316–322,2006.

49. Härröd, M., van den Hark, S., Holmqvist, A. and Moller, P., Hydrogenation atsupercriticalsingle-phaseconditions,ISSAF - 4th International Symposium On High Pressure Process Technology And Chemical Engineering,Venice,Italy,2002.

50. Baiker,A.,Supercriticalfluids inheterogeneous catalysis,Chem. Rev., 99, 453–473,1999.

51. Chouchi,D.,Gourgouillon,D.,Courel,M.,Vital,J.andNunesdaPonte,M.,Theinflu-enceofphasebehavioronreactionsatsupercriticalconditions:Thehydrogenationofalfa-pinene,Ind. Eng. Chem. Res.,40,2551–2554,2001.

7089_C001.indd 23 10/8/07 11:11:21 AM

7089_C001.indd 24 10/8/07 11:11:21 AM

25

2 Supercritical Extraction PlantsEquipment, Process, and Costs

Jose L. Martínez and Samuel W. Vance

Contents

2.1 Introduction...................................................................................................252.2 SupercriticalFluidExtraction:ProcessDescription.....................................26

2.2.1 SupercriticalFluidExtractionofCompoundsfromaSolidMatrix...282.2.1.1 ProcessingParametersintheSupercriticalExtraction

ofSolids................................................................................302.2.2 SupercriticalFluidExtractionofCompoundsfromaLiquidFeed... 31

2.3 SupercriticalFluidProcessingPlants:EquipmentDesign...........................342.3.1 Overview............................................................................................342.3.2 Vessels................................................................................................ 352.3.3 PumpsandCompressors.................................................................... 372.3.4 HeatExchangers................................................................................ 382.3.5 PipingandValves............................................................................... 392.3.6 ControlSystems................................................................................. 41

2.4 IndustrialProcessImplementation............................................................... 422.5 Conclusions...................................................................................................48References................................................................................................................48

2.1 IntroduCtIon

In the last decade, supercritical fluid technology has reemerged, mainly due toa dramatic rise in the research and development activities focused on innovativeapproachesaswellasnewtrendsinthepharmaceutical,food,andchemicalsectors.Inthefoodindustry, thesenewtrendsincludeanincreasedpreferencefornaturalproductsoversyntheticonesandregulationsrelatedtonutritionalandtoxicitylevelsoftheactiveingredients.Ontheotherhand,consumersaretakingamoreproactiveroleinmaintainingtheirhealth,whichhasdrivenanewgenerationofproductsonthemarketaddressingdiseaseprevention.Thesetrendshavemadesupercriticalfluidtechnologyaprimaryalternativetotraditionalsolventextractionfortheextractionandfractionationofactiveingredients.

7089_C002.indd 25 10/15/07 5:22:37 PM

26 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds

Theobjectiveofthischapteristoprovideareviewofsupercriticalfluidextraction,describingtheprocessanddiscussingtheinfluenceoftheprocessparameters.More-over,thischapterisintendedtogiveanoverviewofthemaincomponentsofasuper-criticalextractionplantaswellasthestepsinvolvedinprocesscommercialization.

2.2 superCrItICal FluId extraCtIon: proCess desCrIptIon

Asupercriticalfluidextractionprocessconsistsoftwosteps:extractionofthecom-ponentssolubleinasupercriticalsolventandseparationoftheextractedsolutesfromthesolvent.Theextractioncanbeappliedtoasolid,liquid,orviscousmatrix.Basedontheobjectiveoftheextraction,twodifferentscenarioscanbeconsidered:

1)Carriermaterialseparation.Inthiscase,thefeedmaterialconstitutesthefinal product after undesirable compounds are removed—for example,dealcoholizationofalcoholbeverages,removalofoff-flavors,ordecaffein-ationofcoffee.

2)Extract material separation. The compounds extracted from the feedmaterialconstitutethefinalproduct—forexample,essentialoiloranti-oxidantextraction.

Theseparationofsolublecompoundsfromthesupercriticalfluidcanbecarriedoutbymodifyingthethermodynamicpropertiesofthesupercriticalsolventorbyanexternalagent(Figure2.1).Inthefirstcase,thesolventpowerismodifiedbymanip-ulatingtheoperatingpressureortemperature.Inthesecondcase,theseparationcanbecarriedoutbyadsorptionorabsorption.Themorecommonmethoddecreasestheoperatingpressurebyanisoenthalpicexpansion,whichprovidesareductionofthefluiddensityandthereforeareductionofthesolventpower.Ifseparationtakesplacebymanipulatingthetemperature,twosituationsmayoccur,dependingonthesolubilityof thedissolvedcompounds. If solubility increaseswith temperature atconstantpressure,adecreaseintemperaturewilldecreasethesolubilityandseparatethecompoundsdissolvedinthesupercriticalsolvent.Ifsolubilitydecreaseswithanincreaseintemperatureatconstantpressure,anincreaseintemperaturewillseparatethecompoundsfromthesupercriticalfluidsolvent.Iftheseparationiscarriedoutbyanauxiliaryagent,suchasanadsorbent,nosignificantpressurechangeoccurs,sothedifferentialpressureacrossthepumpismuchlower.Thistypeofprocessimpliesloweroperating costs; however, the recoveryof the extract from the adsorbent isoften very difficult. To overcome this disadvantage of high losses of the extract,theadsorptionstepmaybereplacedbyanabsorptionstep.Theextractdissolvedinthesupercriticalsolventisabsorbedbyawashfluidinacountercurrentflowusingapackedcolumnorspraytowerunderpressure.Separationofsolutesbyadsorptionandabsorptionhasbeenappliedinthedecaffeinationofcoffee[1,2].

Oneofthemainadvantagesofsupercriticalfluidsistheabilitytomodifytheirselectivitybyvaryingthepressureandtemperature(i.e.,modifyingfluiddensity).Therefore,supercriticalfluidsareoftenusedtoextractselectivelyorseparatespecificcompounds from a mixture. One procedure is by a fractional extraction process.

7089_C002.indd 26 10/15/07 5:22:38 PM

Supercritical Extraction Plants 27

In this case, the extraction is carried out in two stages. During the first stage, arelatively lowfluiddensity isselected,whichallowsextractionof thecompoundsthataresolubleatlowpressure.Then,theresidueisfurtherextractedathighfluiddensitytorecoverheaviercompounds(e.g.,dealcoholizationofcider[3]).Anotherexampleoffractionalextractionconsistsofremovalofnonpolarfractionsinthefirststagebyusingasupercriticalsolventandtheremovalofamorepolarfractionfromtheresidueinthesecondstagebyaddingacosolvent(e.g.,extractionofactiveingre-dientsfromgrapeseed[4]).

Anotherprocedure toselectivelyextractorseparatespecificcompoundsfromamixtureissequentialdepressurization[5].Inthiscase,bothfractions(lightandheavy)aresimultaneouslyextractedbyusinghigh-densityfluid.Thenthesupercriti-calsolventandtheextractpassthroughmultipledepressurizationsteps,allowingfrac-tionalseparation.Inthefirstdepressurizationstage,theheavierfractioniscollected;thevolatileorlightfractioniscollectedinthelaststage.Twodepressurizationstepsaregenerallyused,althoughinsomespecificcases,threeseparationstepshavebeenused.Thismethodiscommonlyusedintheextractionofspices,wherethesolubilityofoleoresinandessentialoil fractionsinasupercriticalsolventvarysignificantlywithpressureandtemperature.Generally, theextractiontakesplaceathighpres-sures(40to60MPa),sobothfractionsaresolubleinthesupercriticalsolvent.Theseparationorcollectionoftheoleoresinfractiontakesplaceinthefirstseparatorby

Group I. By Modifying the Thermodynamic Conditions

Group II. By External Agents

Extractor

Pump

Valve

Separator Extractor

Pump

Heat Exchanger

Heat Exchanger

Separator

Extractor Extractor Adsorption Vessel

Absorption Column

Pump

Pump Pump

FIgure 2.1 Basicschemeofsupercriticalextractionprocess.

7089_C002.indd 27 10/15/07 5:22:39 PM


reducingtheextractionpressuretointermediatepressure(15to20MPa).Undersuchoperatingconditions,thearomaticfractionremainsinthesupercriticalphase.Afterleavingthefirstseparation,thepressureisfurtherreducedandtheessentialoilsarecollectedinthesecondseparator.Thistypeofprocesshasbeensuccessfullyappliedinmultipleproducts.Insomecases,bothfractionsaredesirable(e.g.,oleoresinandessentialoils,colorandpungentfraction),whereasinothers,onlyoneofthefrac-tionshascommercialinterest.

2.2.1 Supercritical Fluid extraction oF compoundS From a Solid matrix

Mostofthedevelopmentandindustrialimplementationinsupercriticalfluidextrac-tionhasbeenperformedonsolidfeedmaterials.Figure2.2illustratesageneralflowdiagramofasupercriticalextractionprocessfromsolids.Thesolventissubcooledpriortothepump,assuringaliquidphasetoavoidcavitationissues.Thepressurizedsolventisheatedaboveitscriticaltemperaturetotheextractiontemperaturepriortotheextractionvessel.Theextractionvessel,whichisfilledwiththefeedmaterial,iselectricallyorwaterheatedtotheextractiontemperature.Thesupercriticalsolventflowsthroughthefixedbedandthesolublecompoundsareextractedfromthecarriermaterial.Thesupercriticalfluidplustheextractleavestheextractionvesselfromthetop,throughapressurereductionvalve.Thesolventpowerdecreaseswithpressurereduction,sothecompoundsprecipitate.Toassuretotalprecipitation,thesupercriti-calsolventisheatedabovethesaturationtemperaturetoreachthegasphase.Underthoseconditions,thesolventpowerisnegligible.Thenthematerialiscollectedinaseparatorwhilethesolventingasphaseleavestheseparatorvesselfromthetopand

1 Extraction Vessel 6 Receiver2 Pressure Reduction Valve 7 Pre-cooler3 Vaporizer 8 Pump4 Separator 9 Pre-heater5 Condenser

1

9 8 7

2 4

5

3

6

FIgure 2.2 Flowdiagramofasupercriticalextractionprocessfromsolids.

7089_C002.indd 28 10/15/07 5:22:39 PM


isrecirculatedbacktotheextractionvessel.Oncetherawmaterialisfullyextracted,thefollowingstepsarerequiredintheextractionvessels:

DepressurizationOpeningoftheextractionvesselUnloadingthespentmaterialLoadingwithfreshmaterialClosingtheextractionvesselPressurizingtooperatingconditions

Oneofthemostdifficultaspectsisattainingcontinuousfeedofthesolidsandcontinuousdischargeatahighpressureextractionvessel.Generally,thesolidfeedmaterialishandledbyusingpreloadedbaskets.Fromanindustrialorcommercialpointofview,theuseofonlyoneextractionvessel,evenwithaquick-openingclosurethatallowsforrapidopeningandclosing,isnoteconomicallyviable.Therefore,multi-pleextractionsvesselsoperating inacountercurrentflowarerequired.Figure2.3showsageneralschemeofacascadeextractionwithfourextractionvessels.Inthiscase,oncetherawmaterialinthefirstextractionvesselisfullyextracted,thevesselistakenoutfromtheprocessbyvalving.Oncethevesselisdepressurized,emptied,andrefilled,itenterstheprocesslineasthelastextractionvessel.Thesecondextrac-toristhenextonetobeisolatedoftheprocessline.Operatingthisway,thefreshsupercriticalsolventextractsfirsttherawmaterialthatispartiallyexhaustedandinthefinalextractionstep,thesupercriticalsolventextractsfreshrawmaterial.Thisconfigurationprovideshigher solvent loading (amountofmaterial extract/amountofsolvent).Theobjectiveistomaximizethesolventloading(i.e., tomaintainthesupercriticalsolventsaturatedorclosetothesaturationpoint).

Sincetheextractorsareoperatedbatchwise,acriticalfactoristoshortenthecharge and discharge cycle times. Therefore, a cap automation mechanism with

••••••

From Pre-heater

To Pressure Control

FIgure 2.3 Scheme of cascade operation of multiple extraction vessels for extractionofsolids.

7089_C002.indd 29 10/15/07 5:22:40 PM


quick opening closure, as well as fast depressurization, and unloading/loadingsequencearecriticalinthedesignofasupercriticalextractionplant.

2.2.1.1 processing parameters in the supercritical extraction of solids

ParametersaffectingthesupercriticalfluidextractionofsolidsarelistedinTable2.1.Theinfluenceoftheprocessparameterscanbesummarizedasfollows:

Solubilityofcompoundsincreasesbyincreasingtheextractionpressureatconstanttemperature.Atpressureclosetothecriticalpressure,solubilityofthecompoundsincreasesbydecreasingthetemperature.However,athighpressures,solubilityofcom-poundsincreasesbyincreasingthetemperature.Thiscrossovereffectisduetothecompetingeffectsofthereductioninsolventdensityandtheincreaseofthevaporpressure.Thelatterhasmarkedinfluenceathigherpressures.Thepressureatwhich thecrossovereffectoccursdependson the typeofcompoundstoextract.Thecrossoverrangeformostofthecompoundstakesplacebetween20and35MPa.The separation conditions depend on the solubility of the compounds atdifferentpressuresandtemperaturesaswellaswhetherafractionationofextract is carriedoutby sequential depressurization steps.Generally theseparationpressureiscarriedoutat5–6MPa.Foressentialoilsorvolatilefractions,theseparationtakesplaceat3to5MPaandlowtemperaturestomaximizetherecoveryofthetopnotescomponents.Foroils,theseparationcantakeplaceat15to20MPaduetotheirlowsolubilityinsupercriticalcarbondioxide(CO2)underthoseconditions.Thesolvent-feedratiodependsonmanyfactors,suchasconcentrationofthesoluteinthefeedmaterial,solubilityinthesupercriticalsolvent,type

•

•

•

•

table 2.1processing parameters in the extraction of solidsraw Material related

ParticlemorphologyandsizeMoistureChemicalreactionsforsettingfreetheextractcompoundsCelldestructionPelletization

•••••

operating Conditions

Extractionconditions:PressureTemperatureTimeSolventflowSolvent-feedratio

••••••

Extractionoperation:FractionalextractionConstantconditions

•••

Separationconditions:PressureTemperature

•••

Separationoperation:SinglestageFractionalseparation

•••

7089_C002.indd 30 10/15/07 5:22:40 PM


of feed material, and distribution of the compound in the feed material.Lowsolvent-feedratiosimplyloweroperatingcostsandhigherproductioncapacity.Generally,theindustrialprocessestargetsolvent-feedratioslowerthan30.However,highersolvent-feedratiosare justifiedforhighadded-valueproducts.Inspecificcases,asolvent-feedratiohigherthan100:1hasbeenreachedforcommercialapplications.Highsolventflowrates implyhighoperatingandcapitalcosts.However,theycouldincreaseproductioncapacity.Thesolventflowrateortheresi-dencetimeofthesolventintheextractionvesselmustbeoptimized.Ahighresidencetimeimpliesalongbatchtime.Conversely,ashortresidencetimemayresultinshortercontacttimebetweenthesolventandsolute,resultingina loadingof the solventmuch lower than the saturationconcentrationat the selected operating conditions. Linear velocities ranging from 1 to5mm/sarecommonlyusedinthesupercriticalfluidextractionprocess.Thesizeandmorphologyofthesolidmaterialhaveadirecteffectonthemass transfer rate. In general, increasing the surface area increases theextractionrate.Therefore,smallerparticlesizeorgeometry(suchasflakes)generallyfavorshighermasstransfer,decreasingthebatchtimeaswellasdiffusioncontrolledprocess.Ifthesolublesubstancesarelocatedinrigidstructuresinsideofthesolidmatrix,thesizereductionbreaksthisstructuresoitwillbeeasilyaccessibleforthesolvent.However,verysmallparticlesfavorachannelingeffect,whichdecreasestheextractionrate.Particlesizeneedstobeevaluatedcasebycasebasedonthetypeofmaterialtobepro-cessed.Inthecaseofprocessingofspicesandseeds,particlesizeisgener-allybetween30and60Mesh.Similarlytoparticlesize,moisturecontentmustbeevaluatedcasebycase.High content of moisture is usually not desirable because moisture actsas amass transferbarrier.On theotherhand,moisture expands the cellstructure,facilitatingthemasstransferofthesolventandthesolutethroughthesolidmatrix (e.g., in seedsandbeans).For instance, the influenceofmoisturebetween3% to10%generallyhasno significant impacton themasstransferofedibleoilfromseeds.

2.2.2 Supercritical Fluid extraction oF compoundS From a liquid Feed

Whenfeedmaterialisinaliquidstate,extractionistypicallycarriedoutinacoun-tercurrentcolumn.Thedensematerial(liquid)isintroducedfromthemiddleorthetopofthecolumn,andthematerialwithlowerdensity(solvent)isintroducedfromthebottomofthecolumn.Thiscontinuousprocessleadstoloweroperatingcoststhanthoseincurredwithextractionfromasolidmatrix.Ageneralprocessflowdia-gramisshowninFigure2.4.Theseparationstepsandregenerationofthesolventissimilartotheextractionfromsolids.

Similartotheconventionalcountercurrentcolumnprocesses,thecontactbetweenphasesisfavoredbyrandomorstructuredpackingmaterial.Additionally,refluxofextractimprovesselectivityintheextractionprocess.Theextractandsolventleavethecolumnfromthetop,whiletheheaviermaterial,orraffinate,iscollectedfrom

•

•

•

7089_C002.indd 31 10/15/07 5:22:41 PM


the bottom. The countercurrent column is heated electrically or with a hot waterjacketand theextractionprocesscan takeplaceatconstant temperatureorwithacontrolled temperaturegradient.Thelatterprocessprovidesan internalrefluxthatincreasesselectivity.

Processdesignisbasedonphaseequilibriumdata,whichdeterminethenum-berof theoretical stagesnecessary toperformaspecificseparation;heightof thecolumn,whichisrelatedtomasstransferorheightequivalenttoatheoreticalplate,anddiameterofthecolumn,whichdeterminesthecapacity.Thelatterparameterisrelatedtohydrodynamicbehaviorofthemixtureincontactwiththepacking.

In cases where the viscosity of the liquid is very high, the extraction processrequiresintensiveanduniformcontactbetweenthefeedandthesolvent.Thiscontactcanbecarriedoutbymechanicalmixingorbynebulizingtheviscousmaterialthroughanozzle.Inthecaseofmechanicalmixing,themixercanbemagneticallyormechani-cally coupled. In the latter case, thedriving shaft is actuatedoutsideof thevesseldirectlybyamotor,whereasinthefirstcase, it isactuatedbymagneticfields.Thetorquegeneratedbymagneticcouplingislower;however,thereisnorotarysealandthereisnoneedforlubrication.Directdrivecouplingsrotatetheouterhousingandthemagneticfieldthenrotatesthedrivenmagnetssecuredtothemixershaft.Whenthemixerismechanicallycoupled,ashaftdesignprovidinghightorqueathighpressures,withextendedlifetimeofbearingsandsealsisrequired.Additionallythemixersmustbeproperlydesignedbasedonthespecificapplication.Figure2.5showsamechanicalcouplingdevelopedbyTharTechnologiesthatoperatesat69MPa.

From Pre-heater

Extract

Feed

Raffinate

FIgure 2.4 Flowdiagramofasupercriticalextractionprocessfromliquids.

7089_C002.indd 32 10/15/07 5:22:42 PM


Inamechanicalmixingprocess,theviscousliquidormeltedproductispumpedinto the vessel. By adding the supercritical solvent, the viscosity of the productdecreases, which facilitates mixing and reduces the torque required. The super-criticalsolventflowsthroughtheviscousmaterialextractingthesolublecompounds.Thesupercriticalfluidandtheextractleavetheextractionvesselfromthetop.Oncetheextractioniscomplete,thematerialleftcanbedischargedfromthebottomoftheextractionvessel.Iftheviscosityoftheremainingmaterialisstillhigh,thesuper-criticalfluidassistsinremovingthematerialthroughtheopening.Thisprocesscanbeappliedtomaterialwithveryhighviscosityatatmosphericpressure.

Another alternative for processing viscous liquid material involves intensivecontactbetweenbothphases(i.e.,mixingandnebulizingthemixture).Inthiscase,theviscousmaterial and the supercritical solvent aremixedand sprayed throughanozzle.Thesupercriticalsolvent reduces theviscosityof the feedand thereforedecreasestheinterfacialtension.Bysprayingthroughanozzle,anatomizationtakesplace,creatingveryfinedropletswithaverylargesurfaceareaandahighcontactbetweenbothphases.Thesupercriticalsolventextractsthesolublematerialandtheinsolublesprecipitateinthebottomoftheextractionvessel.Thisprocessisfavoredwhen there is a significant difference in solubility between the compounds to beseparated.Thecriticalparametersinthisprocessarethecontactormixingdevices,spraying devices, vessel design, and solid removal from a pressurized vessel. Aprocessusingthisconcepthasbeensuccessfullydevelopedandindustriallyimple-mentedinthedeoilingofcrudelecithin[6].Thisisacontinuousprocessinwhich

FIgure 2.5 Mechanicalmixingusingamechanicalcoupling.Designpressure:69MPa(CourtesyofTharTechnologies,Pittsburgh).

7089_C002.indd 33 10/15/07 5:22:42 PM


crudelecithinispumpedandmixedwithsupercriticalCO2andthensprayedthroughanozzleintoahighpressurevessel.TheneutrallipidsaresolubilizedintheCO2,while thepolar lipidsareprecipitated in thebottomof theextractionvessel.TheCO2 and the neutral lipids leave the extraction vessel and the oil is recovered inthe separator. The polar fraction in a powder form is continuously transferred toa storage tank (Figure2.6). Some work was done in the early 1980s; however, itwasnever industrially scalable,mainlybecause the solvent-feed ratiosusedwereextremelyhighandabatchprocesswasused,sotheproductioncapacitywasverylow.Therefore,theplantsizerequiredtosatisfycommercialdemandhadtobeverylarge,whichimpliedveryhighcapitalcost.Thenewprocessofferstwosignificantadvantages:(1)theprocessiscontinuousand(2)thesolvent-feedratiorequiredislow.Thisisanexampleofindustrialimplementationofasupercriticalprocessfora commodity product, so the operating costs must be comparable to that at con-ventionalprocessing.Theconventionalprocessisawell-establishedprocessintheoilindustryusingacetoneasasolvent.However,usingacetoneasasolventformsacetonederivatives(mesityloxide)withadverseeffectsforthedeoiledlecithinduetoitstoxicityandoffflavor.Theoilindustryhasbeensearchingforalternativemethodsbuthasnotfoundanalternativeprocessuntilnow.Asimilarprocesscanbeappliedtoremovalofresidualsolventsinthepharmaceuticalindustry[7].

2.3 superCrItICal FluId proCessIng plants: equIpMent desIgn

2.3.1 overview

Designandselectionofequipmentforasupercriticalfluidprocessing(SFP)systemrequiresconsiderationofsomeparametersandspecificationsthatareuniquetothis

7

1

2 3

4

9

5

6

8

10

FIgure 2.6 Scheme foracontinuousprocess forde-oilingofcrude lecithin.1Tankofcrudelecithin,2Lecithinpump,3Preheater,4Mixer,5Recirculationpump,6Backpressureregulator,7Nozzle,8Extractionvessel,9Transfervessel,10CO2inlet.

7089_C002.indd 34 10/15/07 5:22:43 PM


typeofplant.Manyitemsthatwouldbeoff-the-shelfformostplantsaresimplynotavailableornoteasilyfoundforapplicationtotheoperationordesignconditionsoftheSFPenvironmentandprocess requirements.Forexample,manySFPsystemsrequiresanitarydesignforfood,nutraceutical,orpharmaceuticalproductsandoper-atingpressuressubstantiallyhigherthannormallyfoundinfood,nutraceutical,orpharmaceuticalplants.Meetingtheserequirementsentailsinvestigationofvendorswhocanprovideitemsthataresuitable.Uniqueconditionsmaybeencounteredinregularoperationoramajormalfunctionmayoccurthatrequiresspecialmaterialsof construction (e.g., metals can undergo brittle fracture in such environments).Systemcapitalcostmustbecloselycontrolledtobecompetitivewithothersystems.

2.3.2 veSSelS

Ingeneral,SFPvesselsaredesignedandmanufacturedinaccordancewithAmericanSocietyofMechanicalEngineers(ASME)SectionVIIIStandards.Inmanycases,theprocessrequiresone,orsometimestwo,full-diameterquick-openingclosuresforchargingfreshfeedstockordischargingspentfeedstock.Theclosuremechanismisoftenautomatedtominimizedowntimeofavesselwhenfillingoremptying.Thereareanumberofsuchclosuresproprietarytovesseldesignersorsuppliers.Consid-erationmustbegiventomethodsofcleaningvesselsbetweenchargesoremptyingvesselswhen the solidsplugorbridge.Vesselsare jacketedorelectrically tracedfor process temperature control. Vessel shape and aspect ratio must be carefullyevaluatedtominimizevesselcostswithoutaffectingperformance.Themostcriticalvesseldesignintheprocessisthatoftheextractionvessel.Itrequiresthemaximumdesignpressureandmaybethemostcriticalinselectionofmaterialsofconstruc-tion.Inmanycases,specialalloysofstainlesssteelorexoticmetalscanbeused,buttheactualselectionofalloysandthicknessesmayalsodependontheabilitytomachine,forge,andweldvesselload-bearingcomponents.Extractionvesselscanbefabricatedbyforging,machiningsolidbarstock,rollingandweldingofplate,multi-wallrollingandwelding,compositemultilayers,andcasting.

Fulldiameterquick-openingvessel closuresmayutilize self-energizing seals,segmentedrings,breechlocking,flanges,andthreadedcaps.Mostareproprietarydesigns.ExamplesofclosuresareshowninFigure2.7.

Vesselsotherthantheextractionvesselsareusuallydesignedforsubstantiallyloweroperatingpressuresandtemperatures,butthefunctionofeachvesselmustbecarefullyconsideredinselectingthesizeandshapeofseparationvesselsandprocessholdingvessels.Insomecases,thevesselsmayneedspecialdesignstokeepthemcleanandminimizepluggingandcontamination.

Atthebeginningandendofthebatchprocesscycle,thevesselsmayverywellbeatornearambientpressuresandtemperatures,asmaterialsarebeingtransportedtoorfromtheSFPsections.Butevenfortheseareas,thevesselsmaystillrequireadaptationforcleaning-in-place(CIP)orothercleaningmethods.

Inmanycases,thefeedstockisinparticulatesolidorpelletform.Theextrac-tionvesselwouldthenbedesignedfor(usually)batchchargingandemptyingoftheextraction feedstock. In this case, an extractionvesselwouldbefilledwith feed-stock,brought fromambient temperature andpressure to extractionpressure and

7089_C002.indd 35 10/15/07 5:22:44 PM


temperature,extracteduntilthesolutehasbeenremoved,andthendepressurized,emptied,andrecharged.Thebalanceoftheplantwouldbeessentiallycontinuousinoperationwithclosedcyclesolventrecirculation.Multipleextractionvesselswouldbeusedtoapproachacontinuousoperation.

Insomecases,thefeedstockisliquidandtheextractionvesselmaybeapackedcolumnoperatingcontinuously(Figure2.8).Inthesesituations,atrulycontinuousoperationwouldbethenorm,withreductionofpressureonlydoneforproductchangeorsystemshutdownandoverhaulormaintenance.Somesystemsutilizesupercritical

FIgure 2.8 Countercurrent column (10 m) of a supercritical process extraction plant(CourtesyofTharex,Seoul).

(a) (b)

FIgure 2.7 Vesselclosuretypes.a)Automatedsegmentringclosure,b)Automatedclampclosure(CourtesyofTharTechnologies,Pittsburgh).

7089_C002.indd 36 10/15/07 5:22:44 PM


fluidchromatography(SFC)inapackedcolumntoachievetheseparationofcom-ponentstargetingveryhighpurities(95%to99%).Figure2.9showsaprocessscaleSFCusingadynamicaxialchromatographycolumn.

2.3.3 pumpS and compreSSorS

Thesecondmostcriticalequipmentitemsarethepumpsandcompressorsusedforbuildingsystempressuresandtemperaturestothesupercriticalregionsestablishedintheprocessdevelopmentstudies.SFPflowsarerelativelylowandpressuresarerela-tivelyhigh,rangingfrom5to120litersperminuteflowat6to65MPa.Theprocessrequiresclosecontroloftemperatures,pressures,andflows.Pressures,inparticular,requirecriticalcontrolbecausepressurefluctuationsmaymakesubstantialdiffer-enceinprocessingresultsandcanresultinoverpressuredevicesshuttingtheprocessdownandwastingbothsolutesandsupercriticalsolvents.Suchshutdownsresultinpoorproductionratesandunnecessarycostpenaltiesforthesystem.

Mosthigh-pressurepumpsaremultiplunger styles.Flowandpressurecontrolcommonly use some type of speed control, such as variable frequency speed. Afurtherdevelopmentincludesdiaphragmtypepumps,whichareactuatedbyplungersandmorenormalliquidsthatcausethedisplacementandflexingofthefinalpump-ingelement(thediaphragm).Thepumpshaveproprietarydesignfeaturestoprovidesuitableoperationforthepressuresandflowsrequired.Morestandardpumpscanbeusedforprovisionofmakeupsupercriticalfluidsandfinalproduct(extract)pumps.

Compressorsmayalsobereciprocatingpistons.Inraresituations,morestandardrotarycompressorscanbeused,ofteninrecoveryofotherwisewastedsupercriticalfluid.Economicsandprocessvariablesdetermine theextentof recoveryof spentsolventfluids.

Thefluidendsofplungerpumpsorcompressorsrequireclosetolerancemachin-ingof theplungerorpistonand thecylinder tominimize leakage.Nonlubricatedplungersareoftenchosen,withcarefulselectionofmaterialstoensurelowcoeffi-cients of friction and dimensional stability. Lubricants are avoided because they

FIgure 2.9 Dynamicaxialcolumn(30cmID)ofasupercriticalprocesschromatographyplant(CourtesyofTharTechnologies,Pittsburgh).

7089_C002.indd 37 10/15/07 5:22:45 PM


contaminate theprocess solvent and solute.O-rings,gaskets, and seals for recip-rocatingandrotatingpartsmustbecarefullydesigned.Materialsmustbecompat-iblewiththesolventsandsolutes.AbsorptionofsolventintheO-ringmaypresentproblemswhenthesystemisdepressurizedbecausethesolventmayexpandwithintheO-ring,causingdisintegration,especiallyifdepressurizationisrapid.Unusualorexoticmaterials for theplungerandcylindermaybeusedascoatingsor solidsections.Proprietaryinformationisacquiredbysubstantialequipmentdevelopmentandiscarefullyprotectedbydesignersandfabricators.Figure2.10showsamulti-plungerpumpwithadesignpressureof96MPaandaflowrateof30kg/min.

2.3.4 Heat excHangerS

Heat exchange equipment also presents unique problems for supercritical fluidsystemsdue to thehighpressures required inkeypartsof theprocess.Althoughheatexchangersintheprocessindustriesareamatureandverycompetitivetech-nology,designsarenotreadilyavailableatthepressuresencountered.Also,specialconsiderationmustbegiventocleaningoftheprocesssideheatexchangesurfaceintheeventoffoulingwithsolutesandcleaninganddisassemblyoftheexchangerforchangeover toanotherproduct.Another specialconsideration in selectionofheatexchangertypesorstylesistheriskanalysisforheatexchangertubefailure.Aleakorcatastrophicfailuremaycreateadryicepluginthehighpressureside(forCO2asthesolvent),freezingoftheheatexchangefluid,andoverpressureofthelowpressureheattransferfluidpipingsystem.SelectionofoverpressuresafetydevicesmustbecarefullyinvestigatedbyprocessriskanalysisandHazardandOperability(HAZOP)studiestechniquesforthesystem.

Removable heat exchanger heads are often desirable. The supercritical fluidsolventmostoftenisonthetubesideofshell-and-tubeexchangersanddesigncom-promisesmustoftenbemadebetweenmultitubetubediameterandnumberoftubes.Thesmallerthediameterofthetube,themoretubesarerequiredtoprovidesuitable

FIgure 2.10 High-pressure multiplunger pump. Design pressure: 96 MPa, Flow rate:30kg/min(CourtesyofTharTechnologies,Pittsburgh).

7089_C002.indd 38 10/15/07 5:22:46 PM


fluidvelocitythroughtheexchangerbutthelesstubingandmetalrequiredfortheheat transfer area. Smaller diameter tubes present additional problems for clean-ingandforfasteningtothetubesheets(usuallybywelding).Largerdiametertubesimprovethefluidvelocityandtubenumbersbutalsomayresultinlongerexchangers.Asthetubelengthincreases,differentiallinearexpansionoftheshellandtubesmayrequireexpansionjointsorfloatingheads.Thesesituationscomplicatetheexchangerdesignandmayaddtothecostofdesignandfabrication.

In most process designs, ASME Section VIII Unfired Pressure Vessel CodeStandardsandCodeStampsarenecessary.Insomecases,asimplerdesignoftheexchangercanbeaccomplishedifthepossibilityofheatexchangesurfacefoulingandpluggingofthetubinginteriorcanbeminimizedbycarefullycontrollingpro-cessingconditionsinthesystem.

Typesofheatexchangers thatcanbeused includeshell-and-tubeexchangers,double-pipeandmulti-U-tubeexchangers,doublepipecoils,orsimplecoilsintanks.Anexampleofshell-and-tubeheatexchangerdesignisshowninFigure2.11.

2.3.5 piping and valveS

Selectionofpiping,fittings,andvalvesforSFPalsorequiresspecialdesignspecifi-cationsandcriteria.Thematerialtobeusedmustbenonreactivewiththesupercriti-calfluidsolventandsolutesintheprocess.Thepossibilityofreactionsbetweenthesolventandthepipingsurfacesmustbeevaluated.Thesolventmaybesubstantiallymoreaggressiveinthesupercriticalfluidregimethanwouldbetrueatlowerpres-sures,soadditionaltestingofmaterialsmayleadtomoreexpensivealloystomini-mize such reactions. Where possible, high strength alloys (with higher allowablestressthanthetypical300Seriesstainlesssteels)arethechoiceforoverallcostandprocess suitability.Sinceflow rates formost supercriticalfluid systemsaremuchlowerthaninmoreconventionalsystems,pipediametersandsuitablehigh-pressurefittings are smallerwhilemaintainingappropriateflowvelocities in the intercon-nectingpipingortubing.Pipingcostisthusminimized.However,theconventionalthreadedjointsor“standard”flangesarenotcosteffective.Inmostcases,specialhigh-pressurefittings,couplings,andthelikewillbetheselectionofchoice,bothforconvenienceandeconomicreasons.Specialhigh-pressurecouplingsareshowninFigure2.12.

Valving isanotheruniqueareafor theprocess.Twotypesofvalvingmustbeconsidered: (1) isolation valving and (2) flow control valving. Isolation valvingmost often includes plug valves or butterfly valves for leak-proof on-off service.Thesevalvescommonlyhavemetal-on-metalsealingsurfaceswherelowfrictionisdesired.Specialcoatings(sprayed,vapordeposition,orcomposition)maybeusedtoavoidseizingorgalling.Selectionofdissimilarmetalsormetaloxidesorcarbideswithhighhardnessvaluesandgoodmachiningproperties improvesperformance.However,pairingofmaterialsandselectionofdesigns for theoperatingenviron-mentisstillmoreartthanscience.Sospecialtyhigh-pressurevalvecompaniesarethevendorsofchoice.Flowcontrolvalvesarealsoaspecialtyitematsupercriticalfluidoperatingpressures. In somecases, pressuredrop through the control valvemay be at critical flow or with a phase change when passing through the valve.

7089_C002.indd 39 10/15/07 5:22:46 PM


Theseconditionsrequirespecializedknowledgeoftheeffectsofthehighvelocitythroughthevalveorificeandthepossiblepresenceoftwo-phaseflowthroughthevalve.Again, inselectingmaterialsforpackingglandsandvalvestemseals,caremustbetakentoselectanappropriateelastomerorcompositethatwillnotabsorbhighpressuresolventduringoperationandthenfractureorfailwhenthepressureisreleased.ThesupercriticalfluidsolventmayvaporizeandexpandinthepackingorO-ring,withsubsequentdestructionoftheseal.Attherangeofpressuresundercon-sideration,dimensionalstabilityandeliminationofcreepflowarealsonecessary.

As SFP system throughputs become larger, automated control and isolationvalvesbecomemoreattractive.Pneumaticorhydraulicoperatorsand,occasionally,electricallyoperatedmodulatingoperatorsmayberequiredtominimizethedowntimefortheplant.Fail-safeoperationmustbetheorderoftheday.

FIgure 2.11 Shell-and-tubeheatexchanger(CourtesyofTharTechnologies,Pittsburgh).

7089_C002.indd 40 10/15/07 5:22:47 PM


2.3.6 control SyStemS

Asshouldbeobviousbythispoint,manyoftheareasofcontrolforSFPsystemsrequirespecialconsiderationbecauseofthehighoperatingpressures(withtheresult-inghighpotentialenergyintheprocess)andthepossiblehazardsforbothoperatingpersonnelandplantintegrity.Controlsystemfailuresinmoreconventionalprocess-ingplantswouldnotpresent thepossiblehazards tooperatorsanddamage to theequipmentandhazardsbeyondtheplantarea.Relativelysmallvariationsinprocessconditionscanbereflectedinsubstantialvariationsinsystempressuresandphasetransitions.Socontrol response to thesevariationsmustbe rapidandeffective indampingtheresultswithinthesystem.

Selectionofprimarysensorsmustbecarefullymadewithprovisionsforsensorfailure, leakage, or error. Redundancy must be considered and carefully thoughtthrough.Evenpressuregaugesandtemperatureelementsmustbeexamined.Pressuregaugesortransducersmayrequireliquidsealsorthermowellstopermitisolationandreplacementwhilethesystemisoperating.Temperaturesensorscanbethermocouplesorresistancetemperaturedetectors(RTD)sensors,butresponsetimemustbeweighedagainstthermowellisolation.Gaugesshouldhaveblowoutdiscs.Levelsensorsmustbeaccurateandreliable.Pumpandcompressorflowratesarecommonlymeasuredbymassflowmeters(Coriolismeters)andflowcontrolledbyfrequencymodulationoftheconnectedmotor.Overallsystemshutdowniscontrolledbydistributedcontrolwithcomputercapability.SystemconditionsatstartupandshutdownoftheprocessmustbethoroughlythoughtthroughwithaHAZOPreview.

Summarizing,theforegoingdescriptionofthefactorsthatmustbeconsideredinthespecificationandselectionofthehardwarethatgoesintoaSFPplantshowsthattheuniquemechanicalsystemrequirementsmustbecarefullymadeandreviewed.Eachsystemhassomecharacteristicsthatmustbeevaluatedbasedontheparticularenvironmentandprocesschemistryforthatproductorcommodity.Insomecases,the plant will be a multipurpose or multiproduct plant. Each purpose or productmustbeconsideredtoestablishthesingleproductthatwouldcontrolthemechanicalspecificationsofeachitemofequipmenttobeselected.

(a) (b)

FIgure 2.12 High-pressurecouplings:a)DUROLOK(CourtesyofBETEFogNozzle,Inc.,Greenfield,MA),b)Grayloc(CourtesyofGraylocProducts,Houston,TX).

7089_C002.indd 41 10/15/07 5:22:48 PM


2.4 IndustrIal proCess IMpleMentatIon

Processdevelopmentrespondstoaspecificrequirementofacompanyortoamarketdemandforaspecificproduct.Insomecases,thecurrentprocessshowssignificantweaknessevenforawell-positionedproductinthemarket.So,theprocessingcom-panies that are aware of the limitations or constraints search for new processesto strengthen the product, resulting in a leading market position and projectinghigherrevenues.Inothercases,traditionaltechnologiesdonotofferasatisfactorysolutiontoaspecificproblem.Additionally,thereisacontinuoussearchtoreduceproductioncosts.

A general workflow for an industrial process implementation is illustrated inFigure2.13.Thefirststepistoprovethatthetechnologyiscapableofmeetingtheproduct specifications and process requirements defined by the customer or themarket.Intermsofproductspecifications,requirementsgenerallyareaminimumconcentrationorpurityofspecificcompoundsandminimumextractionorrecoveryefficiency. Regarding process requirements, the main constraints are maximumoperatingtemperatures,typeofpretreatmentormaterialconditioning,andaccept-ablecosolventstobeused.

In the case of supercritical fluid extraction, the first point to be addressedis if a compound tobe extracted is soluble in the supercritical solventor if thesolventwillbeselectivetofractionateorseparateamixtureofcompounds.Thethermodynamicdatarequiredare thesolubilityof thespecificcompoundin the

Supercritical FluidExtraction

CustomerRequirements

Lab scaleOptimization of

Process Parameters

Initial Cost Estimate

Scale-upPilot Plant Scale

Semi-industrial Scale

ProductSpecifications

ProcessRequirements

ConventionalTechnologies

Cost Calculation

CommercialPlant

CostComparison

FIgure 2.13 Workflowforindustrialprocessimplementation.

7089_C002.indd 42 10/15/07 5:22:48 PM


supercriticalfluidasafunctionofpressure,temperatureandsoluteconcentration,partitioncoefficients,andselectivityorseparationfactors.Averyextensivedata-baseofphaseequilibriaandsolubilitydataforbinarysystemshasbeengeneratedoverthelasttwodecadesandcanbeusedasareference.However,insomecases,thepublisheddataarequestionable.

Arapidwaytodeterminesolubilitydataorphasetransitionsforabinarymixture(specific compound and supercritical solvent) is by a phase equilibrium analyzer(Figure2.14).Thisisastaticmethodwherebythesoluteandsolventareloadedintoahigh-pressurevessel.Thisvesselconsistsofavariablevolumehigh-pressureviewcellwithanintegralstirrer,waterjacket,andvideosystem.Oncethemixtureiscom-pressedtoasinglephaseforaselectedtemperature,slowmovementofthecellpistonatacontrolledrateslowlydecreasesthepressureuntilasecondphaseappears.Byobservingthevideooutputofthesystem,itispossibletodeterminethecloudpointforthesampleatthecurrentpressureandtemperature.Additionalexperimentaldataareobtainedbymodifyingthetemperatureandrepeatingtheexperimentalprocedure.Themainadvantagesofthismethodarerapidgenerationofdataandvisibleconfir-mationofdissolution;inaddition,nosamplingisrequired,noextractionefficiencyisinvolved,andaminimumamountofsoluteisused.

Once the thermodynamic data are obtained, the next step is to evaluate theextractionofthatspecificcompoundfromtheoriginalsample,whichisgenerallyamulticomponentmixtureinasupercriticalfluidextractionsystematbenchscaletooptimizeprocessparameters.Theprocessparameterstooptimizearelistedhere:

FIgure 2.14 Phaseequilibriumanalyzer(CourtesyofTharTechnologies,Pittsburgh).

7089_C002.indd 43 10/15/07 5:22:49 PM


Conditioningtherawmaterial:moisture,sizeandshape,etc.Kineticdata;pressure,temperature,andsolventflowrateeffect:

ExtractionyieldExtractiontimeQualityoftheextract

Fractionationconditions

Thelaborbenchsystemmustbeproperlydesignedtobeversatileandcoverawiderangeofoperatingconditions.Thesystemmustbeableto:

Cover a wide range of operating conditions: pressure, temperature, andflowratesUsecosolventsPerformsequentialdepressurizationbyusingatleasttwoseparationstagesContinuouslylogprocessparameterdata

Aninitialcostestimateisprovidedoncetheprocessdevelopmentsatisfiesthecustomerproductspecifications.Theinitialcostestimatediscalculatedusingscaleupmethodsbasedonthefollowinginformation:

Customerproductionrequirements(i.e.,amountofmaterialtoprocessperyear,workingdaysperyear,andworkinghoursperday)Rawmaterial(i.e.,particlesizeandshape,concentrationoftheproduct)Optimizedprocessparameters (i.e., extractionpressureand temperature,solventflowrate,residencetime,kineticsoftheprocess,bulkdensityofthefeedmaterial,andseparationpressureandtemperature)

Thiscostestimationprovidestothecustomerthefollowinginformation:operatingcosts ($/kg of feed, or $/kg of final product), plant size, and plant configuration.At thispoint, thecustomerdetermines if thesupercriticalprocesswillmeet theirbudgetandiftheinvestmentandoperatingcostsarecomparablewithorbetterthanconventional technologies. If so, the next step is to scale up the process to pilotplantorsemi-industrialscale.Theobjectivestoaccomplishinthisstage,meetingallproductsspecifications,are:

Verificationof theprocessparametersselected in the labscaleand theiroptimizationifrequiredOptimizationofutilityrequirementsOptimizationofrecirculationparametersofthesolventandaddressinganyissuesrelatedtomaterialhandling

Reducingoperatingcostsrequiresminimizingenergyrequirements,whichalsoimpliesareductionintheassociatedcapitalcostoftheauxiliaryequipment.Thesecostsaredirectlyrelatedtotherecirculatingcostsofthesolvent.Recyclingofthesolventdependsontheseparatingconditionsof thesubstancefromtheextractionfluid.Typically,recyclingisperformedatlowseparationpressuresandthesolvent

••

•••

•

•

•••

•

••

•

••

7089_C002.indd 44 10/15/07 5:22:50 PM


is recycled in the liquid state. Figure2.15 shows the solvent cycle in a pressure-enthalpydiagram.In thiscase, theseparationof thesolublecompoundsfromthesupercriticalsolventisachievedbyisoenthalpicthrottling(a-b)followedbyheating(b-c).Atc,thesolventisingasstate,sothesolventpowerisnegligible.Thenthesolventissubcooled(c-d)andpumpedtotheextractionpressure(d-e).Thesolventisheatedtotheextractiontemperature(e-a).Achillerunitisusedinordertocooldownandcondensetheextractionfluidbeforeitentersthepump.However,ifextractiontakesplaceatpressuresbelow30MPa,therecyclingofthesolventasgas,replac-ingthepumpbyacompressor,generallyresultsinenergysavings.Forinstance,inextractionofessentialoils,wheretheextractionconditionsaregenerallycarriedoutinthepressurerangeof8to20MPaandinthetemperaturerangeof35°Cto50°C,thesolventrecycledinagasstateismoreenergyefficient.

Aspreviouslymentioned, at pressureshigher than30MPa, solvent recyclinginliquidstageismoreefficient.Undertheseoperatingconditions,analternativetoprovideahigherenergyefficiencysolventcycleisadditionofacompressorintothesystemafterseparationoftheextractedcompounds[8].Afterexpandingthemixturetoformatwo-phaseregionandheatingthemixture,sothatthesolventbecomesasinglegasphase,thesolventiscompressedtoapressurehigherthancriticalpressurebyacompressor.Thenthesolventissubcooledbeforeenteringthepump.Twoadvan-tagesareobtainedusingthismethod.Thefirstisthat,insteadofusingachiller,thesolventcanbecooleddownwithwaterfromacoolingtower.Second,themechanicalenergyrequiredofthepumpislower.Theenergysavingsofrecyclingthesolventusingbothapumpandacompressor,comparedwiththemoretraditionalprocess,dependsonextractionconditionsbutcouldbeupto65%.

Incaseswherethesolubilityofthespecificcompoundintheextractionfluidisverylow,typicallylowerthan0.5%,atapressurehigherthanthecriticalpressureofthesolvent,thesolventcanberecycledinasupercriticalstateprovidingadditionalenergysavings.Forinstance,intheextractionofseedoilsusingsupercriticalCO2,theseparationoftheoilintheseparatorshouldbecarriedoutundersupercritical

100

–50,°C

–30,°C –10,°C 10,°C 30,°C 50,°C 70,°C 90,°C 110,°C 130,°C 150,°C 170,°C190,°C1000

100

10

200 300Enthalpy (kJ/kg)

Pres

sure

(bar

)

400 500 600

L-V

L V

SCF

a

bcd

e

FIgure 2.15 DiagramP-Hforalow-pressurerecirculationsolventusingpump.

7089_C002.indd 45 10/15/07 5:22:50 PM


conditionsatapressurelessthan20MPaandthesolventrecycledinsupercriticalstate.Thesolubilityoftheoilatpressuresbelow20MPaisgenerallylessthan0.3%.Additionally,thesupercriticalCO2couldberecompresseddirectlywithouttheneedofsubcoolingtheCO2.Thereisnotachangephasethatcouldcreatecavitationinthepump.Table2.2summarizesthesolventcycleatdifferentextractionconditionsbasedonthesolubilityoftheextract.However,becausethecompressibilityofsuper-criticalCO2ishigherthanthatofliquidCO2,itreducespumpcapacity.Figure2.16showsthesolventcycleofCO2regeneratedundersupercriticalconditions.

ChordiaandMartinez [8]describeanalternativemethodofprovidinghigherenergysavingsforrecyclingthesolventinthesupercriticalstate.Inthiscase,highpressurerecyclingisrealizedbyreplacingtheexpansionvalvewithaturbineandenergeticallycouplingtheturbinetothepump.Basedontheoperatingconditions,energy savings of up to 60% can be reached. Table2.2 summarizes the solvent

100

–50,°C

–30,°C 190,°C1000

100

10

200 300Enthalpy (kJ/kg)

Pres

sure

(bar

)

400 500 600

L-V

LV

SCF

a

bc

–10,°C 10,°C 30,°C 50,°C 70,°C 110,°C90,°C 130,°C 150,°C 170,°C

FIgure 2.16 DiagramP-Hforasupercriticalrecirculationsolvent.

table 2.2solvent Cycle at different operating Conditionssolubility of

solute at p > pc solvent Cycle

extraction pressure

solvent recycling state Main Components

>0.5%Lowpressure

recycling

Pext>30MPaLiquid Pump

Gas Compressor+pump

Pext<30MPaLiquid Pump

Gas Compressor

<0.5%Highpressure

recyclingPext>30MPa

Supercritical Expansionvalve+pump

SupercriticalTurbine+pump

(energeticallycoupling)

7089_C002.indd 46 10/15/07 5:22:51 PM


cyclerecommendedforenergysavingsatdifferentextractionconditionsbasedonsolubilityoftheextract.

An additional factor that must be considered to reduce operating costs is tominimize solvent losses. The losses take place from the extraction vessel duringthedepressurization for unloading the spent material, aswell as in the separatorwhenwithdrawing theextract.Mostof theCO2 lossesoccur in thedepressuriza-tionprocess.Generally,depressurizationinvolvestwosteps:a)Theextractionvesselisdepressurizeddowntothereceiverpressure(5to6MPa)andb)Therestofthesolvent isventedto theatmosphere.AnalternativetoreduceCO2losses is touseacompressorandcondenseadditionalCO2. In thiscase,once thepressure in theextractionvesselequals thepressure in thereceiver(5 to6MPa), thecompressorcompressestheCO2untiltheresidualpressureintheextractionvesselreaches0.2to0.5MPa.ThentheresidualCO2isreleasedintotheatmosphere.InplaceswhereCO2costishigh,investmentincapitalcostofadditionalequipmenttoreduceCO2lossesmakeseconomicsense.

An additional operating cost is labor. The personnel required to operate anindustrialSFEplantdependsonthesize,configurationof theplant,batchtime,andautomationoftheplant.Ingeneral,asupervisorandtwooperatorsarerequiredfor a fullyautomated largeplant.The laborcost, aswell as thecostofCO2, ishighlydependentonthegeographicallocation.Afterscaling-uptheprocessanddefinitionoftheoperatingparametersandconfiguration,thefinalcostestimateismade.Whiletheindustrialplantisbuilt,tollingisapreferablestepinmanycases.Thisstepisusedformultiplepurposes:formulationofdifferentproducts,marketevaluation,andlaunchingtheproductintothemarketwhiletheindustrialplantisunderconstruction.

ThecapitalcostofaSFEplantdependsonmanyfactors,suchasthenumberofvessels,designpressure,sizeofthevessels,flowrate,automation,andGoodManu-facturingPrice(GMP)compliance.Generally,thecapitalcostoftheSFEplantishigherthanthetraditionalorconventionalextractionplant,whiletheoperatingcostsarelower.However,tocompareproperlythecapitalcostsofSFEplantversustradi-tionalextractionprocess,isnecessarytotakeintoaccountalltheassociateequip-mentusedintheconventionalextractionprocess,suchasdistillationorevaporation

table 2.3Case study: Cost and revenue estimates

Flaxseed astaxanthin ginger

Amounttobeprocessed(MT/year) 3,000 50 3,000

Concentrationoftheproduct(%) 13 2.5 5

Numberofdays/year 300 300 300

Numberofhours/day 24 24 24

Estimatedequipmentcost($) 2,500,000 1,500,000 4,500,000

Operatingcosts1($/kgoffeed) 0.23 5.57 0.43

ROI 3.2 1.0 1.61 Includespowerconsumption,CO2losses,maintenance,andlabor

7089_C002.indd 47 10/15/07 5:22:52 PM


systems for solvent recovery; aswell as associate costs inbuilding requirements,instrumentation and electrical connections to meet explosion proof. On the otherhand,thesellingpriceoftheproductsobtainedbySFEisalsohigher(bothextractandraffinate).

Table2.3illustratestheestimatedcostsandrevenuesobtainedforasupercriticalextraction plant used in three different industrial sectors: edible oil, spices, andalgae.Theoperating cost includespower consumption,CO2 losses,maintenance,andlabor.Inallcases,anextractionefficiencygreaterthan95%wasachievedandthe return of investment was less than 4 years. Based on the typical productionrequirementsselected,theplantsizevariessignificantlybetweenthethreeexamples.In thefirstcase, theexampleconsidered is therecoveryofflaxseedoil fromthecakeaftermechanicalpressing.Flaxseedoilisconsideredaspecialtyoil.Specialtyoilsaretypicallyprocessedbymechanicalpressingofseeds.However,thisprocess-ing techniquenormally leavesahighpercentageof residualoil (RO) in thecake(5 to15wt,%). Inmost cases, the cake is used as animal feed.Supercriticalfluidextractionprovidesasolvent-freemethodforrecoveringtheROfromthecake[9].TheoilextractedbysupercriticalfluidextractionhasahighercontentofphytosterolandvitaminEthantheoilobtainedbymechanicalpressing[10].Additionally,themealcanbefurtherprocessedtoobtainconcentratedorisolateproteins.Boththeoilandmealprocessedwithsupercriticalfluidextractionmeetthedemandsofthenutraceuticalandorganicmarkets.Similarprocessescanbeappliedtorecoverotherspecialtyoils,suchasborageandeveningprimrose.Thosespecialtyoilshavehighersalevalues,whichimplyshorterreturnoninvestment(ROI).

Inthesecondcase,thecasestudyistheextractionofastaxanthinfromamicro-algae (Hematococcus pluvialis). Astaxanthin content ranges from 1% to 3%. TheconcentrationofastaxanthinintheextractcanbemuchhigherbySFEthanbycon-ventional solvent (acetoneorhexane)bymanipulating the selectivityof the super-criticalsolvent.Extractswithastaxanthincontentrangingfrom5%to10%canbeobtained. Eventhoughtheoperatingcostsarehigh,theROIisshorterbecausethesalepriceoftheextractisveryhigh.

Inthethirdcase,thespiceselectedwasginger.Theextractionofgingeroleo-resinandessentialoiliscarriedoutbysequentialdepressurization.Inthiscase,thecombinedsalesofbothfractionsprovideaROIinlessthan2years.

2.5 ConClusIons

Supercriticalfluidtechnologyisconsideredbythenutraceuticalandpharmaceuticalsectors as a viable technology to satisfy customer demands by replacing conven-tional technologies as well as providing solutions that traditional technologiescannotprovide.Tosuccessfullyimplementthistechnologyontheindustrialscale,it isnecessarytounderstandthetechnology,focusingontheproperdesignoftheplantcomponentsandoptimizationoftheprocessparametersthatprovideminimumoperatingcosts.Someexampleshavebeenpresented,showingthatthistechnologyhas been successfully applied to commodity products. There is a future trend toimplement this technology as part of a process line combined with traditional

7089_C002.indd 48 10/15/07 5:22:53 PM


processes—forexample,SFE+conventionalextraction+SFC,SFE+SFC,conven-tionalprocess+supercriticaldrying.

reFerenCes

1. Zosel,K.,U.S.Patent,3,806,619,1974. 2. Zosel,K.,U.S.Patent,4,247,570,1981. 3. Medina,I.andMartinez,J.L.,Dealcoholationofciderbysupercriticalextractionwith

carbondioxide,J. Chem. Tech. Biotech.,68(1),14,1997. 4. Martinez,J.L.,Ashraf-Khorassani,M.andChordia,L.,Supercriticalextractionprocess

ofgrape seedoil andphenoliccompounds,AICHEannualmeeting,SanFrancisco,2003.

5. Stahl, E., Quirein, K-W. and Gerar, D., Dense Gases for Extraction and Refining, Springer-Verlag,Berlin,1988.

6. Chordia,L.,Martinez,J.L.andDesai,B.,U.S.Patentapplication20050170063. 7. Martinez, J.L., Removal of residual solvents by supercritical fluids, AAPS 2004,

Baltimore,2004. 8. Chordia,L.andMartinez,J.L.,U.S.Patentapplication20050194313. 9. Chordia,L.andMartinez,J.L.,U.S.Patent7,091,366,2006. 10. Martinez,J.L.,Recovery of residual specialty oils after mechanical press using super-

critical fluid extraction,8thInternationalSymposiumonSupercriticalFluids,Kyoto,2006.

7089_C002.indd 49 10/15/07 5:22:53 PM

7089_C002.indd 50 10/15/07 5:22:53 PM

51

3 Supercritical Fluid Extraction of Specialty Oils

Feral Temelli, Marleny D. A. Saldaña, Paul H. L. Moquin, and Mei Sun

CONTENTS

3.1 Introduction................................................................................................... 523.2 BioactivesinSpecialtyOils.......................................................................... 52

3.2.1 Carotenoids........................................................................................563.2.2 PolyunsaturatedFattyAcids(PUFAs)............................................... 573.2.3 Squalene............................................................................................. 583.2.4 Sterols................................................................................................ 583.2.5 Tocols................................................................................................. 59

3.3 ExtractionofDifferentTypesofSpecialtyOils........................................... 613.3.1 NutOils.............................................................................................. 62

3.3.1.1 FactorsAffectingExtractionYield...................................... 623.3.1.2 CharacterizationofProductsExtractedbySC-CO2............693.3.1.3 ComparisonwithConventionalMethods............................. 72

3.3.2 SeedOils............................................................................................ 723.3.2.1 FactorsAffectingExtractionYield...................................... 763.3.2.2 CharacterizationofProductsExtractedbySC-CO2............ 783.3.2.3 ComparisonwithConventionalMethods.............................80

3.3.3 CerealOils.........................................................................................803.3.3.1 FactorsAffectingExtractionYield......................................803.3.3.2 CharacterizationofProductsExtractedbySC-CO2............ 833.3.3.3 ComparisonwithConventionalMethods.............................84

3.3.4 FruitandVegetableOils....................................................................843.3.4.1 FactorsAffectingExtractionYield......................................863.3.4.2 CharacterizationofProductsExtractedbySC-CO2............ 893.3.4.3 ComparisonwithConventionalMethods.............................90

3.4 FutureTrends................................................................................................903.5 Conclusions................................................................................................... 91References................................................................................................................ 91

7089_C003.indd 51 10/15/07 5:29:08 PM


3.1 INTrOduCTION

Some plant-based oils are classified as specialty oils due to their high concentra-tionsofbioactivecomponentswithdemonstratedhealthbenefits.Ingeneral,theyarecomprisedoftriacylglycerolswithafattyacidcompositionrichinunsaturatesandminorcomponentssuchastocols(tocopherolsandtocotrienols),carotenoids,sterols,andsqualene.Suchoilsincludenutoils(almond,hazelnut,peanut,pecan,pistachio,andwalnut),seedoils(borage,flax,eveningprimrose,grape,pumpkin,androsehip),cerealoils (amaranth, ricebran,andoatandwheatgerm),andfruitandvegetableoils (buriti fruit, carrot,olive, and tomato).Even though thedemand for specialtyoilsisgrowingatarapidpace,theyarestillconsideredanichemarketcomparedtothelarge-volumecommodityoils.Ingeneral,specialtyoilsaresoldintheformofcapsules,targetingthedietarysupplementmarket,aswellasgourmetoils.Similartocommodityoils,specialtyoilsarealsoproducedusingconventionalmethodsofmechanicalpressingand/orsolventextraction.Eventhoughcoldpressingattempera-tures below 60°C is used extensively in the specialty oil market, cold pressing islimitedintermsofoilrecoveryandthehighlevelsofresidualoilleftinthemeal.Ontheotherhand,conventionalsolventextractiondependsontheuseoforganicsolventssuchashexane,whichneeds tobe removedvia subsequent evaporation.Theheatappliedforsolventremovalmaybedetrimentaltoheat-labilebioactivecomponents.Inaddition,governmentregulationsontheuseoforganicsolventsaregettingstricterandthesafetyofresidualorganicsolventsinthefinalproductisbeingquestioned.

Supercriticalfluidextraction technologyisgrowingatarapidpacebecause itcanovercomemanyofthedisadvantagesassociatedwithconventionaltechnologiesand meet the consumer demand for “natural” products. The supercritical solventof choice for food applications has been supercritical carbon dioxide (SC-CO2).AdvantagesofprocessingwithSC-CO2includelowprocessingtemperatures;mini-malthermaldegradationoftheminorcomponentsofinterest;easeofseparationofextractionsolvent, resulting innosolvent residue left in theproduct;and the factthatprocessingintheCO2environmentminimizesundesirableoxidationreactions,whichisespeciallybeneficialforthesensitivebioactivecomponentsofspecialtyoilssuchassterols,tocols,carotenoids,andpolyunsaturatedfattyacids(PUFAs).

TheobjectivesofthischapteraretoreviewsomeoftherecentfindingsrelatedtothehealthbenefitsofbioactivecomponentspresentinspecialtyoilsandtheuseofSC-CO2extractiontechnologyfortherecoveryofspecialtyoilsfromdifferentplantsources,suchasnuts,seeds,cereals,fruits,andvegetables,withanemphasisontheeffects of various samplepreparation and extraction parameters on theyield andcharacteristicsoftheoilsobtained.

3.2 BIOaCTIvES IN SpECIalTy OIlS

Ofthelargevarietyofbioactivecompoundspresentinnaturalsources,thischapterfocusesonlyonthecarotenoids,PUFAs,squalene,sterols,andtocols(tocopherolsand tocotrienols) foundmainly inspecialtyoils.ThemainchemicalandphysicalpropertiesofthesebioactivecomponentsaresummarizedinTable3.1[1,2].

7089_C003.indd 52 10/15/07 5:29:09 PM

Supercritical Fluid Extraction of Specialty Oils 53

TaB

lE 3

.1ph

ysic

al p

rope

rtie

s of

Bio

acti

ve C

ompo

unds

[1,

2]

Bio

acti

ve

Com

poun

dFo

rmul

aM

olec

ular

W

eigh

t

Mel

ting

po

int

(°C

)

Boi

ling

poin

t (°

C)

Solu

bilit

yaSt

ruct

ure

Car

oten

oids

β-

Car

oten

eC

40H

5653

6.87

183

—sl

EtO

H,c

hl;s

eth

.,ac

e,b

z

Ly

cope

neC

40H

5653

6.87

175

—sl

EtO

H,p

eth;

se

th;v

sbz

,chl

,C

S 2

L

utei

nC

40H

56O

256

8.87

196

—vs

bz,

eth

,EtO

H,p

eth

HO

H

OH

Toco

ls

sE

tOH

,eth

,ace

,chl

HO R2

R1

α-to

coph

erol

β-to

coph

erol

δ-

toco

pher

ol

γ-to

coph

erol

R1 CH

3C

H3

CH

3

CH

3H H

H HR2O

α-

Toco

pher

olC

29H

50O

243

0.71

3.0

210b

β-

Toco

pher

olC

28H

48O

241

6.68

—20

5b

δ-

Toco

pher

olC

27H

46O

240

2.65

—15

0c

γ-

Toco

pher

olC

28H

48O

241

6.68

–1.5

205b

cont

inue

d

7089_C003.indd 53 10/15/07 5:29:14 PM


TaB

lE 3

.1 (c

onti

nued

)ph

ysic

al p

rope

rtie

s of

Bio

acti

ve C

ompo

unds

[1,

2]

Bio

acti

ve

Com

poun

dFo

rmul

aM

olec

ular

W

eigh

t

Mel

ting

po

int

(°C

)

Boi

ling

poin

t (°

C)

Solu

bilit

yaSt

ruct

ure

Toco

ls (

cont

inue

d)

sE

tOH

,eth

.,ch

l,ac

e,o

il

HO R2

R1

α-tocotrieno

lβ-tocotrieno

lδ-tocotrieno

lγ-tocotrieno

l

R1 CH

3

O

CH

3

CH

3

CH

3H H

H HR2

α-

Toco

trie

nol

C29

H44

O2

424.

67—

—

β-

Toco

trie

nol

C28

H42

O2

410.

64

δ-

Toco

trie

nol

C28

H42

O2

410.

64

γ-

Toco

trie

nol

C27

H40

O2

396.

01

Ster

ols

C

ampe

ster

olC

28H

48O

400.

6815

7.5

——

HO

St

igm

aste

rol

C29

H48

O41

2.69

170

—vs

bz,

eth

,EtO

H

HO

7089_C003.indd 54 10/15/07 5:29:17 PM


β-

Sito

ster

olC

28H

50O

414.

7113

7—

sE

tOH

,eth

,HO

Ac

H

HO

Hyd

roca

rbon

Sq

uale

neC

30H

5041

0.72

–4.8

280d

slE

tOH

;se

th,a

ce,c

tc

Fatt

y a

cids

L

inol

eic

acid

C18

H32

O2

280.

45–7

229e

vsa

ce,b

z,e

th,E

tOH

O

OH

α -

Lin

olen

ica

cid

C18

H30

O2

278.

43–1

123

0–23

2fs

EtO

H,e

th;s

lbz

O

OH

γ -

Lin

olen

ica

cid

C18

H30

O2

278.

43—

——

OO

H

asl

: slig

htly

sol

uble

, s:s

olub

le, v

s:v

ery

solu

ble;

ace

: ace

tone

,bz:

ben

zene

,chl

: chl

orof

orm

,ctc

: car

bon

tetr

achl

orid

e,E

tOH

: eth

anol

,eth

: die

thyl

eth

er, H

OA

c:a

cetic

aci

d,

peth

:pet

role

ume

ther

,bB

oilin

gpo

inta

t0.0

133

kPa,

cB

oilin

gpo

inta

t0.0

0013

3kP

a,d

Boi

ngp

oint

at2

.266

kPa

,eB

oilin

gpo

inta

t2.1

33k

Pa,f B

oilin

gpo

inta

t0.1

33k

Pa.

7089_C003.indd 55 10/15/07 5:29:21 PM


3.2.1 Carotenoids

Carotenoidsrepresentagroupofover600fat-solublepigments[3].Thesepigmentsareresponsibleforthebrightyellow,orange,andredcolorsoffruits,roots,flowers,fish, invertebrates, birds, algae, bacteria, molds, and yeast. Some carotenoids arealso present in green vegetables, where their color is masked by chlorophyll [4].Carotenoidsaretypicallydividedintotwoclasses:carotenes,whichareC40poly-unsaturatedhydrocarbons, andxanthophylls,oxygenatedderivativesofcarotenes.Carotenoid compounds are colored due to their high level of conjugated doublebonds,whichalsomakesthemquiteunstable.Indeed,eachconjugateddoublebondcanundergoisomerizationtoproducevarioustrans/cisisomers,particularlyduringfoodprocessingandstorage[5].About10%ofcarotenoidsarecalled“provitaminA,”indicatingthattheypossessatleastoneunsubstitutedβ-iononeringthatcanbecon-vertedintovitaminA[4].Thetwomaincarotenoidsthathavebeenheavilystudiedare β-carotene and lycopene. In terms of specialty oils, carotenoids are mainlypresentinburitifruit,carrot,rosehip,tomato,andwheatgermoils.

Of all carotenoids,β-carotene has the highest provitamin A activity, approxi-mately twice thatofα-andγ-carotene[4]. In theearly1980s,evidencesupportedβ-carotene as a chemopreventive agent [6]. Thus,β-carotene was the subject of anumberofstudies,suchastheAlpha-TocopherolBeta-CaroteneCancerPrevention(ATBC),β-CaroteneandRetinolEfficacyTrial(CARET),andthePhysician’sHealthStudy. The ATBC trial concluded that β-carotene supplementation did not helpsmokerswhopreviouslyhadaheartattack;infact,theirriskoffatalcoronaryheartdiseaseactuallyincreased[7,8].TheCARETstudy[9]showedthatsupplementationwithβ-caroteneandvitaminAhadnobenefitforcurrentandrecentex-smokersandmale asbestos-exposedworkers and that itmay increase the incidenceand riskofdeathdue to lungcancer,cardiovasculardisease,andanyothercause.Finally, thePhysician’sHealthStudyconcludedthatβ-caroteneintakerenderedneitherbenefitnorharmintermsofcancer,cardiovasculardisease,stroke,oroverallmortality[10].

Lycopene, although lackingprovitaminAactivity, isknown tobeoneof themost potent antioxidants among the digestible carotenoids. Its highly conjugatedmolecularstructureisresponsibleforthebrightredcolorofripetomatoesaswellasthepigmentationofwatermelons,pinkgrapefruits,apricots,andpapayas[3,11].Anumberofstudieshaveshownthat lycopenecouldplayaprotectiverole in thedevelopmentofatherosclerosis[12,13].Aswell, in vivoand in vitrostudieshaveshown that it has a hypocholesterolemic effect, thereby suggesting that lycopenecould attenuate atherogenesis and reduce the risk of cardiovascular disease [14].Somestudieshavefoundthatlycopeneintakecouldlowertheriskofprostatecancer,whileothers reportednoprotectiveeffect [3].However,acasestudyshowed thathigh consumption of tomatoes and tomato-based food products reduced stomachcancers[15].AccordingtoOmoniandAluko[14],thecomplexinteractionamongthepotentiallybeneficial compounds found in tomatoesmight contribute to theiranticancer properties. According to Rao and Shen [16], the recommended dailyintakeoflycopeneis5to10mg/day.Itisinterestingtonotethatapproximately80%ofdietarylycopenecomesfromtomatoesandthatprocessedtomatoeshaveahigherleveloflycopenethanrawtomatoesbecauseheattreatmentandhomogenizationof

7089_C003.indd 56 10/15/07 5:29:22 PM


tomatoesenhance theavailabilityof lycopene[17–19].Cohnetal. [20]comparedthe consumption of synthetic lycopene and lycopene in processed tomatoes andfound that theavailabilitywas thesame.Concerninghealthclaims, inNovember2005,theU.S.FoodandDrugAdministration(FDA)allowedthefollowingqualifiedhealthclaim:“Verylimitedandpreliminaryscientificresearchsuggeststhateatingone-halftoonecupoftomatoesand/ortomatosauceaweekmayreducetheriskofprostatecancer.FDAconcludesthatthereislittlescientificevidencesupportingthisclaim”[21].

3.2.2 Polyunsaturated Fatty aCids (PuFas)

PUFAsarefattyacidsthatcontaintwoormoredoublebondsinthecarbonchain.MostPUFAsareessentialfattyacidsandhavetobeprovidedtothebodythroughthediet.Theyareusuallyclassifiedasω-3andω-6,dependingonthepositionofthefirstdoublebondfromthemethylendofthecarbonchain.α-linolenic(ALA),eicosapentaenoic(EPA),docosapentaenoicacid(DPA),anddocosahexaenoic(DHA)acidsareexamplesofω-3PUFAs,whereaslinoleicacid(LA)andγ-linolenicacid(GLA)areexamplesofω-6PUFAs.ThemainsourceofEPA,DPA,andDHAarefishoils(seeChapter5).Becausethefocusofthischapterisplant-derivedspecialtyoils,onlyPUFAssuchasLA,ALA,andGLAwillbediscussed.With regard tospecialtyoils,PUFAsarefoundmainlyinalmond,apricot,hazelnut,peanut,walnut,borage,eveningprimrose,pumpkin,andricebranoils.

LA,ALA,andGLAareessentialfattyacidsthathumanenzymescantransforminto the PUFAs required by the body [22]. For instance, LA is converted by thehumanbodyintoarachidonicacid.Thelatter is transformedintoeicosanoidsandprostaglandins,whichareimportantmediatorsincardiovasculardisease[22].AlackofLAcanleadtofattyliver,skinlesions,andreproductivefailure[23].ALA,ontheotherhand,isconvertedtoEPAandDHA[23].DHAisamajorcomponentofthephospholipidmembranesofthebrainandretinas;therefore,alackofDHAcausesabnormalfunction[24].Whenthebodyexperiencesalackofω-3inthedietalongwithanincreaseinω-6,thelackofω-3tendstobeaccentuated,whichmayleadtoinhibitionofthesynthesisofDHAfromALA[25].Thus,itisimportanttokeeptheratioofω-6toω-3balancedinthediet.Somestudiesreportareductionincardio-vascular disease risk associated with higher ALA intake [26–28]. However, someinvestigatorsarestilltryingtoproveotherwise.SuchdebateisclearlyillustratedinthemultipleletterspublishedintheAmerican Journal of Clinical Nutrition [29,30].

It has been well established that intake of LA and GLA increases the tissuebiosynthesisof1-seriesprostaglandins,whichinturnsuppressesinflammation[31].ClinicalstudieshavealsoshownthatadministrationofGLAcanreducepainandswelling in rheumatoidarthritis [32,33].GLA is also said tobea“conditionallyessential fatty acid for the skin” [34]. Furthermore, a diet rich in EPA and GLAwasdeemedbeneficialforpatientswithacutelunginjury[35].BasedontheLyonDietHeartStudy,dietaryintakeofALAshouldbeabout1.8to2g/day[22].ThebestsourcesofALAarecanolaoilandalgae.NutsareagoodsourceofnotonlyALAbutalsoLA.DuetothecloseassociationbetweenALAandLA,careshouldbetakennottoconsumelargeamountsofLA-richoils,suchassoybean,sunflower,

7089_C003.indd 57 10/15/07 5:29:23 PM


andwalnutoils[22].ThedoseofGLAfedduringastudyonrheumatoidarthritiswas 1.4g/day, which was reported to be well tolerated by the patients [32]. TheaveragerecommendedintakeofPUFAsis7%oftotalenergyintake[36].However,consumingexcessivelevelsofPUFAswithoutproperintakeofantioxidantsisnotrecommendedsincePUFAsarepronetooxidation,whichmayplayaroleincarcino-genesis[37]andotherdiseases.

3.2.3 squalene

Squaleneisalipidthatwasoriginallyobtainedfromsharkliveroil.Itisalsofoundinolive,palm,andwheatgermoils[38].Anumberofanimalstudiesshowedthatdietarysqualenehasdistinctanticarcinogeniceffects. Itwasshownthatsqualenepresentsinhibitoryactionincarcinogenesismodelsofskin[39,40],colon[41],andlung[42]cancer.However,itdoesnotpresentchemopreventiveactivity[43].Onereportedsideeffectofsqualeneisthatofa51-year-oldmanwithesophagealcancerwhodevelopedsevereexogenouslipoidpneumoniaaftereatinglargedosesofsqualene[44].

Besides its anticarcinogenic activity, squalene prevents lipid peroxidation inhumanskinsurface[45]andisusefulintreatingconditionsresultingfrominadequateimmuneresponse[46].Itisalsousefulasacytoprotectant(medicationthatcombatsulcersbyincreasingmucosalprotection)incyclophosphamide-inducedtoxicities[47]andlow-dosesqualene(860mg)coadministrationwithlow-dosepravastatin(10mg)furtherenhancestheefficacyofpravastatinasacholesterol-loweringdrug[48].

Inthehumanbody,squaleneistheprecursortoimportantsterolssuchascholes-terol[49,50].Thus,itwasoriginallythoughtthatincreasedsqualeneconsumptionwouldactuallyincreasebloodcholesterollevels.Thisdoesnotseemtobethecasewhen0.5gofsqualeneisconsumedperday.Indeed,MiettinenandVanhanen[51]observedanincreaseintotalbloodcholesterolconcentrationsinmalesubjectsafteradietarysupplementationof1g/dayofsqualenefor9weeks,butwhenthedosewasreducedto0.5g/day,thebloodsterollevelswentbacktonormal.Itistruethat60%to80%ofdietarysqualeneisabsorbedthroughtheintestine[52]andthatasubstan-tialamountofthissqualeneisconvertedtocholesterolinthehumanbody.However,areasonableincreaseinsqualeneconsumptionappearstosignificantlyincreasethefecalexcretionofcholesterol[53].

3.2.4 sterols

Themainsterolsinplantmaterialsaresitosterol,campesterol,andstigmasterol[54].Theyaremainlyfoundinthespecialtyoilsofacorn,hazelnut,walnut,cherry,grape,pumpkin,andricebran.Overtheyears,ithasbeenwellestablishedthatahighdietaryintakeofphytosterolslowersbloodcholesterollevelsbycompetingwithdietaryandbiliarycholesterolduringintestinalabsorption[55–57].However,recently,PlatandMensink[58]speculatedthatbecausephytosterolsaremorereadilyoxidizedbyfreeradicalsthancholesterol,theycouldincreasethelevelofoxidizedlow-densitylipo-proteins(LDLs),whichformatheroscleroticplaquesinarteries.Atthistime,littleinformationisavailabletoprovesuchaclaim.Ontheotherhand,phytosterolsarenotrecommendedforindividualswhoaresufferingfromsitosterolemia,aninher-itable disorder that increases the absorption of cholesterol and phytosterols [55].

7089_C003.indd 58 10/15/07 5:29:24 PM


Fortunately,thisdisorderisquiterare;Björkhemetal.[59]knewof45patientswiththisdiseasein1998.

Phytosterolshavealsobeenthesubjectofmuchinvestigationforpropertiesotherthantheirabilitytolowercholesterol.Inthe1980s,consumptionofsitosterolwasshowntoreducecoloncancerinrats[60];however,thereisstillnostrongandcon-sistentevidencethatthesamewouldbetrueinhumans[61].Somestudiesreportthatsterolsmayhaveaneffectonimmunefunctionandthattheycouldpreventthesubtleimmunosuppression experienced by marathon runners [62, 63]. Finally, animalstudiesreportedthatphytosterolscouldinhibittheinflammatoryresponse[64]andthattheycouldcauseinsulin-releasingpropertiesfordiabetics[65].AccordingtotheScientificCommitteeonFood,theaverageamountofphytosterolsintheWesterndietis150to400mg[66].However,therecommendeddoseofphytosterolstoreduceplasmaLDL-cholesterollevelsby5%to15%is1.3to2g/day[67,68].InordertoachievesuchlevelsandcomplywiththeFDA-approvedhealthclaimontheroleofplantsterolorplantstanolestersinreducingtheriskofcoronaryheartdisease[69],foodmanufacturershave introducedvarious functional foodproductswithaddedphytosterols. In those countries where such products are currently marketed, thesuccessissogreatthatauthoritiesarenowworriedaboutconsumerseatingtoomuchphytosterols.Theconcernislegitimatebecauseastudyshowedthatdailyconsump-tionof3.8to4.0gofplantsterolesterscansignificantlylowerserumconcentrationsofvariouscarotenoidsandtocopherols[70].

3.2.5 toCols

TocopherolsandtocotrienolsmakeupthetocolsfamilyofvitaminEcompounds,whichmustbeobtainedfromthedietbecausehumanscannotsynthesizethem[71].Tocolsarefoundinalmond,hazelnut,pecan,walnut,flax,buritifruit,tomato,ricebran,andwheatgermoils.Thedifferencebetweentocopherolsandtocotrienolsliesin thephytylchainattached toachromanol ring: thephytylchain issaturated intocopherols,whereasthephytylchainintocotrienolshasthreedoublebonds[72].Thesecompoundsrepresentagroupoffourisomerswithvaryingnumbersandposi-tionofmethylgroupsonthechromanolring:α-,β-,γ-,andδ-tocopherolandα-,β-,γ-,andδ-tocotrienol.

Althoughallof these tocol isomers are absorbed through the intestine in thehumanbody, it isbelieved thatonlyα-tocopherolcontributes towardmeeting thehumanvitaminErequirement[73].Thereasoningbehindthisclaimisthatintra-cellular vitamin E content and distribution are regulated by different proteinsbindingspecificallytoα-tocopherol[74].Anotherfactorcontributingtothisschoolofthoughtisthatα-tocopherolisthemostpotentnaturallyoccurringscavengerofreactive oxygen and nitrogen species [72]. However, evidence is building on theimportanceofconsumingamixtureofthewholefamilyofvitaminEcompoundssincetheymayhaveadditiveandsynergisticactivitiesthatsupportbroaderbenefi-cialbiologicalfunctions[75].Theymayalsoactsynergisticallywithothernaturallyoccurringcompoundscommonlyfoundinfruitsandvegetables[72].Anoverviewofthecurrentscientificliteraturerevealstheimportanceoftocopherolsandtocotrienolsasthemajorfat-solubleantioxidants[73].Indeed,thesemoleculescanscavengefree

7089_C003.indd 59 10/15/07 5:29:24 PM


radicalsinthebody,therebypreventingthemfromdamagingcellmembranesandgeneticmaterialandchangingthecharacteroffatsandproteins[76].OneexampleistheprotectionthatvitaminEgrantstoPUFAs,whichareespeciallyvulnerabletodestructiveoxidation[73].EventhoughvitaminEisviewedasapotentantioxidant,in vitro studies have shown that vitamin E may have pro-oxidant effects at highdosages [77,78]. Interestingly, itappears thatα-tocopherolalonecanhaveapro-oxidanteffect[79];however,inthepresenceofγ-andδ-tocopherol,thepro-oxidanteffectofα-tocopherolseemstodiminish[75].

Theantioxidantpropertiesofmixedisomersoftocolscouldalsobebeneficialinpreventingtheonsetofatherosclerosis[80,81].ThismedicalconditionisinpartduetofreeradicalsoxidizingLDLs,whichinturnformatheroscleroticplaquesonthesurfacesofarterywalls.Byscavengingfreeradicals,tocopherolsblockthisprocess[80].Tocotrienolsarealsobelievedtobeusefulinthepreventionandtreatmentofatherosclerosisinpeoplewithtype2diabetes[82].Besidesitsantioxidantproper-ties,α-tocopherol acts as a regulator of gene expression that lowers the build-upofoxidizedLDLsinarteries[83,84].Unliketheliteratureonatherosclerosis,thescientificliteratureontheeffectsofvitaminEoncardiovasculardiseaseisdivided.SomeplainlystatethatthereisnoconcreteevidencethatvitaminEreducescardio-vascular-relatedmortality,particularlyinhigh-riskindividuals[85],whereasothersshow a reduction in mortality [86, 87] or no effect on cardiovascular events anddeath[75,88–92].

TheapplicationofvitaminEincancertreatmenthasalsobeenstudied.AlthoughanimalstudiesweresuccessfulinshowingthatvitaminEinhibitedcarcinogenesisandultraviolet-induceddeoxyribonucleicaciddamage[93–96]andpreclinicaldatarevealedthatitmightstimulateanantitumorimmuneresponse[97],clinicaltrialshavegivenmixedresults[72].SomestudiesonpatientswithstageIandIIheadandneckcancerwhowerefedsupplementsofα-tocopherolshowedahigherincidenceofsecondprimarycancersandalowerdegreeofcancer-freesurvival[98].Also,afewstudiesshowedthatα-tocopherolwasoflittletonobenefitinpreventinglungcancer[99].However,α-tocopherylsuccinatewasfoundtobetumorspecificinprostateandbreasttissues[100].Thereisalsoevidencethatγ-tocopherolmayhelppreventandtreatcolon[101]andprostate[102]cancer.

Anumberofstudiesreport thatvitaminEcanbenefit individualswithosteo-arthritis[103–106].Supplementationof400mg/dayofvitaminEhadabeneficialanalgesicandanti-inflammatoryeffect,withalowincidenceofsideeffects[107].Aswell,supplementationwithmixedisomersofvitaminEwasadvantageous[108].Some research has shown that α-tocopherol supplementation delays or preventsAlzheimer’sdisease(AD)diagnosisofelderlyindividualswithsignsofmildcogni-tiveimpairment[109];however,othersreportnoclearbenefitsofvitaminEinthetreatmentofpeoplewithAD[110–112].UnrelatedtoAD,astudyonnursinghomeresidentsshowedthatsupplementalvitaminEreducedtheincidenceanddurationofrespiratoryinfections[113].

The FDA has approved the following health claim for dietary supplementscontaining vitamin E and/or vitamin C [114]: “Some scientific evidence sug-gests that consumption of antioxidant vitamins may reduce the risk of certainformsofcancer.However,FDAhasdeterminedthatthisevidenceislimitedand

7089_C003.indd 60 10/15/07 5:29:25 PM


notconclusive.”Someworkhasalsobeenperformed toachievequalifiedhealthclaims for vitaminE supplements against cardiovascular disease. However, thishealthclaimwasrefusedbasedoninsufficientevidence[115,116].Theoptimumconsumption levelofvitaminE for thegeneralpopulation is an interestingcon-sideration that is stillopen todebate.According toHealthandWelfareCanada,the Recommended Daily Intake (RDI) for vitamin E should be 5 to 10 mg; theAmericanDieteticAssociationsetstheRecommendedDietaryAllowance(RDA)at15mg/dayofα-tocopherolforadultsand19mg/dayforwomenwhoarebreast-feeding[73].BecausenotallsourcesofvitaminEhavethesamebiologicalactivity,one has to keep in mind that 1international unit (IU) of natural vitamin E cor-responds to0.67mgofα-tocopherol,whereas1IUofsyntheticvitaminEcorre-spondsto0.45mgofα-tocopherol[73].Inaddition,thegrowingevidenceshowingthebenefitsofmixed isomersof tocopherolsand tocotrienols shouldprobablybereflectedintheRDIandRDA.AlthoughvitaminEwasconsideredtobenontoxicformanyyears, it isnowknownthatanoverdoseofvitaminEcaninterferewithbloodclotting,especiallywhentakenalongwithanticoagulantmedicationorwithacetylsalicylicacid[117].Furthermore,arecentmeta-analysisincluding19studieswithmorethan135,000patientsshowedthatmorethan400IU(270mg)perdayofvitaminEsupplementationtopatients(ages47to84years)whomostlyhadchronicdiseasesmighthaveincreasedall-causemortality[118].

3.3 ExTraCTION OF dIFFErENT TypES OF SpECIalTy OIlS

The specialty oils rich in bioactives can be extracted from many plant sources.Theseextractedoilsaremainlymixturesoftriglycerides,freefattyacids(includingPUFAs),monoglycerides,diglycerides,andotherminorcomponents,suchastocols,carotenoids, sterols, and squalene. For specialty oils to be extracted from differ-entplantmaterialsusingSC-CO2,theyhavetobesolubleinSC-CO2.Thesolubil-itybehaviorofmajorandminorlipidcomponentsinSC-CO2hasbeenpreviouslyreviewedandcorrelated[119,120].SolubilityisastrongfunctionofSC-CO2densityandthepropertiesofthesolute,suchasmolecularweight,polarity,andvaporpressure.AllthelipidcomponentsofinterestinspecialtyoilsaresolubleinSC-CO2todiffer-entextents,dependingontemperatureandpressureconditions.Generally,solubilityof lipids inSC-CO2decreaseswithan increase inpolarityandmolecularweight,thusfollowingtheorder:fattyacidesters,fattyacids,andtriglycerides[120].

SC-CO2extractionofspecialtyoilsfromvarioussourceshasbeenstudiedquiteextensively. The extraction efficiency and the characteristics of the products areaffectedbyseveralparameters,suchasparticlesizeandmoisturecontentofthefeedmaterial,extractiontemperatureandpressure,solventflowrate,extractiontime,andtheuseofacosolvent.Therefore,thefollowingdiscussionemphasizestheimpactoftheseprocessingparametersontheyieldandcompositionofspecialtyoilsobtainedfromnuts,seeds,cereals,fruits,andvegetables.Eventhoughsomenutsareseedswithin the fruit of the plant, they are classified in this chapter as nuts based onconsumption;forexample,almondisclassifiedasanutbecauseitisconsumedasasnackuponroasting,whereasapricotkernelisclassifiedasaseed.Inaddition,some

7089_C003.indd 61 10/15/07 5:29:26 PM


fruitsandvegetablesliketomatoesandgrapesarealsoincludedinthediscussionofseedsbecausetheirseedswereusedinextractionstudies.

3.3.1 nut oils

Nutsprovideprotein,fiber,essentialfattyacids,andvitamins.Theirpleasantflavorand aroma and unique texture lead to their popularity as snacks. However, theirhighfatcontentlimitstheirconsumptionduetoconsumerconcernsabouttheirhighcaloric content. Besides, nuts are prone to oxidation due to the presence of highlevelsofPUFAs.Eventhoughtheyhavelowoxidationstability,nutoilshavebeencommercializedinsomecountriesashighlynutritiousspecialtyoils.Defattednutsalsohavemarketvalueaslow-fatproducts;therefore,thepropertreatmentofnutsbeforeandafterextractionneeds tobeconsidered.NumerousstudiesonSC-CO2extraction of nuts, including acorn [121], almond [122–125], hazelnut [126–128],peanut[129–133],pecan[134–136],pistachio[137],andwalnut[138–140],havebeenreportedandaresummarizedinTable3.2.

3.3.1.1 Factors affecting Extraction yield

3.3.1.1.1 Sample PreparationMost of the nuts were extracted fresh, but some were roasted [122, 141]. Feme-niaetal. [122]reported that theroastednutshadhigheroilcontentdue towaterlossandpartialdegradationofproteinandprobablypectinaswellduringroasting.However,itwasdifficult toextracttheoilfromroastedalmonds,possiblyduetotheformationofnewlinksamongcellwallpolymers,thusreducingporosityandstrengtheningthewallstructure[122].

a) Particlesize:Thenutsaregenerallygroundtosmallparticlestoincreasesurfaceareaandshortenthepathlengthsoverwhichthesolutesmusttraveltoreachthebulkfluidphaseandthereforefacilitatetheextractionofthenutoil.Thus,particlesizeimpactsextractionkineticsoftheoil,whichispres-entasreleasedoilonthesurfaceoftheparticlesaswellasunreleasedoilinsidetheparticles.TheextractionrateisdictatedinitiallybythesolubilityofthefreeoilinSC-CO2(fastextractionperiod)andlaterbythediffusionofoilinsidetheparticles(slowextractionperiod).Ingeneral,whenfreshsolventcomesincontactwiththefeedmaterial,thefreeoilonthesurfaceisquicklysolubilizedandextracted.Extractionrateisfastandlimitedbyequi-libriumsolubility,asrepresentedbytheinitiallinearportionoftheextrac-tioncurve.Whentheoilonthesurfaceoftheparticlesisdepleted,SC-CO2hastodiffuseintotheparticlesandsolubilizetheoilandSC-CO2+oilhastodiffuseout,whichisaslowprocessdrivenbytheoilconcentrationgradi-ent;thustheextractioncurveapproachesaconstantvalueasymptotically.Özkaletal.[126]reportedayieldof0.51goil/ghazelnutattheendofthefastextractionperiodandonly0.01goil/ghazelnutduringtheslowextrac-tionperiod.Therefore,theextractioncouldbestoppedafterthefastextrac-tionperiod.Theoilyieldobtainedattheendofthefastextractionperiod

7089_C003.indd 62 10/15/07 5:29:27 PM


TaB

lE 3

.2Ex

trac

tion

of B

ioac

tive

Com

poun

ds fr

om N

uts

usi

ng S

C-C

O2

raw

M

ater

ial

Feed

(g)

Sam

ple

prep

arat

ion

Bio

acti

ve

Com

poun

d

Extr

acti

on C

ondi

tion

sr

ecov

erya

(%)

ref

.pa

rtic

le S

ize

(mm

)H

2O (

%)

T (°

C)

p (M

pa)

Flow

rat

eTi

me

(min

)C

osol

vent

Aco

rnn.

i.0.

27n.

i.O

leic

aci

d,

linol

eic

acid

,β-

sito

ster

ol,

stig

mas

tero

l,ca

mpe

ster

ol,

toco

pher

ols

4018

1.5

×1

0–2 m

/min

bn.

i.N

one

n.i.

121

Alm

ond

1500

–200

0n.

i.n.

i.U

nsat

urat

edo

il50

3333

3.3–

666.

7g/

min

n.i.

Non

en.

i.12

2

4000

n.i.

n.i.

Uns

atur

ated

oil

6048

.2n.

i.n.

i.N

one

n.i.

123

3000

–400

0M

illed

,bro

ken,

w

hole

n.i.

Toco

pher

ols,

ol

eic

acid

,lin

olei

cac

id

35,4

0,5

035

,45,

55

166.

7,3

33.3

,50

0g/

min

n.i.

Non

en.

i.12

4

n.i.

0.3,

0.7

,1.9

n.i.

Uns

atur

ated

oil

4035

12,2

3.8

g/m

inn.

i.N

one

n.i.

125

Haz

elnu

t4

<0

.85

3O

leic

aci

d,

linol

eic

acid

40,5

0,6

030

,37.

5,4

51,

3,5

g/m

in10

Non

e34

126

51–

23

Ole

ica

cid,

lin

olei

cac

id40

,50,

60

30,4

5,6

02

×1

0–3L

/min

250–

300

Non

e59

127

500.

7n.

i.U

nsat

urat

edo

il,

β-si

tost

erol

,α-

toco

pher

ol

35–4

818

–23.

42.

7–4.

3×

10–2

m/m

in24

0N

one

>95

128

cont

inue

d

7089_C003.indd 63 10/15/07 5:29:28 PM


TaB

lE 3

.2 (c

onti

nued

)Ex

trac

tion

of B

ioac

tive

Com

poun

ds fr

om N

uts

usi

ng S

C-C

O2

raw

M

ater

ial

Feed

(g)

Sam

ple

prep

arat

ion

Bio

acti

ve

Com

poun

d

Extr

acti

on C

ondi

tion

sr

ecov

erya

(%)

ref

.pa

rtic

le S

ize

(mm

)H

2O (

%)

T (°

C)

p (M

pa)

Flow

rat

eTi

me

(min

)C

osol

vent

Pean

ut10

00(

h.e.

),

500

(v.e

.)h.

e.:0

.86–

1.68

v.

e.:0

.86–

1.19

,3.

35–4

.75

n.i.

Uns

atur

ated

oil

h.e.

:25–

100;

v.e

.:25

–120

h.e.

:27.

5–69

;v.

e.:1

4–55

h.e.

:20

L/m

in;

v.e.

:40

L/m

inv.

e.:1

80N

one

h.e.

:50;

v.

e.:9

912

9

5,57

90.

864–

1.18

,1.

18–1

.7,1

.7–2

.36,

2.

36–3

.35,

3.

35–4

.75

5,9

,15

Uns

atur

ated

oil

25,5

5,7

5,

9527

.5,4

1.5,

55

40a

nd6

0L

/min

180

Non

e99

130

50,8

60G

roun

d:0

.864

–1.1

8,

1.18

–1.7

,1.7

–2.3

6,

2.36

–3.3

5,

3.35

–4.7

5Fl

akes

:1.2

7

n.i.

Uns

atur

ated

oil

2555

40L

/min

180

Non

en.

i.13

1

1n.

i.n.

i.U

nsat

urat

edo

il40

–80

13.8

–55.

1n.

i.n.

i.N

one

n.i.

132

n.i.

n.i.

4.2–

5.1

Uns

atur

ated

oil

50–6

535

–50

n.i.

n.i.

Non

en.

i.13

3

Peca

n5–

6H

alve

s4

Uns

atur

ated

oil,

to

coph

erol

s40

,80

41.3

,55.

1,

68.9

n.i.

160

Non

e77

134

20H

alve

s4.

9,6

.4,7

.4,

11U

nsat

urat

edo

il75

623

L/m

in60

Non

en.

i.13

5

90H

alve

s,p

iece

sH

alve

s:4

.8;

Piec

es:4

.1U

nsat

urat

edo

il45

,62,

75

41.3

,55.

1,

62,6

6.8

1,1

.5,2

,2.5

,3,4

,7.

5L

/min

60N

one

n.i.

136

Pist

achi

o10

1–1.

68n.

i.U

nsat

urat

edo

il50

,60,

70

20.7

,27.

6,

34.5

2.6

g/m

inn.

i.10

%E

tOH

+66

.1c

137

7089_C003.indd 64 10/15/07 5:29:28 PM


Wal

nut

n.i.

0.01

,0.0

5,0

.1,0

.5n.

i.L

inol

eic

acid

,ol

eic

acid

,lin

olen

ica

cid,

β-

sito

ster

ol,

cam

pest

erol

,α-

toco

pher

ol

35,4

0,4

5,

4818

,20,

22,

23

.4n.

i.n.

i.N

one

9513

8

n.i.

Piec

es3

Lin

olen

ica

cid,

un

satu

rate

doi

l80

68.9

150

g/m

inn.

i.N

one

n.i.

139

n.i.

Piec

esn.

i.U

nsat

urat

edo

il80

68.9

150

g/m

inn.

i.N

one

n.i.

140

T:t

empe

ratu

re,P

:pre

ssur

e;n

.i.:n

otin

dica

ted;

h.e

.:ho

rizo

ntal

ext

ract

ion,

v.e

.:ve

rtic

ale

xtra

ctio

n,a

Rec

over

y(g

ext

ract

/go

ilin

fee

dm

ater

ial×

100

),b s

uper

ficia

lvel

ocity

,c yie

ld(

g/10

0g

feed

mat

eria

l).

+

Cos

olve

nta

dded

con

tinuo

usly

into

SC

-CO

2at

the

leve

l(%

,w/w

)in

dica

ted.

7089_C003.indd 65 10/15/07 5:29:29 PM


wasdependentonparticlesize[126].Forexample,theoilyieldforhazelnutwithaparticlesizeof1.5mmwasabouthalf(0.22goil/gnut)ofthatfromparticleslessthan0.85mm(0.51g/g).Inanotherstudy,usingwhole,bro-ken(4to8mm),andmilled(0.5to3mm)almond,Leoetal.[124]observedthattheextractionyieldincreasedwithreducingparticlesize(50,350,and800g/kg,respectively)undersimilaroperatingconditions.Inaddition,oilrecoveryincreasedfrom36%to82%whentheparticlesizeofpeanutwasreduced from 3.35 and 4.75 mm to 0.86 and 1.19 mm [130]. There is apracticallimittogrindingtosmallerparticlesduetotheoilynutparticlesstickingonthesieves[130].

b) Moisture and equilibration time: Moisture has a great impact on oilextractionbecausethekernelexpandswithmoistureabsorption,resultingin a more permeable cell membrane, allowing both oil and CO2 to passmorereadily.However,excesswatercanalsoimpedethediffusionofoiland have a negative effect on oil accessibility. The amount of pecan oilextractedafter48hoursofmoistureequilibrationwasapproximately30%higher thanthatobtainedafter1hour[135].Waterwascoextractedwiththeoiland increased linearlywith the initialmoisturecontentofpecans[135]. The moisture content of the extracted oil was 0.7% and 11.7% atinitial moisture levels of 3.5% and 12%, respectively, in the pecan. Theoilobtainedwascloudywithayellowishcolor.Ahigh levelofwater inthe extracted oil is not desirable since it negatively impacts its stability.Moistureaffectsnotonlyextractionefficiencyandyieldbutalsothephysi-calstructureofthenuts[135].Breakageofthekernelsduringtheextrac-tioncanbeavoidedbyadjustingthemoisturecontenttoacertainlevel,forexample,8%to11%(w/w)forpeanuts[133].Passeyetal.[133]testedsoak-ing,steaming,andhumidificationaspretreatmentmethodsforpeanutsandfoundthatsoakingandsteamingwereaseffectiveashumidificationinpre-ventingthebreakageofthekernels,buttheycausedbrowning,lossofwatersolubles, and low rate of extraction. Moisture content also affects pecanbreakage[135].Withmoistureabsorption,thekernelbecomessoftandpli-able,inpartbecausethemoistureaffectstheplasticizationofproteinsandcarbohydratesandaltersthephysicalpropertiesofthetissues.Extractionofpecanafter48hoursequilibrationproducedlessbreakagethanextractionat1houratmoisturecontentsof6.1%and7.7%[135].Thiswasduetothewaterinthekernelbeingmoreevenlydistributedafteralongerequilibra-tiontime.However,theeffectoftheequilibrationtimedecreasedat8.5%moistureandwasnegligibleat11.6%[135].Thiscanbeexplainedbytheosmoticpressurecausedbythewateraroundthekernel.

3.3.1.1.2 Extraction Parameters a) Temperature and pressure: Most of the nut oil extractions were per-

formed at a temperature range of 35°C to 100°C and a pressure rangeof9to70MPa.Thesolubilityofoil inSC-CO2ismainlydeterminedbytheSC-CO2density and thevolatilityof theoil components. Ingeneral,

7089_C003.indd 66 10/15/07 5:29:29 PM


SC-CO2 density increases with pressure at constant temperature anddecreaseswithtemperatureatconstantpressure,wherethedensitydecreasebecomessmallerathigherpressures.Ontheotherhand, thevolatilityofoilcomponentsincreaseswithtemperature.Thesetwoopposingeffectsoftemperatureondensityandvolatilityleadtothewell-establishedcrossoverbehaviorof solubility isotherms.A temperature increasemayalsocausebreakdown of cell structure and increase the diffusion rate of the oil intheparticles,thereforeacceleratingtheextractionprocess[130].Itisalsoimportanttoconsiderthatoilcompositionvarieswidelyamongthediffer-enttypesofnutsandthusdifferencesinfunctionalgroupsandfattyacidcompositionare responsible for thedifferences involatility, solubility inSC-CO2,andcrossoverpressure.Solubilityincreaseswithtemperatureandpressureabovethecrossoverpressure.Thesegeneraltrendsoftheeffectsoftemperatureandpressureonoilsolubilityandyieldarereflectedinsomestudies.Forexample,thesolubilityofpeanutoilinSC-CO2decreasedwithtemperatureatpressuresbelow35MPaandincreasedathigherpressures[130].Increasingthetemperaturefrom29°Cto91°Cincreasedtheinitialextractionratefrom15to129mgpeanutoil/LCO2at55MPa;however,at27.5MPa, increasingthe temperaturefrom27°Cto100°Cdecreasedtheinitialextraction rate from7.6 to0.5mgoil/LCO2[130]. Increasing thepressurefrom41.3to55.1MPaincreasedthepecanoilyieldfrom14.3%to 21.3% at 45°C and from 17.5% to 31.5% at 75°C. However, a furtherincrease inpressure to66.8MPaonly slightly increased theoil yield to21.5%and32.4%at45°Cand75°C,respectively[136].Thepositiveeffectofpressureonoilyieldwasalsoobservedathightemperatures.Similarly,raisingthepressurefrom17.7to68.9MPayielded100%and200%moreoilfrompecansat40°Cand80°C,respectively[134].

The rate of depressurization following an extraction affects thebreakage of the nut kernel, as demonstrated for the pecan kernel [135].When the vessel was depressurized from 62 MPa in 20 min there wasnobreakage,whereasasignificantamountofbreakageoccurredduringthe10-mintest.Duringthe20mindepressurization,thepressureintheextractionvesselwasdropped from62MPa toabout7MPawithin thefirstminuteandwasaround2MPaafter10minand0MPaafter20min.This suggested that the final stages of depressurization were crucial incausingpecanbreakage.When theextractionvesselwasopened imme-diatelyafterdepressurizingthereactor,theparticlesjumpedaroundandpoppingsoundswereheard,suggestingthatmostofthebreak-upoccurredastheCO2-saturatedparticlesweredepressurized[130].Gradualpressur-izationanddepressurizationwerealsonecessarytominimizedamagetothewalnutpieces[139].TheruptureorbreakageofthecellsoccursduetothephasechangeofCO2.ArapiddepressurizationtoatmosphericpressureformsliquidCO2aswellasdryice.Therefore,theCO2trappedinsidethesolidmatrixexpands,causingbreakageinthecells.Slowdepressurizationwith appropriate level ofheating toovercome the Joule-Thomsoneffect

7089_C003.indd 67 10/15/07 5:29:30 PM


andtomaintainthetemperatureabove31°CassuresthattheCO2willbeingasphaseandavoidanybreakage.

b) Flowrateandflowdirection: Increasing solventflowrate results inanincreasedsolvent-to-feedratioanddecreasedmasstransferresistance.Theextractedpecanoilyieldincreasedfrom8.8%to21.5%withanincreaseinflowratefrom1to4L/min(measuredatstandardtemperatureandpressure,STP);however,withafurtherincreaseto7.5L/min,theoilyieldchangedonlyfrom21.5%to21.7%[136].Theeffectofflowratewasalsostudiedfortheextractionofhazelnutoil[127].Atalowpressure(15MPa),anincreaseinflowratefrom0.5to2mL/min(measuredatextractorpressureand10°C)didnotcauseasignificantdifferenceintheextractionyieldofhazelnutoil;however, at a highpressure (30MPa), theoil yield increasedmore thanthree-fold[127].ThismightbeduetothelowsolubilityofoilinSC-CO2atlowpressures.AnotherimportantfactorthataffectsextractionyieldisthedirectionoftheCO2flow.Theverticalextractorproducedahighertotaloilrecoveryandamoreuniformlyextractedpeanutmealsamplethanthehorizontal extractor [130]. This might be caused by the meal settling inthehorizontalextractorandleavingalowerresistanceflowpathalongthetop of the meal, leading to channeling and insufficient contact betweenthe fresh solvent and peanut meal. Similarly, the flow direction greatlyaffected thesolubilityof thepeanutoil inSC-CO2atboth55MPa/75°Cand55MPa/95°C,withthedownwardflowresultinginahighersolubility[130].Thisisprobablybecauseofthelargetemperaturegradientbetweenthetopandbottomoftheextractorintheupwardsystem,whichmightbedue to the incoming fresh CO2 cooling the inside of the extractor. Withdownwardflow,itwaspossibletomaintainthetemperaturethroughouttheexperiment,whichwasattributedtothebalancingofdensity-inducedcon-vectioneffectswithdownwardflow[130].Thus,itwaspossibletomaintaintheconstantextractionrateforalongerperiod.

c) Extractiontime:Duetothephysicalstructureofthenut,thepenetrationof thesolventand thediffusionof theunreleasedoil in theparticlesareveryslow.Therefore,extractiontimeisusuallylimitedtothefastextrac-tionperiodsincetheamountofoilrecoveredintheslowextractionperiodis negligible. The duration of the fast extraction period is also inverselyrelated to particle size. However, the extraction rate and the duration ofthefastextractionperiodarealsoaffectedbytemperature,pressure,flowrate,andcosolventaddition.Forexample,thedurationsofthefastextrac-tionperiodforthehazelnutoilextractionsconductedat50°Cand45MPawere50and60minforparticlesizesoflessthan0.85mmand1.5mm,respectively[126].Whentheextractionconditionswerechangedto40°C,37.5MPa,and5g/minflowrate,thefastextractionperiodwas50minforthenutparticlesoflessthan0.85mminsize[126].Fastextractionperioddecreasedfrom183to64and32minwithapressureincreasefrom30to45and60MPaat40°C.Ontheotherhand,itdecreasedfrom64to33minwithatemperatureincreasefrom40°Cto50°Cat45MPa[127].

7089_C003.indd 68 10/15/07 5:29:31 PM


d) Useofcosolvent:Ahigheryieldcanbeobtainedbyaddingasmallamountofcosolvent,suchasethanol.Forexample,when10%ethanolwasaddedtoCO2(w/w),theextractionyieldofpistachiooilat60°Cincreasedby230%at34.5MPaandby750%at20.7MPacomparedtothatobtainedwithCO2alone[137].Inaddition,theextractionyieldofpistachiooilusing10%etha-nolasacosolventat60°Cwashigherat34.5MPathanthatat20.7MPa.

3.3.1.2 Characterization of products Extracted by SC-CO2

3.3.1.2.1 Chemical CompositionThefattyacidcompositionofthenutoilsispresentedinTable3.3.Themainfattyacidsintheextractednutoilsarelinolenic(Ln),linoleic(L),oleic(O),andpalmitic(P)acidsformingtriglycerideslikeLLL,OLL,LLLn,OOO,andPOO,theamountsofwhicharedependentonthetypeofnut.ThefattyacidcompositionofSC-CO2-extractedoilsexhibitedminordifferencesincomparisontooilsobtainedfromthefeedmaterial.However,asmallincreaseinthepercentageofoleicandstearicacidswasdetectedwhenabout65%ofthealmondoilwasextracted[122],indicatingthatSC-CO2 extraction may result in minor modifications of the fatty acid profile oftheextractedoils.Ontheotherhand,nofractionationwasdetectedduringextrac-tionsofhazelnutoil,asthefattyacidcompositionofthethreehazelnutoilfactionsobtainedduringthefirst30min,between70and120min,andafter120minwassimilartothatoftheoilextractedwithhexane[127].Therewasalsonosignificantdifferenceinthefattyacidcompositionofthepecanoilsobtainedatextractiontimesbetween15and480min[136].Thetocopherolcontentofthefat-reduced(25%and40%)walnutswassignificantlylowerthanthat inthefull-fatnuts[139].Thenutsafterextractionhadincreasedprotein,mineral,andcarbohydratecontentduetothereductionintheiroilcontent.Theextractedalmondflakeswith86.5%oilremovalhad approximately twice as much protein, carbohydrates, and minerals as rawalmonds[123].Theproteincontentof25%and40%fat-reducedwalnutsincreasedfrom14%inthefull-fatnutto21%and27%,respectively[139].Thecellstructureofthealmondwasgraduallymodifiedastheextractionprogressed[122].When15%oftheoilwasextracted,thepectinwasaffectedwithnomodificationofcelluloseandhemicelluloses.When35%oftheoilwasextracted,markedchangescouldbeobservedinbothpectinandhemicelluloses;whileat57%and64%oilextraction,the sample exhibited major cell wall disruption [122]. Similar observations werereportedforwalnuts,wherelipidextractionbeyond40%resultedincollapsedcellwallsandfractureandpowderingofwalnutpieces.Mineralssuchascalciumandmagnesiumwerealsoaffectedbytheextraction,especiallyafter65%oftheoilwasextracted.Whenthesedivalentcationswereremovedfromthecalcium-pectincom-plex,thecross-linksbetweenthegalacturonicacidunitsofadjacentpectinchainsorbetweenthepectinsandotherpolymersweredestroyed.Amassbalancelossof2%to20%wasreportedbyGoodrumandKilgo[130],whoattributedittothelossofvolatileorganicsandwatervaporintheexhaustCO2stream.Ingroundpeanuts,thehigherthemoisturecontent,temperature,andpressure,thegreaterwasthemassloss[130].Asexpected,agreatermassofvolatilecomponentswaslostintheexhaust

7089_C003.indd 69 10/15/07 5:29:31 PM


TaB

lE 3

.3N

ut F

at C

onte

nt a

nd F

atty

aci

d C

ompo

siti

ona o

f Nut

Oils

raw

M

ater

ial

Fat

Con

tent

(%

, w/w

)

Fatt

y a

cid

Con

tent

a–d

C16

:0C

16:1

C18

:0C

18:1

C18

:2C

18:3

ref

.

Aco

rn

12.1

13.4

2–13

.44a

0.07

–0.0

82.

564

.81–

65.4

216

.43–

17.0

70.

52–0

.57

121

Alm

ond

57.0

7.87

–8.4

8b0.

56–0

.63

1.58

–1.6

869

.25–

70.3

119

.65–

20.1

6—

122

54.5

6.60

–7.1

0b0.

50–0

.60

1.7–

2.2

68–7

317

.7–2

2—

124

Haz

elnu

t66

.25.

27–6

.01c

0.17

–0.2

02.

19–2

.45

82.6

5–85

.18

6.27

–8.4

20.

08–0

.09

128

56.0

5.86

–5.9

9c—

2.14

–2.1

779

.34–

79.6

211

.37–

11.4

5—

127

Peca

n58

.56.

20–1

0.10

c—

2.9–

3.2

60.4

–65.

723

.1–2

5.5

1–3.

313

6

Wal

nut

69.0

e6.

90–8

.10d

—1.

5–2.

016

.5–1

6.7

60.9

–61.

212

.5–1

3.7

140

71.0

6.08

–6.4

9a0.

072.

1–2.

1320

.98–

21.2

256

.46–

56.8

813

.16–

13.4

113

8

Palm

itic

acid

(C

16:0

),P

alm

itole

ica

cid

(C16

:1),

Ste

aric

aci

d (C

18:0

),O

leic

aci

d (C

18:1

),L

inol

eic

acid

(C

18:2

),L

inol

enic

aci

d (C

18:3

).a m

ol%

,b wt %

,c not

indi

cate

d,d G

CA

rea

%,e F

atc

onte

ntr

epor

ted

in[

139]

.

7089_C003.indd 70 10/15/07 5:29:32 PM


CO2streamathighertemperatures.Aswell,lossesduetoinefficienciesinthecollec-tionofsolutespriortoCO2exhaustshouldnotbeoverlooked.

3.3.1.2.2 Other Quality AttributesThe color of the residual meal and the extracted oil are affected by the degreeof extraction. With higher oil removal, the color of the pecan and peanut kernelresidueswaslighter,asmostofthepigmentsarefatsoluble[134,141].TheL-value(ameasureoflightness)ofthefat-reducedwalnutswashigherthanthatofthefull-fatnuts, indicatingthat thefat-reducedwalnutshadawhiterappearance[139].Inanotherexample,agradualcolorchangeontheresidualmealwasobserved,withmeallocatedatthesolventoutletbeingdarkerandhavingmoreoil[130].Thecolorofmealresidueisalsoaffectedbytemperatureandcosolventaddition.Thepeanutmealbecamedarkerwith temperature increaseatconstantpressure.At75°C, themeallocatedatthereactorinletchangedfromchalkwhitetolightbrownandfinallytodarkbrownwhentemperaturereached95°C[130].Pecankernelsappearedmorered and less yellow after extraction, and this trend increased with temperature[134].WhenpureSC-CO2wasused,onlyasmallchangeinoriginalpistachiocolorwasobserved.However,whenethanolwasaddedasacosolvent,analmostwhitepistachioresiduewasproducedduetotheextractionofchlorophyll[137].Coloroftheresidueisalsoaffectedbypretreatmentofthesample.Comparedwithsoakedpeanuts,humidifiedpeanutshadtheclosestcolorofoilandpeanuts[133].

Thecoloroftheextractedoilisaffectedbytemperature,pressure,andcosolventaddition.PalazogluandBalaban [137] showed increasingcolor intensities for theextractedpistachiooilswithanincreaseinpressure.Thecoloroftheoilextractedat60°Cand34.5MPawasdarkyellow,whereasthatobtainedat50°Cand27.5MPawas lighter. Oil extracted at 70°C and 20.7 MPa with 5% ethanol addition wasyellowishgreenandthatat70°Cand34.5MPawasgreen.Thecrudepeanutoilcolorrangedfromyellowtodarkbrownasextractiontemperatureincreasedfrom25°Cto120°C[130].Theextractedpecanandwalnutoilswereallamberincolor[134,139],whereasthealmondoilwasyellow[124].

Thetexture(hardness)ofthepistachionutchangedsignificantlyafterSC-CO2extraction.Asensorycrunchiness test indicated that theharder thepistachio, thecrunchieritis[137].Theshear-compressionforceofpeanutsincreasedwithextrac-tion time,which indicates that thedefattedpeanuthasacrispy texture[141].Thehardnessofthewalnutdecreasedwiththefatcontentofthenut(fullfat,25%and40%fat-reduced)[139].Also,flavorintensitywasreducedafterSC-CO2extraction,whichmightbeattributedtotheremovaloftheflavorcompounds.Thearomaandflavorintensity,fracturability,andmoistnessofpeanutsalldecreasedwithincreas-ingextractiontime[141].

Thepeanutmealvolumedecreasedafterextractionandthedecreaseinvolumeincreasedwithtemperatureandpressure.Thisbulkvolumereductionwasattributedtothecompactionofthemealbyhighpressureandtheeliminationofoilfromthemeal.At25°C,themealcrumbledwhentouched.At100°Cand55MPa,themealwasacoherentmass,whichwasdifficulttofracturebyhand[130].Thebulkdensityoftheextractedalmondswasconsiderablylessthanthatoftherawmaterials,about41%lessforthedicedalmondsand54%to59%lessfortheflakedalmonds[123].

7089_C003.indd 71 10/15/07 5:29:33 PM


Fewstudiesevaluated theoxidative stabilityofnuts afterSC-CO2extraction.Thefat-reducedwalnuts(25%and40%)hadalowerperoxidevalue(PV)thanthatofthefull-fatwalnutswhenstoredat25°Cand40°Cfor8weeks.After5weeksat40°C,the25%fat-reducedwalnutshadahigherPVthanthe40%fat-reducednuts,buttherewasnosignificantdifferencethroughoutthe25°Cstoragecondition[139].

3.3.1.3 Comparison with Conventional Methods

Nutoils are traditionally recoveredmechanical pressingof organic solvents. Themechanicalpressingprocesscausedconsiderablesplitting(12%to43%)andbreak-age(3.6%to46%)ofpeanuts[133].Furthermore,about5%ofwater-solublesugarsandproteinswerelostinthesoakingstepfollowingthepressing,whichcanexpandthepeanutbacktoitsoriginalsizeandshape.Similarly,aconsiderableamountoffat-solublevitaminsandothervaluableconstituents,suchasphospholipids,areremovedduringhexaneextraction[134].Ithasbeendemonstratedthatpolarphospholipidsplay an important role in lipid stability. Organic solvent extraction also leavesundesirablesolventresidueinthefinalproducts[134,139].Ontheotherhand,thewalnutoilextractedbySC-CO2hadahigheramountoftocopherols(405.7μg/goil)comparedwiththeoilextractedwithhexane(303.2μg/goil)[138].TheoilextractedbySC-CO2wasalsoclearerthanthatobtainedbyhexane,indicatingtheneedforlessrefining[121,128,138].However,theSC-CO2-extractedoilshowedgreatersus-ceptibilitytooxidation.

SC-CO2extraction isalsoused toobtainsheanutoil.The traditionalprocessinvolvespouringhotmoltensheaoilinto10%Fuller’searthcontaininghotacetone,coolingandprecipitatingthepolyisoprenoidgumontotheearth,andthenfilteringtoremovetheearthandgum[142].However,withSC-CO2extraction,acleanandhigh-qualityoilcanbeobtainedwiththehigh-molecular-weightpolyisoprenoidsleftintheextractionvesselasarubber-likemass[142].Inaddition,theextractedoilhaslowlevelsoffreefattyacids,monoglycerides,diglycerides,iron,triterpeneacetate,andtriterpenecinnamate,whichisamajoradvantageforitsuseintheconfectionaryindustryasacocoabuttersubstitute.

3.3.2 seed oils

Numerous studies on the extraction of specialty oils from seeds, such as apricot[143–145],borage[146–148],cherry[149],echium[148],eveningprimrose[150–152],flax[153],grape[154–158],hiprose[159],Hybrid hibiscus[160],milkthistle[161],munch [162],pumpkin [163], rosehip [164–168], seabuckthorn [169–171], sesame[172],andtomato[173],usingSC-CO2havebeenreportedandaresummarizedinTable3.4.InadditiontoSC-CO2,somestudiesalsousedpropaneasthehigh-pressuresolvent.Forexample,Silybum marianumseedoilextractionrateusingpropanewasfoundtobehigherthanthatobtainedwithCO2,whileusing10timeslesspropanethanCO2[161].However,thetocopherolcontentoftheoilobtainedbyCO2(0.085%)washigherthanthatobtainedbypropane(0.02%)[161].Thus,propanemightnotbeanappropriatesolvent for tocopherolextraction.Toattaincompleteoilextractionfromrosehipseeds,asolvent-to-feedratioof3wasusedforpropane/CO2mixtureat

7089_C003.indd 72 10/15/07 5:29:34 PM


TaB

lE 3

.4Ex

trac

tion

of B

ioac

tive

Com

poun

ds fr

om S

eeds

usi

ng S

C-C

O2

raw

M

ater

ial

Feed

(g)

Sam

ple

prep

arat

ion

Bio

acti

ve

Com

poun

d

Extr

acti

on C

ondi

tion

s

rec

over

ya

(%)

ref

.pa

rtic

le S

ize

(mm

)H

2O (

%)

T (°

C)

p (M

pa)

Flow

rat

eTi

me

(min

)C

osol

vent

Apr

icot

5<

0.8

53.

9O

leic

aci

d,

linol

eic

acid

40,5

0,6

015

,30,

45,

52

.5,6

0<

0.5

g/m

inn.

i.N

one

n.i.

143

5n.

i.3.

9O

leic

aci

d,

linol

eic

acid

40,5

0,6

030

,37.

5,4

52,

3,4

g/m

in15

0,1

.5,3

%

EtO

H+

8514

4

5<

0.4

25,

<0

.85,

0.9

2,

1.5

3.9

Lin

olei

cac

id40

,50,

60,

70

30,3

7.5,

45,

52

.5,6

01,

2,3

,4,5

g/m

inn.

i.0,

0.5

,1,

1.5,

3%

E

tOH

+

n.i.

145

Bor

age

10n.

i.n.

i.L

inol

enic

aci

d,

stea

rido

nic

acid

4010

–35

0.5

L/m

inn.

i.0,

0.5

–2%

ca

pryl

ic

acid

met

hyl

este

r+

51.5

c14

6

400.

5,0

.75,

1,

1.5

0,1

.8,7

.4L

inol

enic

aci

d10

,40,

60

5–35

0.5–

2L

/min

180

Non

e29

c14

7

150

0.65

n.i.

Lin

olen

ica

cid

10,2

5,4

0,5

56,

10,

20,

30

0.03

–0.2

g/m

inn.

i.N

one

n.i.

148

Che

rry

n.i.

n.i.

10.8

8L

inol

eic

acid

,st

erol

s40

,60

18,2

0,2

21.

2,3

.6,

4.8×

10–2

m/m

inb

n.i.

Non

en.

i.14

9

Ech

ium

150

0.65

n.i.

Stea

rido

nic

acid

,lin

olen

ica

cid;

10

, 25,

40,

55

6,1

0,2

0,3

00.

03–0

.2g

/min

n.i.

Non

en.

i.14

8

Eve

ning

pr

imro

se0.

8<

0.5

n.i.

γ-L

inol

enic

aci

d35

–60

8–71

1×

10–3

L/m

in10

0N

one

9515

0

50<

0.35

5n.

i.γ-

Lin

olen

ica

cid

40,5

0,6

020

,30,

50,

70

18g

/min

n.i.

Non

e>

95

151

cont

inue

d

7089_C003.indd 73 10/15/07 5:29:35 PM


TaB

lE 3

.4 (c

onti

nued

)Ex

trac

tion

of B

ioac

tive

Com

poun

ds fr

om S

eeds

usi

ng S

C-C

O2

raw

M

ater

ial

Feed

(g)

Sam

ple

prep

arat

ion

Bio

acti

ve

Com

poun

d

Extr

acti

on C

ondi

tion

s

rec

over

ya

(%)

ref

.pa

rtic

le S

ize

(mm

)H

2O (

%)

T (°

C)

p (M

pa)

Flow

rat

eTi

me

(min

)C

osol

vent

50<

0.35

5n.

i.γ-

Lin

olen

ica

cid

40,5

0,6

020

,30,

50,

70

9,1

8,2

7g/

min

Non

en.

i.15

2

Flax

5n.

i.n.

i.L

inol

enic

aci

d,

toco

pher

ols

50,7

021

,35,

55

1,3

L/m

in18

0N

one

7415

3

Gra

pe17

6<

1.25

n.i.

Lin

olei

cac

id,

toco

pher

ols,

ph

ytos

tero

ls,

squa

lene

6537

60g

/min

360

Non

e13

.6c

154

20n.

i.6.

8n.

i.40

280.

5–1

L/m

inn.

i.N

one

n.i.

155

400.

35,0

.75,

1.

5,2

.83

0.3,

1.1

,2.

4,6

.3U

nsat

urat

edo

il10

,40,

60

5,1

0,2

0,3

00.

5,1

,1.5

,2L

/min

300

Non

e92

156

3n.

i.n.

i.Ph

enol

ics

4020

,30

n.i.

n.i.

Non

en.

i.15

7

4,5

000.

25–0

.42,

0.

42–0

.841

,0.

841–

2

n.i.

Uns

atur

ated

oil

(lin

olei

cac

id)

35,4

0,4

520

,25,

30,

40

0.4

mL

/min

;4

L/m

in12

0,2

10,

300

Non

e10

015

8

Hip

rose

130.

42,0

.79,

1.

03n.

i.L

inol

eic

acid

40,5

0,7

010

.3,2

0.6,

41

.3,6

8.9

1,2

,4,6

g/m

inn.

i.N

one

7.4c

159

Hyb

rid

hibi

scus

50.

1n.

i.Ph

ytos

tero

ls80

53.7

2×

10–3

mL

/min

50N

one

20c

160

Milk

this

tle30

n.i.

n.i.

Toco

pher

ols

25,4

0,6

0,8

010

,20,

30

n.i.

n.i.

Non

e20

.5c

161

Mun

ch15

00.

05–0

.25

n.i.

β-D

imor

phec

olic

ac

id(

DA

)

45

30n.

i.n.

i.N

one

>9

516

2

7089_C003.indd 74 10/15/07 5:29:36 PM


Pum

pkin

200.

36n.

i.PU

FAs,

ste

rols

,to

coph

erol

s35

–45

18–2

00.

03–0

.1m

/min

b

120

Non

en.

i.16

3

Ros

ehip

10n.

i.n.

i.C

arot

enoi

ds,

unsa

tura

ted

oil

28,3

510

,25

1–1.

5L

/min

n.i.

Prop

ane+

n.i.

164

260.

85–2

.36,

0.

425–

0.85

,0.

15–0

.425

n.i.

Uns

atur

ated

oil

40

3011

.4g

/min

n.i.

Non

e39

.2c

165

100

n.i.

n.i.

Uns

atur

ated

oil,

fla

vono

ids,

ca

rote

noid

s

40,5

0,6

030

,40,

50

21g

/min

n.i.

Non

e7.

1c16

6

210

n.i.

n.i.

Lin

olei

cac

id,

α-lin

olen

ica

cid

40,6

0,8

030

,50,

70

n.i.

n.i.

Non

en.

i.16

7

26n.

i.n.

i.U

nsat

urat

edo

il40

,50

30,4

04,

8,1

2,1

8,

24g

/min

60–9

0N

one

n.i.

168

Sea buck

thor

n3–

40n.

i.n.

i.U

nsat

urat

edo

il25

,40,

60

9.6,

17.

4,2

71

L/m

inn.

i.N

one

n.i.

169

120

<0.

491,

0.

491–

0.64

3,

0.64

3–1.

033,

>

1.03

3

n.i.

Lin

olei

cac

id30

,35,

40,

45

15,2

0,2

5,3

00.

83–3

.33

L/m

in27

0N

one

n.i.

170

n.i.

0.5–

113

.1L

inol

eic

acid

30,3

5,4

0,5

015

,20,

25,

30

1.7,

3.3

, 5,6

.7L

/min

300

Non

en.

i.17

1

Sesa

me

10n.

i.n.

i.O

leic

aci

d,

linol

eic

acid

50,6

0,7

020

.7,2

7.6,

34

.5n.

i.n.

i.0,

5,

10%

EtO

H+

89.4

172

Tom

ato

4.5

0.27

n.i.

Uns

atur

ated

oil

40,5

5,7

010

.8,1

7.6,

24

.52.

8g/

min

480

Non

en.

i.17

3

aR

ecov

ery

(ge

xtra

ct/g

oil

infe

edm

ater

ial×

100

),b s

uper

ficia

lvel

ocity

, c yie

ld(g

/100

gfe

edm

ater

ial)

,+ cos

olve

nta

dded

con

tinuo

usly

into

SC

-CO

2ath

e le

vel(

%,w

/w)i

ndic

ated

.

7089_C003.indd 75 10/15/07 5:29:37 PM


28°Cand10.0MPa,whilearatioofonly1wassufficientforpropanealoneat25°Cand5.0MPa[174].


3.3.2.1.1 Sample PreparationUnlikeextractingoilfromnuts,whichhavesphericalandelongatedhexagonalcellstructurescontainingoil,itisnearlyimpossibletoextractoilfromuncrackedseedsduetotheiruniquephysicalstructures.Theoil innutsiseasilyaccessiblebydif-fusioninto thecellulosicstructure;however, theseedcoat isalmost impermeable[155].Theoilintherosehiporhiproseseeds,forexample,iscontainedintheoil-bearingstructures,whichareenclosedinathickandhighlylignifiedtesta[159,165].Thehiprosemaybecontainedin longmicroscopicchannels,whichareprotectedby a lignin structure that is probably too compact to alloweffective diffusion ofthesupercriticalfluid ina reasonable time[159].Therefore,grinding theseeds isanimportantstepofsamplepreparation,whichcanbreaktheintactchannelsandexposetheoilinthechannelstotheextractionsolvent.

a) Particlesize:Asexpected,theoilyieldincreasedwithdecreasedparticlesize.Forexample,whenhiproseseedparticlesrangingfrom1.03to0.79and0.42mmwereused, theoil yield increased from4.9% to5.2%and7.4%,respectively[159].Similarparticlesizeeffectswereobservedwhengrape[156]andborage[147]oilswereextractedfromtheirseeds.Particleswithdiameters less than0.35mmweresuggested forgrapeseedextrac-tion[156].Withadecreaseintheparticlesizeofseabuckthornseedsfrombetween0.50–2.36to0.43–1.00mm,thedurationoftheextractionprocesswasreducedfrom6to3hours[175].

b) Moistureandequilibrationtime:Themoisturecontentofthegrapeseed(6.3%,2.4%,1.1%,0.3%)wasmodifiedbydryingthegroundsamplefordif-ferentlengthsoftime(0,2,4,6.3hours)[156].Theextractionyieldwasnotsignificantlyaffectedbythemoisturecontentofthegrapeseeds.However,the sample exposed to the longer drying time (6.3 hours) had a slightlyloweroilyield,whichmightbeduetotheevaporationofvolatileconstitu-ents[156].Similarly, theextractionyieldof theborageseedoilwasalsonotsignificantlyaffectedbythedryingprocess(partiallyandalmostfullydehydrated);however,themoisturecontentoftheborageseed(0%,1.8%,7.4%)hadanegativeimpactontheextractionyield,withthehighermois-turecontentsamplesresultinginlowerextractionyields[147].

3.3.2.1.2 Extraction Parameters a) Temperature and pressure: In general, extraction yield increased with

pressure at constant temperature. For example, increasing the pressurefrom10to15,20,and25MPaat40°Cresultedinanincreaseinborageoilyieldfrom0.1%to5.6%,15.2%and21.9%,respectively.However,onlyaslightincreaseintheyield(21.6%to24.3%)wasobservedwithapres-sureincreasefrom30to35MPa[146].Asimilartrend(34.4%to91.3%,

7089_C003.indd 76 10/15/07 5:29:37 PM


respectively) was observed during extraction of evening primrose seedsat60°Candpressuresof20and30MPa[151].Atconstant temperature,increasingthepressureto50MPaimprovedtheoilyieldslightlyto97.2%,whileafurtherincreaseinpressureto70MPahadalmostnoeffectontheyield (97.7%) [151]. Not only the extraction yield but also the extractionrateincreasedwithpressure.Inaddition,withanincreaseinpressurefrom20.6to41.2and68.9MPa,theextractiontimeforhiprosedecreasedfromabout30to15and5min,respectively[159],sincetriglyceridesaresolubi-lizedtoagreaterextentathigherpressuresinSC-CO2.Morethan94%oftheavailableoilfromeveningprimroseseedswasextractedinonly14min[151].AsdiscussedinSection3.3.1.1.2,temperaturehasanegativeeffectonsolubilityandextractionyieldatlowpressuresbutapositiveeffectathigherpressuresdue to thecrossoverof the solubility isotherms.Moreover, thenegativeeffectoftemperatureatlowpressuresseemstobegreaterthanthepositiveeffectathighpressures.Forexample,withanincreaseintempera-turefrom40°Cto50°Cat20MPa,theeveningprimroseoilyielddroppedfrom66.1%to59.6%,withafurtherdropto34.4%at60°C.However,atahigherpressureof50MPa, theoilyield increasedfrom96.8%to97.5%withatemperatureincreasefrom40°Cto50°Candthenslightlydroppedto 97.2% at 60°C [151]. A similar trend was reported for the extractionof Silybum marianum oil using SC-CO2. In this case, the seed oil yielddecreasedfrom19.9%to5.2%withatemperatureincreasefrom25°Cto80°Cat20MPabut increasedfrom15.3%to20.5%at30MPa[161]. Infact,thepressureincreaseseemstohaveamoresubstantialeffectontheoil yield than the temperature increase. Sesame oil yield increased onlyfrom5.4%to11.8%whentemperaturewasincreasedfrom50°Cto70°Cat27.6MPa;however,theyieldincreasedfrom3.6%to31.3%whenpressurewasincreasedfrom20.7to34.5MPa[172].

b) Flowrateandflowdirection:Theextractionyieldofborageoilincreasedwithflowrate[147].Similarly,66%and74%offlaxoilwasobtainedataflowrateof1and3L/min(measuredatSTP),respectively[153].Similartothenutoils,flowdirectionwasreportedtoaffecttheextraction,withdown-wardflowbeingmorefavorable.Sovovaetal.[155]reportedthatgrapeseedoilextractionwasretardedwhenthesupercriticalsolventflowwasupwardthroughthebed(duetonaturalconvection).Itisthereforeadvantageoustooperatelaboratoryextractionunitsinthedownwardflowmode.

c) Extractiontime:Thedurationofthefastextractionperioddependsontheplanttypeandvariety.Forexample,twovarietiesofgrapeseedsresultedinlowoilyields(5.9%and6.1%)throughoutthefastextractionperiodof60 min with a very little amount of additional oil extracted at extendedextractiontimes[154].Theotherfourgrapevarietiescontainingintermedi-ate levelsofoil (9.4%to10.7%)werenearlycompletelyextractedwithin60min,whereasthehighoilcontentvariety(13.6%)required120min.

d) Useofcosolvent:Additionof10%ethanolasacosolventgreatlyenhancedthe extraction yield of sesame oil. At 27.6 MPa and 50°C, the recoveryincreasedfrom5.4%to74.1%uponadditionofethanolintoSC-CO2,whereas

7089_C003.indd 77 10/15/07 5:29:38 PM


at70°C, theyield increased from11.8% to89.4%, respectively [172].At40°C,whencaprylicacidmethylesterwasusedasacosolvent(0%,0.5%,1%,and2%),theborageoilyieldincreasedfrom0.1%to2.9%,3.1%and6.7%at10MPa;from15.2%to20.7%,30.0%and36.6%at20MPa;andfrom21.6%to30.7%,38.2%and51.5%at30MPa[146].Atalowpressureof10MPa,theamountofcoextractedsolventwashigh,rangingfrom51.1%to73.0%and79.7%ofthetotalfattyacidmethylesterat0.5,1%,and2%additionofcaprylicacidmethylester[146].Whenpressurewasincreasedto20MPa,theamountofcoextractedsolventwasreducedto11.9%,20.8%,and39.8%ofthetotalfattyacidmethylesterat0.5%,1%,and2%,respec-tively,due to thehigher recoveryof triglycerides. Itwasshown that thiscosolventcanbeeasilyremovedfromthefinalextractatlowpressures.


3.3.2.2.1 Chemical CompositionThefattyacidcompositionoftheseedoilsissummarizedinTable3.5;wheretheunsaturatedfattyacids(mainlyC18:1,C18:2,andC18:3)accountedformorethan90%ofthetotalfattyacids.FattyacidcompositionoftheoilobtainedbySoxhletextractionwassimilartothatoftheSC-CO2extractforgrapeseed[154].Inaddi-tion, fatty acidprofilesof thegrape seedoilsobtainedatdifferent temperatures(35°C and 40°C) and pressures (30 and 40 MPa) were also similar [158]. How-ever,Szentmihalyietal.[164]foundthatthelowertemperatureSC-CO2extractionresultedinhigherlevelsofoleicandlinoleicacidsintherosehipoilcomparedtothoseintheSoxhletextract.Dauksasetal.[146]foundthatthelinolenicacidcon-tentoftheborageseedoilincreasedfrom16.2%to20.1%withapressureincreasefrom10to20MPa;however,itslightlydecreasedto18.5%withafurtherincreaseto 35MPa.The fatty acid compositionof theborageoil obtainedusingvariousconcentrationsofcaprylicacidmethylesterasacosolventatdifferentpressureswasdifferent;however,nocleartrendswereestablished[146].Thefattyacidcomposi-tionsaswellastheratiosbetweenthesaturatedandunsaturatedfattyacids(11:89)of the Hippophae rhamnoides L. seed oils extracted using SC-CO2 and hexaneweresimilar[177].Althoughtherewasnodifferencebetweenthefattyacidcompo-sitionsofSC-CO2-andhexane-extractedgrapeseedoils,SC-CO2-extractedfrac-tionsobtainedat30,60,120and180minweredifferent[156].Theα-linolenicacidcontentoftheSC-CO2-extractedflaxoilwashigherthanthatofsolvent-extractedoil; however, the saturated and monounsaturated fatty acids were higher in thesolventextract[153].

3.3.2.2.2 Other Quality AttributesThe SC-CO2-extracted evening primrose oil had a yellow color and its intensityincreasedwithpressure,approachingthedeepyellowcolorofthehexane-extractedoil[151].Theyellowintensityoftheborageoilextractsalsoincreasedwithpressure[146].Similarly,theSC-CO2extractedseabuckthornseedoilwasaclear,yellow-brownliquidatroomtemperature,whereasthepulpflakeoilwasredandsemisolid[175].OdabasiandBalaban[172]foundthatthesesameoilfromSC-CO2extractionappearedclear.However,when5%ethanolwasusedasacosolvent,theoilextract

7089_C003.indd 78 10/15/07 5:29:39 PM


TaB

lE 3

.5Se

ed F

at C

onte

nt a

nd F

atty

aci

d C

ompo

siti

on o

f See

d O

ils

raw

Mat

eria

lFa

t C

onte

nt

(%, w

/w)

Fatt

y a

cidy

Con

tent

a–d

C16

:0C

16:1

C18

:0C

18:1

C18

:2C

18:3

ref

.

Apr

icot

48.1

5.22

–5.7

1c0.

6–0.

781–

1.3

67.3

7–68

.07

24.8

4–25

.11

––14

4

Bor

age

29.0

13.3

2c0.

194.

5819

.78

39.5

722

.56

147

Che

rry

8.5

5.26

a0.

272.

1532

.64

40.8

41.

114

9

Ech

ium

e30

.06–

8b––

3–5

15–1

914

–18

37–4

514

8

Eve

ning

pri

mro

se27

.54.

75–6

.7c

––1.

46–1

.87

4.85

–5.4

75.2

7–77

.07

(γ-)

9.2

2–10

.36;

(α

-)0

.13–

0.16

150

Flax

38.0

5.7–

5.8d

––4.

0–4.

214

.1–1

4.4

12.8

–13.

460

.5–6

115

3

Gra

pe10

–15

6.28

–8.2

6b0.

06–0

.15

3.6–

5.22

12.7

1–18

.47

67.5

6–73

.223

0.44

–1.1

215

4

Hyb

rid

hibi

scus

8.5–

2014

.8–2

7.0b

0–0.

70–

4.1

17.8

–47.

342

.6–6

40–

0.7

160

Lun

aria

e37

.01–

3b––

1–2

16–2

08–

102–

414

8

Mun

ch22

.81.

9–2.

4b––

1.5–

1.9

17.5

–22.

112

.3–1

4.9

0.7–

116

2

Nee

me

45.0

13–1

5c––

14–1

950

–60

8–16

––17

6

Pum

pkin

43.5

14.9

c0.

253.

1913

.559

.91.

6416

3

Ros

ehip

8.0

3.6–

7.87

d––

2.45

–3.2

716

.25–

22.1

135

.94–

54.7

520

.29–

26.4

816

4

18.5

8.85

b––

2.42

21.7

540

.09

––17

7

Sesa

me

––9.

23–1

0.53

c––

4.17

–5.3

37.6

–38.

4844

.17–

45.8

5––

172

Tom

ato

2910

.9–1

5.79

b1.

15–3

.81

4.97

–11.

9819

.9–2

4.13

39.6

9–53

.24

4.58

–5.5

173

Palm

itic

acid

(C

16:0

),P

alm

itole

ica

cid

(C16

:1),

Ste

aric

aci

d (C

18:0

),O

leic

aci

d (C

18:1

),L

inol

eic

acid

(C

18:2

),L

inol

enic

aci

d (C

18:3

).a m

ol%

,b wt %

,c not

indi

cate

d,d G

C%

Are

a,a

nde F

atty

aci

dco

mpo

sitio

nre

port

edf

orth

e ne

wm

ater

ial.

7089_C003.indd 79 10/15/07 5:29:40 PM


wasclearatlowtemperaturebutcloudyathightemperature.With10%ethanoladdi-tion,allsampleswerecloudyregardlessofthetemperature.Thiscouldbeattributedtothecoextractionofphospholipids,waxes,andpigmentsthatoccurswithethanoladdition.Therefore, theselectionandconcentrationofcosolventadditionmustbeoptimizedbasedondesirableproductqualityattributes.


HexaneextractiontendstogiveahigheroilyieldthanSC-CO2extractionduetotheextractionofundesirablecompounds thatmustbe removedduring refining [147].Gomezandde laOssa [156] reported that thegrape seedoilyield fromSC-CO2extractionwas6.9%,while thatwithhexanewas7.5%.This isdue to thehexaneextraction being nonselective for triglycerides since hexane can also extract freefattyacids,phospholipids,pigments,andunsaponifiables.BozanandTemelli[153]alsofoundthattheoilyieldobtainedfromflaxseedwithSC-CO2aftera3hextrac-tionwas21–25%,whereasthepetroleumetherextractiongavea38%yield.Inaddi-tion,theSC-CO2-extractedoilsfrompumpkinandHippophae rhamnoides L.seedswere clearer than those extracted by hexane [163]. Bernardo-Gil et al. [149] alsoreported that cherry seedoil extractedwithSC-CO2wasclearer than thehexaneextract,minimizingtheneedforfurtherrefining.Nevertheless,thepumpkinseedoilextractedwithhexanewasbetterprotectedagainstoxidation(inductiontime=8.3h)comparedwiththeSC-CO2–extractedoil(inductiontime=4.2h)[163].

3.3.3 Cereal oils

Todate,thecerealoilsthathavebeenstudiedareamaranth[178–180],oat[181],ricebran[182–187],wheatgerm[188–191],andwheatplumule[192]oil,whicharesum-marizedinTable3.6.


3.3.3.1.1 Sample preparationCereal grains, which in general have a moisture content of 3% to 12%, may notrequireadryingsteppriortoextraction.

a) Particlesize:Extractionyieldsobtainedusingoriginal(0.75mm)andthemilled (0.3 mm) wheat germ were similar [188]. However, according toPanfilietal. [190],wheatgermoil recovery increasedfrom57%to92%whentheparticlesizewasreducedfrom0.5to0.35mm.Asimilartrendwas reported for the SC-CO2 extraction of oil from Amaranthus grain,whereverylittleoilwasextractedfromwholegrains[179].

b) Moisture:InthecaseofAmaranthusgrain,moisturecontentsof0%,5%,and10%hadnosignificanteffectontheoilandsqualeneextractionyields[179]. As the maximum moisture content of the harvested Amaranthusgrain is 10%, drying is not a necessary step. However, in wheat germextraction, the tocopherol yield increased with a decrease in moisturecontentfrom11.5%to8.2%and5.1%[189],butdecreasedwithafurther

7089_C003.indd 80 10/15/07 5:29:40 PM


cont

inue

d

TaB

lE 3

.6Ex

trac

tion

of B

ioac

tive

Com

poun

ds fr

om C

erea

ls u

sing

SC

-CO

2

raw

Mat

eria

lFe

ed (

g)

Sam

ple

prep

arat

ion

Bio

acti

ve

Com

poun

d

Extr

acti

on C

ondi

tion

s

rec

over

ya

(%)

ref

.pa

rtic

le S

ize

(mm

)H

2O (

%)

T (°

C)

p (M

pa)

Flow

rat

eTi

me

(min

)C

osol

vent

Am

aran

th

(Am

aran

thus

cr

uent

us)

40n.

i.n.

i.L

inol

eic

acid

,ol

eic

acid

40,4

5,5

010

,20,

25,

30

3.3,

6.8

,8.5

g/m

inn.

i.N

one

n.i.

178

60n.

i.n.

i.Sq

uale

ne40

,50,

60,

70

15,2

0,2

5,3

01,

2,3

,5L

/min

120

Non

e4.

8c17

9

(Am

aran

thus

ca

udat

us)

5<

0.2

n.i.

Toco

pher

ols,

sq

uale

ne40

20.2

,40.

52

L/m

in15

Non

en.

i.18

0

Oat

1500

,185

n.i.

n.i.

Ole

ica

cid,

lin

olei

cac

id40

25,3

5n.

i.30

020

%

EtO

H+

+

n.i.

181

Ric

ebr

an20

n.i.

3.1

Toco

pher

ols

4014

.7–3

4.3

n.i.

n.i.

Non

e22

c18

2

300

n.i.

10.1

Uns

atur

ated

oil,

to

coph

erol

s,

ster

ols,

ory

zano

l

0,2

0,4

0,6

017

,24,

31

41.7

g/m

in36

0N

one

96.8

183

300

n.i.

8.48

Uns

atur

ated

oil,

to

coph

erol

s,

ster

ols,

ory

zano

l

40,4

5,5

08.

6,9

.9,1

1.2

58.3

g/m

in24

0N

one

4.1c

184

300.

5n.

i.PU

FAs,

to

coph

erol

s,

squa

lene

40,5

0,7

020

.7,2

7.6,

34

.5,4

1.3

n.i.

480

Non

e80

185

20n.

i.n.

i.α-

Toco

pher

ol,

γ-or

yzan

ol

40,6

0,8

034

.5,5

1.7,

68

.91.

1g/

min

n.i.

Non

e24

.7c

186

7089_C003.indd 81 10/15/07 5:29:41 PM


TaB

lE 3

.6 (c

onti

nued

)Ex

trac

tion

of B

ioac

tive

Com

poun

ds fr

om C

erea

ls u

sing

SC

-CO

2

raw

Mat

eria

lFe

ed (

g)

Sam

ple

prep

arat

ion

Bio

acti

ve

Com

poun

d

Extr

acti

on C

ondi

tion

s

rec

over

ya

(%)

ref

.pa

rtic

le S

ize

(mm

)H

2O (

%)

T (°

C)

p (M

pa)

Flow

rat

eTi

me

(min

)C

osol

vent

Ric

ebr

an80

00.

25,0

.5,0

.75

n.i.

Lin

olei

can

dpa

lmiti

cac

ids,

si

tost

erol

and

ca

mpe

ster

ol

50,6

010

,20,

30,

40

300

g/m

in18

0N

one

20c

187

Whe

atg

erm

250.

3,0

.75

n.i.

Toco

pher

ols,

lin

olei

cac

id10

–60

5–30

0.5–

2L

/min

180

Non

e>

95c

188

5n.

i.n.

i.To

coph

erol

s35

,40,

45,

50

13.7

,20.

7,

27.6

,34.

5,

41.3

1–3×

10–3

L/m

in90

Non

e2.

2c18

9

600–

700

0.35

–0.5

n.i.

Lin

olen

ic,l

inol

eic

acid

s,β

-car

oten

e,

lute

in, a

nd

zeax

anth

in,

toco

pher

ols,

to

cotr

ieno

ls

5525

,38

0.5

L/m

in18

0N

one

9219

0

20Fl

akes

:2.1

n.

i.To

coph

erol

s10

,25,

40,

50

20.2

300–

400

L/m

inn.

i.N

one

9519

1

Whe

at

plum

ule

n.i.

n.i.

n.i.

PUFA

s35

, 45,

55,

65

10,1

5,2

0,3

01.

2,1

.8,2

.4,

3L

/min

60–4

80N

one

n.i.

192

T:t

empe

ratu

re,P

:pre

ssur

e,n

.i.:n

otin

dica

ted.

aR

ecov

ery

(ge

xtra

ct/g

oil

infe

edm

ater

ial×

100

), b s

uper

ficia

lvel

ocity

, c yie

ld(g

/100

gfe

edm

ater

ial)

,++C

osol

vent

add

edto

sam

ple

befo

ree

xtra

ctio

nat

the

leve

l (%

,w/w

)ind

icat

ed.

7089_C003.indd 82 10/15/07 5:29:42 PM


reductioninmoistureto4.3%.Thismightbeattributedtotheshrinkingofthegermparticles.

3.3.3.1.2 Extraction Parameters a) Temperature and pressure: During wheat germ extraction, tocopherol

yieldincreasedslightlywithapressureincreasefrom13.8to27.6MPaat40°C, while a further increase in pressure to 34.5 and 41.4 MPa did notimprove theyieldsignificantly [189].On theotherhand, tocopherolyielddecreased with temperature at pressures below 26.2 MPa and increasedwithtemperatureatpressuresabove26.2MPa.Similarly,amaranthoilyieldincreasedwithpressureintherangeof15to25MPa.Also,temperaturehadanadverseeffecton theamaranthoilyieldat thepressure rangeof15to25MPabuthadapositiveeffectat30MPa[179].Similarly,anotherstudyonamaranthseedoilshowedthatextractionyieldandratedecreasedwithtemperatureat10MPaandincreasedwithtemperatureat20and30MPa[178].Althoughtheamaranthoilyieldvariedsignificantlywithpressure,theyieldsofsqualeneintheamaranthoilat40°Canddifferentpressureswereveryclose,rangingfrom0.24to0.27g/100ggrain[179].Ontheotherhand,theamountofsqualeneextractedfromricebrandecreasedwithCO2den-sityandthemaximumamountwas2.9%at70°Cand20.7MPa[185].TheSC-CO2densityhasadifferenteffectonfattyacids.Asthereduceddensitywasincreasedatconstanttemperature,theamountoflinoleicacidextractedincreasedbutthatofoleicaciddecreased[185].

b) Flowrate:Theamaranthoilyieldandinitialextractionratebothincreasedwithflowratefrom1to2L/min,buttherewerenodifferencesatflowratesof2,3and5L/min(measuredatSTP)underthesameextractionconditions[179].Atflowratesabove2L/min,theextractionratewasreducedmarkedlyafter1hofextractionandonlyasmallamountofoilwasextractedinthefollowing2h.Similarly,inthewheatgermoilextractions,theoilyieldandextractionratebothincreasedwithflowratefrom0.5to1and1.5L/min(measuredatSTP),whileafurtherincreaseofflowrateto2L/mindidnotchangeeithertheoilyieldortheextractionrate[188].

c) Extraction time: Not many studies involving cereal oils evaluated theextractionyieldasfunctionofextractiontime.Asexpected,theextractionyieldincreasedwithextractiontimeforwheatplumuleoil[192].

d) Useofcosolvent:Theadditionofethanolasacosolventslightlydecreasedthe free fatty acid content from 5.6% to 4% in the case of oat oil, butincreasedthephosphoruscontentfromlessthan1ppmto80ppmprobablyduetotherecoveryofpolarphospholipids[181].


3.3.3.2.1 Chemical CompositionFattyacidcompositionoftheSC-CO2extractedcerealoilsispresentedinTable3.7.Themainfattyacids in thericebranandwheatgermoilsareoleic, linoleic,andpalmiticacids[185,187,188].ThefattyacidcompositionsofSC-CO2-andhexane-extractedoilsweresimilarandtherewerenodifferencesamongtheextractsobtained

7089_C003.indd 83 10/15/07 5:29:42 PM


atdifferentextractionconditions[188].Crudericebranoilcontains70%triglycer-ides,7%freefattyacids,3.6%fattyacidesters,andsomeminorcomponents,suchasoryzanol[193],α-tocopherol,andβ-sitosterol.Theα-tocopherolcontentofricebranoilwasreportedtobe284mg/kg[182]and1050.8to1279.3mg/kg[186].Inaddition,Shenetal.[184]foundthattheSC-CO2-extractedricebranoilcontained0.23mg/goilofα-tocopherol and18.5mg/goilofβ-sitosterol.Tocopherol andcarotenoidsaretwoimportantminorcomponentsinwheatgerm.α-Tocopherolwasreportedtobethemostabundanttocopherolisomerat1329mg/100gwheatgerm[189]andat2123mg/kgwheatgermoil[190].Luteinandzeaxanthinwerethemostabundantcarotenoidsinwheatgerm,at47.7and37.3mg/kgoil,respectively[190].Squalene,animportantminorcompoundinamaranthoil,wasreportedtobepresentat5.27%(40°C,25MPa)to15.3%(50°C,20MPa)intheextractsobtained[179].

3.3.3.2.2 Other Quality AttributesTheSC-CO2-extractedricebranandwheatgermoilsbothhadlightcolorcomparedwiththoseextractedbyhexane[182,191].However,theSC-CO2-extractedoilwasveryunstablecomparedwithhexane-extractedoil[182].


Because solvent extraction is less selective than SC-CO2 extraction, it generallyresultsinahightotalextractyield,leadingtoreducedconcentrationsofdesirablebioactives.Forexample,wheatgermoilyieldfromSC-CO2extractionwasslightlylower(7.3%to8.0%)thanthatobtainedwithhexane(8.6%),whereasthetocopherolcontentoftheSC-CO2-extractedoilwashigher[188].TheyieldoftotaltocopherolsfromwheatgermobtainedbySC-CO2washigher thanthoseobtainedbysolventextraction [189]. Extraction of vitamin E by hexane and chloroform/methanoltook about 960 and 140 min, respectively, whereas SC-CO2 extraction requiredonly90min.Theoperating temperature forSC-CO2extractionwas lower (40°C)than thoseofhexaneandchloroform/methanol (70°Cand65°C)extraction [189].By choosing suitable extraction conditions, some compounds can be selectivelyextractedbySC-CO2,butnotbysolventextraction.SqualenecanbeextractedfromricebranbySC-CO2butnotbyusingchloroformandmethanol[185].About80%ofPUFAswasextractedbySC-CO2,whereasonly60%recoverywasobtainedwithsolventextraction[185].Ahighsqualeneyield(0.31g/100gAmaranthusgrain)andconcentration(15.3%inextract)wasobtainedat50°Cand20MPausingSC-CO2,whilethesqualeneconcentrationinthesolventextractwasonly6%[179].

3.3.4 Fruit and Vegetable oils

Buritifruit[194],carrot[195-198],cloudberry[199],hiprosefruit[174],olivehusks[200],tomato[201-207]arethemainfruitsandvegetablesstudied(Table3.8).Lyco-pene is the dominant carotenoid (85% to 90% of total carotenoids) in the tomatoextract[207],whereasβ-carotene(60%to80%)isthemajoroneincarrotextract.Squalene,tocopherol,andsterolsarethemainbioactivecomponentsfoundinolives.

7089_C003.indd 84 10/15/07 5:29:43 PM


TaB

lE 3

.7Fa

tty

aci

d C

ompo

siti

on o

f Cer

eal O

ils

raw

Mat

eria

l

Fatt

y a

cid

Con

tent

a

C14

:0C

16:0

C16

:1C

18:0

C18

:1C

18:2

C18

:3C

20:0

C20

:1r

ef.

Am

aran

th—

12.3

2–17

.94

—2.

71–4

.66

23.8

5–32

.88

43.6

6–47

.48

—0.

38–1

.54

—18

0

Oat

0.1–

0.2

13.6

–15.

7—

1.4–

1.6

38.3

–43.

739

.1–4

2.2

1.0–

1.5

—0.

8–0.

918

1

Ric

ebr

an—

16.5

–17.

9—

1.1–

1.4

38.8

–41.

437

.8–4

0.4

1.5–

1.7

0.3–

0.6

0.4–

0.6

183

Whe

atg

erm

—18

.09–

18.9

50.

22–0

.24

0.49

–0.7

313

.69–

16.5

257

.1–5

8.99

6.46

–8.5

1—

—18

8

Palm

itic

acid

(C

16:0

),P

alm

itole

ica

cid

(C16

:1),

Ste

aric

aci

d (C

18:0

),O

leic

aci

d(C

18:1

),L

inol

eic

acid

(C

18:2

),L

inol

enic

aci

d (C

18:3

),A

rach

idic

ac

id(

C20

:0),

Eic

osen

oic

acid

(C

20:1

).a

Uni

tsn

otin

dica

ted.

7089_C003.indd 85 10/15/07 5:29:44 PM



3.3.4.1.1 Sample PreparationDryingofsamplespriortoextractionisnecessary,ascarrotsandtomatoescontain80%to95%moisture.Grindingisalsoneededtoachievesmallparticlesize.Olivepomaceandhuskareby-productsofoliveoilproduction.Tofurtherrecoverthevalu-ablebioactivecompoundsbySC-CO2extraction,pretreatmentofsuchby-productstheconventionalfruitandvegetableprocessingindustryisnecessary.

a) Particle size:Duringextractionof freeze-driedcarrots, ahigherextrac-tionyieldwasobtainedwithsmallercarrotparticles[196,198].Thetotalcarotenoid yield increased from 1109.9 to 1369.6 and 1503.8 μg/g drycarrotwhentheparticlesizewasdecreasedfrom1–2mmto0.5–1mmand0.25–0.5mm,respectively[198].

b) Moisture: Moisture had different effects on the carotenoids yield. Theα- andβ-carotene yields increased with decreasing level of moisture inthefeedmaterial,whiletheluteinyielddecreased[198].Theluteinyielddecreasedfrom55.3to29.9,19.3and13.0μg/gdrycarrotwithadecreaseinmoisturefrom84.6to48.3,17.5and0.8%,whiletheα-andβ-caroteneyields increased from 184.1 to 323.0, 442.3 and 599.0 μg/g, and from354.2to547.8,668.3and891.7μg/gdrycarrot,respectively[198].Ontheotherhand,onlytraceamountsoflycopenewereextractedwhenthetomatofeedmaterialcontained50%to60%moisture[207].Thiscanbeexplainedbythefactthatwatercanactasacosolventfortheextractionofrelativelypolarcompounds,likelutein,whereasthepresenceofwaterisnotfavorablefortherelativelynonpolarlycopeneandcarotenes.

3.3.4.1.2 Extraction Parameters a) Temperature and pressure: Lycopene extraction yield increased with

pressurefrom33.5MPato45MPaataconstanttemperatureof66°Candincreasedwith temperaturefrom45°Cto66°Cataconstantpressureof45MPa[207],becauseSC-CO2densityincreaseswithpressureatconstanttemperatureandsolubilityincreaseswithtemperatureabovethecrossoverpressure.Temperaturegreatlyaffectstheextractionrateatpressuresabovethecross-overpressure.Usingtomatoskin[202],theextractionrateandyieldweregreatlyincreasedat110°C,resultingin96%lycopenerecoveryin40minand100%recoveryin50min.However,onlyabout20%and30%recoverywereachieved in80minat60°Cand85°C, respectively.Pressurealsoaffectedthecompositionoftheextractsastherecoveryoftrans-lycopene increased and that of cis-lycopene decreased with CO2density [205]. Therefore, the fractionation of trans-lycopene is possiblewhenoptimumCO2densityischosenasthelycopeneisomershavediffer-entsolubilitiesinSC-CO2.

b) Flowrate:Totalcarotenoidsyieldincreasedwithflowrate[198],rangingfrom934.8to1332.3µg/gand1973.6µg/gdrycarrotatCO2flowratesof0.5,1,and2L/min(measuredatSTP),respectively.However,thelycopene

7089_C003.indd 86 10/15/07 5:29:44 PM


TaB

lE 3

.8Ex

trac

tion

of C

ompo

unds

from

Fru

its

and

vege

tabl

es u

sing

SC

-CO

2

raw

M

ater

ial

Feed

(g)

Sam

ple

prep

arat

ion

Bio

acti

ve

Com

poun

d

Extr

acti

on C

ondi

tion

sr

ecov

erya

(%)

ref

.pa

rtic

le S

ize

(mm

)H

2O (

%)

T (°

C)

p (M

pa)

Flow

rat

eTi

me

(min

)C

osol

vent

Bur

itif

ruit

n.i.

n.i.

11C

arot

enoi

ds,

toco

pher

ols

40,5

520

,30

18.6

,25

.8g

/min

n.i.

Non

e7.

8c19

4

Car

rot

20.

5–1

0.8

α-,β

-Car

oten

e,

lute

in

40,5

012

–33

1.2

L/m

in48

0N

one

n.i.

195

n.i.

0.26

,0.4

7,1

.12

n.i.

Car

oten

oids

40,5

0,6

07.

8–29

.4n.

i.n.

i.1,

3,5

%E

tOH

+n.

i.19

6

2000

n.i.

n.i.

caro

tene

s,

phen

olic

s,

phyt

oste

rols

,lin

olen

ica

cid

45–5

035

–38

n.i.

120–

180

Non

en.

i.19

7

20.

25–0

.5,0

.5–1

,1–

20.

8,1

7.5,

48

.7,8

4.6

α-,β

-car

oten

e,

lute

in

40,5

5,7

027

.6,4

1.3,

55

.10.

5,1

,2L

/min

240

0,2

.5,5

%

cano

lao

il+

0.2c

198

Clo

udbe

rry

42n.

i.n.

i.U

nsat

urat

edo

il,

β-ca

rote

ne,

toco

pher

ols

40,6

09,

10,

12,

15

,30

n.i.

n.i

Non

en.

i.19

9

Hip

rose

fru

itn.

i.0.

36n.

i.To

coph

erol

s,

caro

teno

ids

3525

1–1.

5L

/min

n.i.

Non

e10

017

4

Oliv

ehu

sks

n.i.

0.4

n.i.

Uns

atur

ated

oil

(ole

ica

cid)

35–5

710

.4–1

8n.

i.n.

i.N

one

n.i.

200

cont

inue

d

7089_C003.indd 87 10/15/07 5:29:45 PM


TaB

lE 3

.8 (c

onti

nued

)Ex

trac

tion

of B

ioac

tive

Com

poun

ds fr

om F

ruit

s an

d ve

geta

bles

usi

ng S

C-C

O2

raw

M

ater

ial

Feed

(g)

Sam

ple

prep

arat

ion

Bio

acti

ve

Com

poun

d

Extr

acti

on C

ondi

tion

sr

ecov

erya

(%)

ref

.pa

rtic

le S

ize

(mm

)H

2O (

%)

T (°

C)

p (M

pa)

Flow

rat

eTi

me

(min

)C

osol

vent

Tom

ato

0.5

0.05

–0.2

5n.

i.Ph

ytoe

ne,

phyt

oflue

ne,

ξ-ca

rote

ne,

β-ca

rote

ne,

lyco

pene

40,5

0,6

08–

264×

10–3

L/m

in30

Non

en.

i.20

1

0.3

n.i.

n.i.

Lyco

pene

60,8

5,1

1040

.51.

5×

10–3

L

/min

50A

ceto

ne,M

eOH

,E

tOH

,hex

ane,

di

chlo

rom

etha

ne,

wat

er+

+

100

202

n.i.

n.i.

n.i.

Lyco

pene

45–8

035

–38

n.i.

120–

180

Non

e55

203

3n.

i.n.

i.Ly

cope

ne,

toco

pher

ols

32–8

613

.8–4

8.3

2.5

×1

0–3

L/m

in

n.i.

Non

e61

204

0.5

n.i.

n.i.

Lyco

pene

408–

284

×1

0–3

L/m

in

n.i.

Non

en.

i.20

5

20n.

i.n.

i.Ly

cope

ne40

32n.

i.n.

i.N

one

n.i.

206

3000

1n.

i.Ly

cope

ne45

–70

33.5

–45

133.

3–33

3.3

g/m

in12

0–48

01–

20%

ha

zeln

uto

il++

6020

7

T:t

empe

ratu

re,P

:pre

ssur

e,n

.i.:n

otin

dica

ted.

aR

ecov

ery

(g e

xtra

ct/g

oil

in f

eed

mat

eria

l ×1

00),

b sup

erfic

ialv

eloc

ity,c y

ield

(g/

100

gfe

edm

ater

ial)

,+co

solv

enta

dded

to s

ampl

ebe

fore

ext

ract

ion

atth

ele

vel (

%,w

/w)

indi

cate

d,+

+co

solv

enta

dded

tos

ampl

ebe

fore

ext

ract

ion

atth

ele

vel(

%, w

/w)

indi

cate

d.

7089_C003.indd 88 10/15/07 5:29:46 PM


yielddecreasedasflowratewasincreasedfrom2.5to15mL/min(measuredatextractiontemperatureandpressure)[204].Comparedwiththelycopenerecoveryof38.8%(oryieldof4.59μg/grawmaterial)obtainedataflowrateof2.5mL/min,only8%recovery(~1μg/gorlessyield)wasobtainedataflowrategreaterthan10mL/min(measuredatextractiontemperatureandpressure)[204].Withflowratesfrom0.875to1.25L/min(measuredatSTP),theolivehuskoilyieldincreased,whereastheyielddecreasedathigherflowrates[208].ThedecreasemaybeattributedtotheshortresidencetimeofCO2intheextractorandthereforetheCO2leavingtheextractornotbeingsaturatedwithoil.A lowflowrate (1.8g/min)producedasmalleramountofsqualenebutatahigherconcentration,whereasahighflowrate(5.4g/min)producedahigheramountofsqualeneatalowerconcentrationintheextract[209].

c) Useofcosolvent:Acetone,ethanol,methanol,hexane,dichloromethane,and water have been compared as cosolvents in SC-CO2 by mixing thecosolventwiththesamplepriortoextraction[202]anditwasshownthatall cosolvents tested except water increased lycopene recovery. In fact,water showed a negative effect, decreasing lycopene recovery to 2%.Ethanol increased recovery but decreased extraction rate. All the othercosolventsstudiednotonlyincreasedthelycopeneyieldbutalsoimprovedtheextractionratetovaryingdegrees[202].Theuseofvegetableoilsasacosolventfortherecoveryofcarotenoidsfromvegetableswasrecentlydeveloped [198,203, 207]. For example, hazelnut oil was chosen byVasapolloetal. [207]becauseof its lowacidity,whichcanprevent thedegradationoflycopeneduringextraction.Lycopeneyieldincreasedwithhazelnutoiladditionasacosolvent,buttheextractwasmoredilutedathigheramountsofoil[207].For theextractionwithoutcosolventaddi-tion, the lycopene recovery was practically maintained below 10%from2to5hoursextractiontime,whileinthepresenceofhazelnutoil,the lycopene recovery increased to about 20% in 5 hours and 30% in8hours.SunandTemelli[198]addedcanolaoilinacontinuousmannerintoSC-CO2fortherecoveryofcarotenoidsfromcarrot.TheextractionyieldwithSC-CO2withoutcanolaoiladditionforα-carotenewas137to330.4μg/gandβ-carotenewas171.7to386.6μg/gfeedmaterialatdif-ferent temperatures and pressures, while the yields more than doubleto288.0–846.7μg/gand333.8–900.0μg/g feed forα-andβ-carotene,respectively,uponadditionofcanolaoil.Themajoradvantageofusingvegetableoilsascosolvents is theeliminationoforganic solventaddi-tion,whichneedstoberemovedlater,andthefactthattheoilenrichedinbioactivescanbeusedasisinavarietyofproductapplications.


3.3.4.2.1 Chemical CompositionFattyacidcompositionofoilsextractedfromvariousfruitsandvegetablesisshowninTable3.9.Trilinolein(LLL)isthemaintriglyceridepresentincarrotoilfollowed

7089_C003.indd 89 10/15/07 5:29:46 PM


byLLP,LLO,POL,andOOP[197].Linoleicacidisthemainfattyacid,followedbypalmiticacidinbothcarrotandtomatooils[197,204].ThefattyacidcompositionoftomatoextractobtainedbySC-CO2wassimilartothatofchloroformextract.ButtheextractsobtainedbySC-CO2atdifferenttemperatureandpressureconditionshaddifferentfattyacidcompositions,whichwereduetothedifferencesinthesolubili-tiesoflinoleicandpalmiticacidsatdifferentconditions[204].

3.3.4.2.2 Other Quality AttributesTheyellow-orangecolorofcarrotoilwasmainlycontributedbythecarotenes,whicharefat-solublepigments[197].


CarrotoilextractedbySC-CO2hadhighercarotenes(1,850mg/kg)thanthatofcom-mercialcarrotoil(170mg/kg)[197].Italsohadahighsterolcontent(30.2mg/kg),which was 17-fold higher than that in commercial carrot oil (1.7 mg/kg). ThesqualeneconcentrationofoliveoilintheSC-CO2extractwas10timeshigherthanthatobtainedwithsolventextraction.However, thisenrichmentwasaccompaniedbyadropintheoverallextractedsqualenequantities[209].SC-CO2extractionpro-duced superiorolivehuskoil in termsofoil acidity,PV,andphosphoruscontent[208];therefore,asimplerrefiningprocesswouldberequired.

3.4 FuTurE TrENdS

TheliteraturereviewedinthischapterdemonstratesthefeasibilityofusingSC-CO2fortherecoveryofspecialtyoilsfromavarietyofplantmaterials.Asshown,it isessentialtostudyeachplantmaterialindividuallybecausethepretreatmentoffeedmaterialandoptimumextractionconditionsaredependentonthestructureandcom-positionofthespecificplantmaterial.Themajorityofthesestudieshavebeencar-riedoutat laboratoryscale,andpilot-scaleSC-CO2extractionstudiesare lacking.Even thoughsomeapplicationshavealready reachedcommercial scale,additionalpilot-scalestudieswouldprovideimportantdatanecessaryforscale-upandeconomicfeasibilityassessment.ForSC-CO2technologytobeadoptedmorewidely,itseco-nomic viability and advantages over conventional techniques must be proven foreachapplication.Pilot-scalestudiesmayshowthatdespiteinitialhighcapitalcosts,

TaBlE 3.9Fatty acid Composition of vegetable Oils

raw Material

Fatty acid Conent

C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:1 ref.

Carrot 0.4 16.6 1.8 1.8 11.6 60.1 4.9 0.4 197

Tomato 1.0b 1.5 0.6 69.0 5.8 1.3 — 11.4 206

1.2c 3.8 3.5 18.6 4.4 3.4 — 18.9 206

Myristicacid(C14:0),Palmiticacid(C16:0),Palmitoleicacid(C16:1),Stearicacid(C18:0),Oleicacid(C18:1),Linoleicacid(C18:2),Linolenicacid(C18:3),Eicosenoicacid(C20:1).a wt%,bSeparationvessel1,cSeparationvessel2.

7089_C003.indd 90 10/15/07 5:29:47 PM


operatingcostswouldbelowerandtheoverallfeasibilitycanbeprovenatcertainscalesofoperation.Inaddition,supercriticaltechnologyallowsthepossibilityofcou-plinganextractionoperationwithcolumnfractionationundersupercriticalconditionstofurtherconcentratethebioactivecomponentsofinterest.Aswell,theresidualmealfollowingextractionofspecialtyoilscanbeevaluatedforotherhigh-valueendusessincedegradationofmealisminimizedwhenSC-CO2isusedasthesolvent.Ontheotherhand,moreresearchisneededtoinvestigatethequalityattributesofSC-CO2-extractedspecialtyoils,suchasoxidativestability,chemicalcomposition,stabilityofbioactivecomponents throughoutextractionandstorage,andtheflavorprofileandconsumeracceptabilityofsuchoils.TheadvantagesofSC-CO2extractionovercon-ventionalsolventextractionneedtobebettercommunicatedtoconsumers.

3.5 CONCluSIONS

Specialtyoilsaretraditionallyrecoveredbymechanicalpressingorextractionusingorganic solvents.Thedisadvantagesof theseconventional techniquesare thehighlevelofresidualoilinthepressedmeal,undesirablesolventresidueleftintheproduct,anddegradationoffat-solublebioactivecomponents.SC-CO2extractionisaprom-ising technology that overcomes these disadvantages for the recovery of specialtyoilsrichinbioactivecomponentssuchascarotenoids,PUFAs,squalene,sterols,andtocolsfromdifferentplantsources.Extensiveresearchcarriedoutwithalargevarietyofplantmaterials—suchasnuts,seeds,cereals,fruits,andvegetables—hasshownthatSC-CO2 is effective in recovering specialtyoils rich inbioactive compounds.Theextractionefficiencyintermsofyieldandrecoveryaswellasthecompositionofspecialtyoilsareaffectedbydifferentfactors,suchassamplepreparation(particlesizeandmoisturecontent)andextractionparameters(temperature,pressure,solventflow rate, extraction time, anduseof a cosolvent).Theseparametersalsohaveanimpactonvariousqualityattributes,suchascolor,flavor,andoxidativestabilityoftheextractedoilandtextureoftheresidualmeal.Ingeneral,thecoloroftheresidualmealbecamelighterasmoreoilwasremovedbecausemostofthepigmentsarefatsolu-ble.SC-CO2extractionproducedsuperioroilwithrespecttooilacidityandperoxidevalue. However, more research on quality attributes like oxidative stability wouldbebeneficialtobetterelucidatetheeffectoftheuseofSC-CO2ontheextractionofspecialtyoils.Ethanolhasbeenusedasacosolventinnumerousstudiestoenhancetheefficiencyofextraction;however,thefactthatadditionalheattreatmentisneededtoremoveethanolfromthefinalproductshouldnotbeoverlookedbecauseheattreat-mentcanbedetrimentaltothesensitivebioactivecomponentsofinterest.

rEFErENCES

1. Physicalconstantsoforganiccompounds,inCRC Handbook of Chemistry and Physics, Internet Version 2007, (87th Edition),Lide,D.R.,Ed.,TaylorandFrancis,BocaRaton,FL,2006.

2. Kamal-Eldin, A. and Appelqvist, L., The chemistry and antioxidant properties oftocopherolsandtocotrienols,Lipids, 31,671,1996.

3. Giovannucci,E.,Areviewofepidemiologicstudiesoftomatoes,lycopene,andprostatecancer,Exp. Biol. Med., 227,852,2002.

7089_C003.indd 91 10/15/07 5:29:48 PM


4. Groff,J.L.,Gropper,S.S.andHunt,S.M.,Advanced Nutrition and Human Metabo-lism.WestPub.Co.,Minneapolis,MN,p.575,1995.

5. Rao,A.V.andAgarwal,S.,Roleofantioxidantlycopeneincancerandheartdisease,J. Am. Coll. Nutr., 19,563,2000.

6. Goodman,G.E.etal.,Thebeta-caroteneandretinolefficacytrial:incidenceoflungcancer and cardiovascular disease mortality during 6-year follow-up after stoppingβ-caroteneandretinolsupplements,J. Natl. Cancer Inst., 96,1743,2004.

7. Heinonen, O.P. and Albanes, D., The effect of vitamin E and beta carotene on theincidenceoflungcancerandothercancersinmalesmokers,New Engl. J. Med., 330,1029,1994.

8. Rapola, J.M. et al., Randomised trial ofα-tocopherol and β-carotene supplementson incidence of major coronary events in men with previous myocardial infarction,Lancet, 349,1715,1997.

9. Omenn,G.S.etal.,EffectsofacombinationofbetacaroteneandvitaminAonlungcancerandcardiovasculardisease,New Engl. J. Med., 334,1150,1996.

10. Hennekens,C.H.etal.,Lackofeffectoflong-termsupplementationwithbeta-caroteneon the incidence of malignant neoplasms and cardiovascular disease, New Engl. J. Med., 334,1145,1996.

11. Shi,J.andLeMaguer,M.,Lycopeneintomatoes:Chemicalandphysicalpropertiesaffectedbyfoodprocessing,Crit. Rev. Food Sci., 40,1,2000.

12. Klipstein-Grobusch,K.etal.,Serumcarotenoidsandatherosclerosis:TheRotterdamStudy,Atherosclerosis, 148,49,2000.

13. Rissanen,T.H.etal.,Serumlycopeneconcentrationsandcarotidatherosclerosis:TheKuopioIschaemicHeartDiseaseRiskFactorStudy,Am. J. Clin. Nutr., 77,133,2003.

14. Omoni,A.O.andAluko,R.E.,Theanti-carcinogenicandanti-atherogeniceffectsoflycopene:Areview,Trends Food Sci. Technol., 16,344,2005.

15. Franceschi,S.etal.,Tomatoesandriskofdigestive-tractcancers,Int. J. Cancer, 59,181,1994.

16. Rao,A.V.andShen,H.,Effectoflowdoselycopeneintakeonlycopenebioavailabilityandoxidativestress,Nutr. Res., 22,1125,2002.

17. Stahl,W.andSies,H.,Uptakeoflycopeneanditsgeometricalisomersisgreaterfromheat-processed than from unprocessed tomato juice in humans, J. Nutr., 122, 2161,1992.

18. Van Het Hof, K.H. et al., Carotenoid bioavailability in humans from tomatoes pro-cessedindifferentwaysdeterminedfromthecarotenoidresponseinthetriglyceride-richlipoproteinfractionofplasmaafterasingleconsumptionandinplasmaafterfourdaysofconsumption,J. Nutr., 130,1189,2000.

19. Porrini, M., Riso, P. and Testolin, G., Absorption of lycopene from single or dailyportionsofrawandprocessedtomato,Brit. J. Nutr., 80,353,1998.

20. Cohn,W.etal.,Comparativemultipledoseplasmakineticsoflycopeneadministeredintomatojuice,tomatosoup,orlycopenetablets,Eur. J. Nutr., 43,304,2004.

21. CenterforFoodSafetyandAppliedNutrition,Qualified health claims: Letter regarding tomatoes and prostate cancer (lycopene health claim coalition), http://www.cfsan.fda.gov/~dms/qhclyco2.html,U.S.FoodandDrugAdministration,Rockville,MD,2005.

22. DeLorgeril,M.etal.,Alpha-linolenicacidinthepreventionandtreatmentofcoronaryheartdisease,Eur. Heart J., 3,2001.

23. Connor,W.E.,Neuringer,M.andReisbick,S.,Essentialfattyacids:Theimportanceofn-3fattyacidsintheretinaandbrain,Nutr. Rev., 50,21,1992.

24. Neuringer, M., Connor, W.E. and Lin, D.S., Biochemical and functional effects ofprenatalandpostnatalω-3fattyaciddeficiencyonretinaandbraininrhesusmonkeys,Proc. National Academy of Sci. U.S.A., 83,4021,1986.

7089_C003.indd 92 10/15/07 5:29:49 PM


25. Connor, W.E.,α-Linolenic acid in health and disease, Am. J. Clin. Nutr., 69, 827,1999.

26. Julius,U.,Fatmodificationinthediabetesdiet,Exp. Clin. Endocrinol. Diabetes, 111,60,2003.

27. De Lorgeril, M. et al., Mediterranean alpha-linolenic acid-rich diet in secondarypreventionofcoronaryheartdisease,Lancet, 343,1454,1994.

28. Ascherio,A.etal.,Dietaryfatandriskofcoronaryheartdiseaseinmen:CohortfollowupstudyintheUnitedStates,Brit. Med. J., 313,84,1996.

29. Lanzmann-Petithory, D. et al., Primary prevention of cardiovascular diseases byα-linolenicacid(multipleletters),Am. J. Clin. Nutr., 76,1456,2002.

30. Renaud,S.C.etal.,Thebeneficialeffectofα-linolenicacidincoronaryarterydiseaseisnotquestionable(multipleletters),Am. J. Clin. Nutr., 76,903,2002.

31. Iliev, E., Tsankov, N. and Broshtilova, V., Short communication: Omega-3, -6 fattyacidsintheimprovementofpsoriaticsymptoms,Semin. Integr. Med., 1,211,2003.

32. Leventhal,L.J.,Boyce,E.G.andZurier,R.B.,Treatmentofrheumatoidarthritiswithgamma-linolenicacid,Ann. Intern. Med., 119,867,1993.

33. Zurier,R.B.etal.,Gamma-linolenicacidtreatmentofrheumatoidarthritis:Arandom-ized,placebo-controlledtrial,Arth. Rheum., 39,1808,1996.

34. Muggli,R.,Systemiceveningprimroseoilimprovesthebiophysicalskinparametersofhealthyadults,Int. J. Cosmetic Sci., 27,243,2005.

35. Singer, P. et al., Benefit of an enteral diet enriched with eicosapentaenoic acid andgamma-linolenicacidinventilatedpatientswithacutelunginjury,Crit. Care Med., 34,1033,2006.

36. Zock,P.L.andKatan,M.B.,Linoleicacidintakeandcancerrisk:Areviewandmeta-analysis,Am. J. Clin. Nutr., 68,142,1998.

37. Broitman,S.A.etal.,Polyunsaturatedfat,cholesterol,andlargeboweltumorigenesis,Cancer, 40,2455,1977.

38. Merck Research Laboratories, The Merck Index, 12th ed., Whitehouse Station, NJ,1741,1996.

39. Murakoshi,M.etal.,Inhibitionbysqualeneofthetumor-promotingactivityof12-O-tetradecanoylphorbol-13-acetateinmouse-skincarcinogenesis,Int. J. Cancer, 52,950,1992.

40. Desai,K.N.,Wei,H.andLamartiniere,C.A.,Thepreventiveandtherapeuticpotentialof the squalene-containing compound, Roidex, on tumor promotion and regression,Cancer Lett., 101,93,1996.

41. Rao,C.V.,Newmark,H.L. andReddy,B.S.,Chemopreventive effectof squaleneoncoloncancer,Carcinogenesis, 19,287,1998.

42. Smith,T.J.etal.,Inhibitionof4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-inducedlungtumorigenesisbydietaryoliveoilandsqualene,Carcinogenesis, 19,703,1998.

43. Scolastici,C.,Ong,T.P.andMoreno,F.S.,SqualenedoesnotexhibitachemopreventiveactivityandincreasesplasmacholesterolinaWistarrathepatocarcinogenesismodel,Nutr. Cancer, 50,101,2004.

44. Choi,H.S.etal.,Severeexogenouslipoidpneumoniafollowingingestionoflargedosesqualene:Successfultreatmentwithsteroid,Tuber. Respir. Dis., 60,235,2006.

45. Kohno,Y.etal.,Kineticstudyofquenchingreactionofsingletoxygenandscavengingreactionoffreeradicalbysqualeneinn-butanol,Biochim. Biophys. Acta-Lipids Lipid Metab., 1256,52,1995.

46. Lewkowicz,N.etal.,Biologicalactionandclinicalapplicationofsharkliveroil,Polski Merkuriusz Lekarski, 20,598,2006.

47. Senthilkumar,S.etal.,Effectofsqualeneoncyclophosphamide-inducedtoxicity,Clin. Chim. Acta, 364,335,2006.

7089_C003.indd 93 10/15/07 5:29:50 PM


48. Chan,P.etal.,Effectivenessandsafetyoflow-dosepravastatinandsqualene,aloneandincombination,inelderlypatientswithhypercholesterolemia,J. Clin. Pharmacol., 36,422,1996.

49. Goodwin, T.W., Biosynthesis of sterols, in Lipids: Structure and Function, Vol. 4,Stumpf,P.K.andConn,E.E.,Eds.,AcademicPress,London,pp.485–507,1980.

50. Lehninger,A.L.,Nelson,D.L.andCox,M.M.,Lipids,inPrinciples of Biochemistry, 2nded.,WorthPublishers,NewYork,pp.240–267,1993.

51. Miettinen,T.A.andVanhanen,H.,Serumconcentrationandmetabolismofcholesterolduringrapeseedoilandsqualenefeeding,Am. J. Clin. Nutr., 59,356,1994.

52. Smith, T.J., Squalene: Potential chemopreventive agent, Expert Opin. Inv. Drug., 9,1841,2000.

53. Strandberg,T.E.,Tilvis,R.S.andMiettinen,T.A.,Metabolicvariablesofcholesterolduring squalene feeding in humans: Comparison with cholestyramine treatment,J. Lipid Res., 31,1637,1990.

54. Fernandez,P.andCabral,J.M.S.,Phytosterols:Applicationsandrecoverymethods—Review,Bioresour. Technol.,98,2335,2007.

55. Piironen,V.etal.,Plantsterols:Biosynthesis,biologicalfunctionandtheirimportancetohumannutrition,J. Sci. Food Agric., 80,939,2000.

56. Normén,L.etal.,Soysterolestersandβ-sitostanolesterasinhibitorsofcholesterolabsorptioninhumansmallbowel,Am. J. Clin. Nutr., 71,908,2000.

57. Jones,P.J.H.etal.,Cholesterol-loweringefficacyofasitostanol-containingphytosterolmixturewithaprudentdietinhyperlipidemicmen,Am. J. Clin. Nutr., 69,1144,1999.

58. Plat,J.andMensink,R.P.,Plantstanolandsterolestersinthecontrolofbloodcholes-terollevels:Mechanismandsafetyaspects,Am. J. Cardiol., 96,2005.

59. Björkhem,I.,Boberg,K.M.andLeitersdorf,E.,Inbornerrorsinbileacidbiosynthesisandstorageofsterolsotherthancholesterol,inThe Online Metabolic and Molecular Bases of Inherited Disease, Scriver,C.R.etal.,Eds.,McGraw-Hill,NewYork,2002.

60. Raicht,R.F.,Cohen,B.I. andFazzini,E.P.,Protectiveeffectofplant sterols againstchemicallyinducedcolontumorsinrats,Cancer Res., 40,403,1980.

61. DeJong,A.,Plat,J.andMensink,R.P.,Metaboliceffectsofplantsterolsandstanols(Review),J. Nutr. Biochem., 14,362,2003.

62. Bouic,P.J.D.etal.,Theeffectsofβ-sitosterol(BSS)andβ-sitosterolglucoside(BSSG)mixtureonselectedimmuneparametersofmarathonrunners:Inhibitionofpostmara-thonimmunesuppressionandinflammation,Int. J. Sports Med., 20,258,1999.

63. Bouic,P.J.D.andLamprecht,J.H.,Plantsterolsandsterolins:Areviewoftheirimmune-modulatingproperties,Altern. Med. Rev., 4,170,1999.

64. Gupta, M.B., Gupta, G.P. and Bhargava, K.P., Anti-inflammatory and anti-pyreticactivitiesofβ-sitosterol,Planta Med., 39,157,1980.

65. Ivorra, M.D. et al., Antihyperglycemic and insulin-releasing effects of β-sitosterol3-β-D-glucosideand its aglycone,β-sitosterol,Arch. Int. Pharmacodyn. Ther., 296,224,1988.

66. ScientificCommitteeonFood,Generalviewofthescientificcommitteeonfoodonthelong-termeffectsoftheintakeofelevatedlevelsofphytosterolsfrommultipledietarysources,withparticularattentiontotheeffectsonβ-carotene,vol.SCF/CS/NF/DOS/20ADD1Final,EuropeanCommission,Brussels,Belgium,2002.

67. Hallikainen, M.A., Sarkkinen, E.S. and Uusitupa, M.I.J., Plant stanol esters affectserumcholesterolconcentrationsofhypercholesterolemicmenandwomeninadose-dependentmanner,J. Nutr., 130,767,2000.

68. Law,M.,Plantsterolandstanolmargarinesandhealth,Brit. Med. J., 320,861,2000.

7089_C003.indd 94 10/15/07 5:29:51 PM


69. U.S.DepartmentofHealthandHumanServices,FDA authorizes new coronary heart disease health claim for plant sterol and plant stanol esters, http://www.fda.gov/bbs/topics/ANSWERS/ANS01033.html, Food and Drug Administration, Fishers Lane,Rockville,MD,2000.

70. Plat, J. and Mensink, R.P., Effects of diets enriched with two different plant stanolester mixtures on plasma ubiquinol-10 and fat-soluble antioxidant concentrations,Metabolism, 50,520,2001.

71. Rupérez,F.J.etal.,Chromatographicanalysisofα-tocopherolandrelatedcompoundsinvariousmatrices,J. Chromatogr. A, 935,45,2001.

72. Tucker,J.M.andTownsend,D.M.,Alpha-tocopherol:Rolesinpreventionandtherapyofhumandisease,Biomed. Pharmacother., 59,380,2005.

73. Insel,P.,Turner,E.R.andRoss,D.,Vitamins:Vitalkeystohealth,inDiscovery Nutrition,2nded.,JonesandBartlettPublishers,Sudbury,MA,pp.317–365,2006.

74. Hacquebard,M.andCarpentier,Y.A.,VitaminE:Absorption,plasmatransportandcelluptake,Curr. Opin. Clin. Nutr., 8,133,2005.

75. Saldeen,K.andSaldeen,T.,Importanceoftocopherolsbeyondα-tocopherol:Evidencefromanimalandhumanstudies,Nutr. Res., 25,877,2005.

76. Jacob, R.A. and Burri, B.J., Oxidative damage and defense, Am. J. Clin. Nutr., 63,1996.

77. Abudu,N.etal.,VitaminsinhumanarteriosclerosiswithemphasisonvitaminCandvitaminE,Clin. Chim. Acta, 339,11,2004.

78. Bowry,V.W.andStocker,R.,Tocopherol-mediatedperoxidation.TheprooxidanteffectofvitaminEontheradical-initiatedoxidationofhumanlow-densitylipoprotein,J. Am. Chem. Soc., 115,6029,1993.

79. Witting,P.K.,Bowry,V.W.andStocker,R., Inversedeuteriumkinetic isotopeeffectforperoxidationinhumanlow-densitylipoprotein(LDL):Asimpletestfortocopherol-mediatedperoxidationofLDLlipids,FEBS Lett., 375,45,1995.

80. InstituteofMedicine,Food,andNutritionBoard,Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids, NationalAcademyPress,Washington,D.C.,2000.

81. Naito,Y.,Shimozawa,M.andYoshikawa,T.,Tocotrienolsandatherosclerosis,J. Clin. Biochem. Nutr., 34,121,2004.

82. Baliarsingh,S.,Beg,Z.H.andAhmad,J.,Thetherapeuticimpactsoftocotrienolsintype2diabeticpatientswithhyperlipidemia,Atherosclerosis, 182,367,2005.

83. Teupser,D.,Thiery,J.andSeidel,D.,α-Tocopheroldown-regulatesscavengerreceptoractivityinmacrophages,Atherosclerosis, 144,109,1999.

84. Devaraj,S.,Hugou,I.andJialal,I.,α-TocopheroldecreasesCD36expressioninhumanmonocyte-derivedmacrophages,J. Lipid Res., 42,521,2001.

85. Vivekananthan,D.P.etal.,Useofantioxidantvitaminsforthepreventionofcardio-vasculardisease:Meta-analysisofrandomisedtrials,Lancet, 361,2017,2003.

86. Knekt,P.etal.,Antioxidantvitaminintakeandcoronarymortalityinalongitudinalpopulationstudy,Am. J. Epidemiol., 139,1180,1994.

87. Kushi,L.H.etal.,Dietaryantioxidantvitaminsanddeathfromcoronaryheartdiseaseinpostmenopausalwomen,New Engl. J. Med., 334,1156,1996.

88. Stephens,N.G.etal.,RandomisedcontrolledtrialofvitaminEinpatientswithcoro-narydisease:CambridgeHeartAntioxidantStudy(CHAOS),Lancet, 347,781,1996.

89. Virtamo,J.etal.,EffectofvitaminEandbetacaroteneontheincidenceofprimarynonfatalmyocardial infarctionandfatalcoronaryheartdisease,Arch. Intern. Med., 158,668,1998.

90. Tognoni,G.etal.,Low-doseaspirinandvitaminEinpeopleatcardiovascularrisk:Arandomisedtrialingeneralpractice,Lancet, 357,89,2001.

7089_C003.indd 95 10/15/07 5:29:52 PM


91. Valagussa,F.etal.,Dietarysupplementationwithn-3polyunsaturatedfattyacidsandvitaminEaftermyocardialinfarction:ResultsoftheGISSI-Prevenzionetrial,Lancet, 354,447,1999.

92. Yusuf,S.,VitaminEsupplementationandcardiovasculareventsinhigh-riskpatients,New Engl. J. Med., 342,154,2000.

93. Shklar, G. et al., Regression by vitamin E of experimental oral cancer, J. National Cancer Inst., 78,987,1987.

94. Krol, E.S., Kramer-Stickland, K.A. and Liebler, D.C., Photoprotective action oftopicallyappliedvitaminE,Drug Metab. Rev., 32,413,2000.

95. Neuzil,J.etal.,Inductionofcancercellapoptosisbyα-tocopherylsuccinate:Molecularpathwaysandstructuralrequirements,FASEB J., 15,403,2001.

96. Woutersen,R.A.,Appel,M.J.andVanGarderen-Hoetmer,A.,Modulationofpancreaticcarcinogenesisbyantioxidants,Food Chem. Toxicol., 37,981,1999.

97. Shklar,G.andSchwartz,J.,Tumornecrosisfactorinexperimentalcancerregressionwithalpha-tocopherol,beta-carotene,canthaxanthinandalgaeextract,Eur. J. Cancer Clin. Oncol., 24,839,1988.

98. Taylor,P.R.andGreenwald,P.,Nutritionalinterventionsincancerprevention.J. Clin. Oncol., 23,333,2005.

99. Caraballoso,M.etal.,Drugsforpreventinglungcancerinhealthypeople.Cochrane Database Syst. Rev., 2003.

100. Israel,K.etal.,VitaminEsuccinateinducesapoptosisinhumanprostatecancercells:RoleforFasinvitaminEsuccinate–triggeredapoptosis,Nutr. Cancer, 36,90,2000.

101. Campbell,S.E.etal.,ComparativeeffectsofRRR-alpha-andRRR-gamma-tocopherolonproliferationandapoptosisinhumancoloncancercelllines,BMC Cancer, 6,13,2006.

102. Moyad,M.A.,Brumfield,S.K.andPienta,K.J.,VitaminE,alpha-andgamma-tocopherol,andprostatecancer,Semin. Urol. Oncol., 17,85,1999.

103. Hirohata,K.etal.,Treatmentofosteoarthritisofthekneejointatthestateofhydro-arthrosis,Kobe Med. Sci., 11(supplement),65,1965.

104. Doumerg,C.,Etudecliniqueexperimentaledel’alpha-tocopheryle-quinoneenrheuma-tologieetenreeducation,Therapeutique, 45,676,1969.

105. Machtey,I.andOuaknine,L.,Tocopherolinosteoarthritis:Acontrolledpilotstudy,J. Am. Geriatr. Soc., 26,328,1978.

106. Blankenhorn,G.,ClinicalefficacyofSpondyvit®(vitaminE)inactivatedarthroses.Amulticenter,placebo-controlled,double-blindstudy,Zeitschrift fur Orthopadie und Ihre Grenzgebiete, 124,340,1986.

107. Scherak,O.etal.,TherapywithhighdosesofvitaminEinpatientswithosteoarthritis,Zeitschrift fur Rheumatologie, 49,369,1990.

108. Jordan, J.M. et al., A case-control study of serum tocopherol levels and the alpha-togamma-tocopherolratioinradiographickneeosteoarthritis:TheJohnstonCountyOsteoarthritisProject,Am. J. Epidemiol., 159,968,2004.

109. Grundman,M.,VitaminEandAlzheimerdisease:Thebasis for additional clinicaltrials,Am. J. Clin. Nutr., 71,2000.

110. Tabet,N.,Birks,J.andGrimley,E.J.,VitaminEforAlzheimer’sdisease.Cochrane Database Syst. Rev., 2000.

111. Luchsinger,J.A.etal.,AntioxidantvitaminintakeandriskofAlzheimerdisease,Arch. Neurol-Chicago, 60,203,2003.

112. Kalmijn,S.etal.,Polyunsaturatedfattyacids,antioxidants,andcognitivefunctioninveryoldmen,Am. J. Epidemiol., 145,33,1997.

113. Meydani,S.N.,Han,S.N.andHamer,D.H.,VitaminEandrespiratoryinfectionintheelderly,Ann. N.Y. Acad. Sci., 1031,214,2004.

7089_C003.indd 96 10/15/07 5:29:53 PM


114. Center for Food Safety and Applied Nutrition, Qualified health claims subject to enforcement discretion, http://www.cfsan.fda.gov/~dms/qhc-sum.html,U.S.FoodandDrugAdministration,Rockville,MD,2005.

115. Center forFoodSafety andAppliedNutrition,Letter regarding dietary supplement health claim for vitamin E and heart disease, U.S.FoodandDrugAdministration,Rockville,MD,2001.http://www.cfsan.fda.gov/~dms/ds-ltr16.html

116. Trumbo, P.R., The level of evidence for permitting a qualified health claim: FDA’sreview of the evidence for selenium and cancer and vitamin E and heart disease,J. Nutr., 135,354,2005.

117. Horwitt,M.K.,CritiqueoftherequirementforvitaminE,Am. J. Clin. Nutr., 73,1003,2001.

118. Miller III, E.R. et al., Meta-analysis: High-dosage vitamin E supplementation mayincreaseall-causemortality,Ann. Intern. Med., 142,2005.

119. Güclü-Üstündag,O.andTemelli,F.,Correlatingthesolubilitybehaviorofminorlipidcomponentsinsupercriticalcarbondioxide,J. Supercrit. Fluids, 31,235,2004.

120. Güclü-Üstündag,O.andTemelli,F.,Correlatingthesolubilitybehavioroffattyacids,mono-, di-, and triglycerides, and fatty acid esters in supercritical carbon dioxide,Ind. Eng. Chem. Res., 39,4756,2000.

121. Lopes, I.M.G. and Bernardo-Gil, M.G., Characterisation of acorn oils extracted byhexaneandbysupercriticalcarbondioxide,Eur. J. Lipid Sci. Technol., 107,12,2005.

122. Femenia,A.etal.,Effectsofsupercriticalcarbondioxide(SC-CO2)oilextractiononthecellwallcompositionofalmondfruits,J. Agric. Food Chem., 49,5828,2001.

123. Passey,C.A.andGros-Lois,M.,Productionofcalorie-reducedalmondsbysupercriticalextraction,J. Supercrit. Fluids, 6,255,1993.

124. Leo, L. et al., Supercritical carbon dioxide extraction of oil andα-tocopherol fromalmondseeds,J. Sci. Food Agric., 85,2167,2005.

125. Marrone, C. et al., Almond oil extraction by supercritical CO2: Experiments andmodelling,Chem. Eng. Sci., 53,3711,1998.

126. Özkal, S.G. et al., Response surfaces of hazelnut oil yield in supercritical carbondioxide,Eur. Food Res. and Technol., 220,74,2005.

127. Özkal, S.G., Salgin, U. and Yener, M.E., Supercritical carbon dioxide extraction ofhazelnutoil,J. Food Eng., 69,217,2005.

128. Bernardo-Gil,M.G.etal.,Supercriticalfluidextractionandcharacterisationofoilfromhazelnut,Eur. J. Lipid Sci. Technol., 104,402,2002.

129. Goodrum,J.W.andKilgo,M.B.,PeanutoilextractionusingcompressedCO2,Energy Agric., 6,265,1987.

130. Goodrum, J.W. andKilgo,M.B.,Peanut oil extractionwithSC-CO2:Solubility andkineticfunctions,Trans. ASAE., 30,1865,1987.

131. Goodrum,J.W.andKilgo,M.B.,ModelingofliquidCO2extractionofpeanutoilinafixedbed,Trans. ASAE,31,926,1988.

132. Chiou,R.R.Y.etal.,Partialdefattingof roastedpeanutmealsandkernelsbysuper-criticalCO2usingsemicontinuousandintermittentlydepressurizedprocesses,J. Agric. Food Chem., 44,574,1996.

133. Passey, C.A. and Patil, N.D., Process for preparing low-calorie nuts, U.S. Patent5290578,1994.

134. Zhang,C.etal.,Feasibilityofusingsupercriticalcarbondioxideforextractingoilfromwholepecans,Trans. ASAE, 38,1763,1995.

135. Li,M.,Bellmer,D.D.andBrusewitz,G.H.,PecankernelbrekageandoilextractedbysupercriticalCO2asaffectedbymoisturecontent,J. Food Sci., 64,1084,1999.

136. Alexander,W.S.,Brusewitz,G.H.andManess,N.O.,Pecanoilrecoveryandcomposi-tionasaffectedbytemperature,pressure,andsupercriticalCO2flowrate,J. Food Sci., 62,762,1997.

7089_C003.indd 97 10/15/07 5:29:54 PM


137. Palazoglu,T.K.andBalaban,M.O.,SupercriticalCO2extractionoflipidsfromroastedpistachionuts,Trans. ASAE, 41,679,1998.

138. Oliveira, R., Rodrigues, M.F. and Bernardo-Gil, M.G., Characterization and super-criticalcarbondioxideextractionofwalnutoil,J. Am. Oil Chem. Soc., 79,225,2002.

139. Crowe,T.D.andWhite,P.J.,Oxidation,flavor,andtextureofwalnutsreducedinfatcontentbysupercriticalcarbondioxide,J. Am. Oil Chem. Soc., 80,569,2003.

140. Crowe,T.D.andWhite,P.J.,Oxidativestabilityofwalnutoilsextractedwithsuper-criticalcarbondioxide,J. Am. Oil Chem. Soc., 80,575,2003.

141. Santerre,C.R.,Goodrum,J.W.andKee,J.M.,Roastedpeanutsandpeanutbutterqualityareaffectedbysupercriticalfluidextraction,J. Food Sci., 59,382,1994.

142. Turpin,P.E.,Coxon,D.T.andPadley,F.B.,Thefractionationofsheanutoilusingsuper-criticalcarbondioxide:Afeasibilitystudyfortheextractionofoilfromgum,Fett Wiss. Technol., 92,179,1990.

143. Özkal, S.G., Yener, M.E. and Bayindirli, L., The solubility of apricot kernel oil insupercriticalcarbondioxide,Int. J. Food Sci. Technol., 41,399,2006.

144. Özkal,S.G.,Yener,M.E.andBayindirli,L.,Responsesurfacesofapricotkerneloilyieldinsupercriticalcarbondioxide,Lebensm.–Wiss. Technol., 38,611,2005.

145. Özkal,S.G.,Yener,M.E.andBayindirli,L.,Masstransfermodelingofapricotkerneloilextractionwithsupercriticalcarbondioxide,J. Supercrit. Fluids, 35,119,2005.

146. Dauksas,E.,Venskutinis,P.R.andSivik,B.,Supercriticalfluidextractionofborage(Borago officinalis L.)seedswithpureCO2anditsmixturewithcaprylicacidmethylester,J. Supercrit. Fluids, 22,211,2002.

147. Gomez,A.M.anddelaOssa,E.M.,Qualityofborageseedoilextractedbyliquidandsupercriticalcarbondioxide,Chem. Eng. J., 88,103,2002.

148. Gaspar,F.etal.,Solubilityofechium,borage,andlunariaseedoilsincompressedCO2,J. Chem. Eng. Data,48,107,2003.

149. Bernardo-Gil,G.etal.,Extractionof lipids fromcherryseedoilusingsupercriticalcarbondioxide,Eur. Food Res. Technol., 212,170,2001.

150. Gawdzik,J.etal.,Supercriticalfluidextractionofoilfromeveningprimrose(Oenothera paradoxaH.)seeds,Chemia Analityczna,43,695,1998.

151. Favati, F., King, J.W. and Mazzanti, M., Supercritical carbon dioxide extraction ofeveningprimroseoil,J. Am. Oil Chem. Soc., 68,422,1991.

152. King,J.W.,Cygnarowicz,M.L.andFavati,F.,Supercriticalfluidextractionofeveningprimroseoilkineticandmasstransfereffects,Ital. J. Food Sci.,9,193,1997.

153. Bozan,B.andTemelli,F.,SupercriticalCO2extractionofflaxseed,J. Am. Oil Chem. Soc., 79,231,2002.

154. Beveridge,T.H.J.etal.,Yieldandcompositionofgrapeseedoilsextractedbysuper-criticalcarbondioxideandpetroleumether:Varietaleffects,J. Agric. Food Chem., 53,1799,2005.

155. Sovova,H.,Kucera,J.andJez,J.,RateofthevegetableoilextractionwithsupercriticalCO2-II.Extractionofgrapeoil,Chem. Eng. Sci., 49,415,1994.

156. Gomez,A.M.,Lopez,C.P.anddelaOssa,E.M.,Recoveryofgrapeseedoilbyliquidandsupercriticalcarbondioxideextraction:acomparisonwithconventionalsolventextraction,Chem. Eng. J., 61,227,1996.

157. Murga,R.etal.,Extractionofnaturalcomplexphenolsandtanninsfromgrapeseedsbyusingsupercriticalmixturesofcarbondioxideandalcohol,J. Agr. Food Chem.,48,3408,2000.

158. Cao, X.L. and Ito, Y.C., Supercritical fluid extraction of grape seed oil and subse-quent separation of free fatty acids by high-speed counter-current chromatography,J. Chromatogr. A, 1021,117,2003.

159. Reverchon,E.,Kaziunas,A.andMarrone,C.,SupercriticalCO2extractionofhiproseseedoil:Experimentsandmathematicalmodelling,Chem. Eng. Sci., 55,2195,2000.

7089_C003.indd 98 10/15/07 5:29:55 PM


160. Holser,R.A.andBost,G.,Hybrid hibiscusseedoilcompositions,J. Am. Oil Chem. Soc., 81,795,2004.

161. Hadolin, M. et al., High pressure extraction of vitamin E–rich oil from Silybum marianum,Food Chem., 74,355,2001.

162. Muuse, B.G., Cuperus, F.P. and Derksen, J.T.P., Extraction and characterization ofDimorphotheca pluvialis seedoil,J. Am. Oil Chem. Soc., 71,313,1994.

163. Bernardo-Gil, M.G. and Lopes, L.M.C., Supercritical fluid extraction of Cucurbita ficifolia seedoil,Eur. Food Res. Technol., 219,593,2004.

164. Szentmihalyi,K.etal.,Rosehip(Rosa caninaL.)oilobtainedfromwastehipseedsbydifferentextractionmethods,Bioresour. Technol., 82,195,2002.

165. delValle,J.M.andUquiche,E.L.,ParticlesizeeffectsonsupercriticalCO2extractionofoil-containingseeds,J. Am. Oil Chem. Soc., 79,1261,2002.

166. del Valle, J. M. et al., Comparison of conventional and supercritical CO2-extractedrosehipoil,Brazil. J. Chem. Eng.,17,335,2000.

167. Eggers,R.,Ambrogi,A.andvonSchnitzler,J.,SpecialfeaturesofSCFsolidextrac-tionofnaturalproducts:Deoilingofwheatglutenandextractionofrosehipoil,Brazil. J. Chem. Eng.,17,329,2000.

168. delValle,J.M.etal.,SupercriticalCO2processingofpretreatedrosehipseeds:effectofprocessscaleonoilextractionkinetics,J. Supercrit. Fluids, 31,159,2004.

169. Stastova, J. et al., Rate of the vegetable oil extraction with supercritical CO2 - III.Extractionfromseabuckthorn,Chem. Eng. Sci.,51,4347,1996.

170. Yin,J.etal.,ModelingofsupercriticalfluidextractionfromHippophae rhamnoidesL.seeds,Sep. Sci. Technol., 38,4041,2003.

171. Yin,J.Z.etal.,ExperimentsandnumericalsimulationsofsupercriticalfluidextractionforHippophae rhamnoidesL.seedoilbasedonartificialneuralnetworks,Ind. Eng. Chem. Res., 44,7420,2005.

172. Odabasi,A.Z.andBalaban,M.O.,SupercriticalCO2extractionofsesameoilfromrawseeds,J. Food Sci. Technol., 39,496,2002.

173. Roy,B.C.,Goto,M.andHirose,T.,TemperatureandpressureeffectsonsupercriticalCO2extractionoftomatoseedoil,Int. J. Food Sci. Technol., 31,137,1996.

174. Illés,V.etal.,ExtractionofhiprosefruitbysupercriticalCO2andpropane,J. Supercrit. Fluids, 10,209,1997.

175. Yakimishen, R., Cenkowski, S. and Muir, W.E., Oil recoveries from sea buckthornseedsandpulp,Appl. Eng. Agric., 21,1047,2005.

176. Mongkholkhajornsilp, D. et al., Supercritical CO2 extraction of nimbin from neemseeds-Amodellingstudy,J. Food Eng., 71,331,2005.

177. Yin,J.Z.etal.,Analysisoftheoperationconditionsforsupercriticalfluidextractionofseedoil,Sep. Purif. Technol., 43,163,2005.

178. Westerman,D.etal.,ExtractionofAmaranthseedoilbysupercriticalcarbondioxide,J. Supercrit. Fluids, 37,38,2006.

179. He,H.P.,Corke,H.andCai, J.G.,SupercriticalcarbondioxideextractionofoilandsqualenefromAmaranthus grain,J. Agric. Food Chem., 51,7921,2003.

180. Bruni,R. et al.,WildAmaranthus caudatus seedoil, anutraceutical resource fromEcuadorianflora,J. Agric. Food Chem., 49,5455,2001.

181. Fors,S.M.andEriksson,C.E.,Characterizationofoilsextractedfromoatsbysuper-criticalcarbondioxide,Lebens.–Wiss. Technol.,23,390,1990.

182. Zhao,W.etal.,Fractionalextractionofricebranoilwithsupercriticalcarbondioxide,Agric. Biol. Chem., 51,1773,1987.

183. Shen,Z.etal.,Pilotscaleextractionofricebranoilwithdensecarbondioxide,J. Agr. Food Chem.,44,3033,1996.

184. Shen, Z. et al., Pilot scale extraction and fractionation of rice branoil using super-criticalcarbondioxide,J. Agric. Food Chem., 45,4540,1997.

7089_C003.indd 99 10/15/07 5:29:56 PM


185. Kim,H.J.etal.,CharacterizationofextractionandseparationofricebranoilrichinEFAusingSFEprocess,Sep. Purif. Technol., 15,1,1999.

186. Perretti,G.etal.,Improvingthevalueofriceby-productsbySFE,J. Supercrit. Fluids, 26,63,2003.

187. Danielski,L.etal.,Aprocesslinefortheproductionofraffinatedriceoilfromricebran,J. Supercrit. Fluids, 34,133,2005.

188. Gomez,A.M.anddelaOssa,E.M.,Qualityofwheatgermoilextractedbyliquidandsupercriticalcarbondioxide,J. Am. Oil Chem. Soc., 77,969,2000.

189. Ge,Y.etal.,ExtractionofnaturalvitaminEfromwheatgermbysupercriticalcarbondioxide,J. Agric. Food Chem., 50,685,2002.

190. Panfili,G.etal.,ExtractionofwheatgermoilbysupercriticalCO2:Oilanddefattedcakecharacterization,J. Am. Oil Chem. Soc., 80,157,2003.

191. Taniguchi, M. et al., Extraction of oils from wheat germ with supercritical carbondioxide,Agric. Biol. Chem., 49,2367,1985.

192. Zhang,X.W.etal.,Supercriticalcarbondioxideextractionofwheatplumuleoil,J. Food Eng., 37,103,1998.

193. Dunford,N.T.andKing,J.W.,Phytosterolenrichmentofricebranoilbyasupercriticalcarbondioxidefractionationtechnique,J. Food Sci., 65,1395,2000.

194. deFranca,L.F. et al.,Supercritical extractionofcarotenoidsand lipids fromburiti(Mauriti flexuosa), a fruit from the Amazon region, J. Supercrit. Fluids, 14, 247,1999.

195. Saldaña,M.D.A.etal.,Comparisonofthesolubilityofβ-caroteneinsupercriticalCO2basedonabinaryandamulticomponentcomplexsystem,J. Supercrit. Fluids, 37,342,2006.

196. Goto,M.,Sato,M.andHirose,T.,Supercriticalcarbondioxideextractionofcarotenoidsfromcarrots,inYano,T.,Matsuno,R.andNakamura,K.,Eds.,Developments in Food Engineering, Proceedings of the Sixth International Congress on Engineering and Food,BlackieAcademic&Professional,London,1994,835–837.

197. Ranalli,A. et al.,Characterizationof carrot rootoil arising fromsupercriticalfluidcarbondioxideextraction,J. Agric. Food Chem., 52,4795,2004.

198. Sun,M.andTemelli,F.,Supercriticalcarbondioxideextractionofcarotenoidsfromcarrotusingcanolaoilasacontinuousco-solvent,J. Supercrit. Fluids, 37,397,2006.

199. Manninen,P.,Pakarinen,J.andKallio,H.,Large-scalesupercriticalcarbondioxideextraction and supercritical carbon dioxide countercurrent extraction of cloudberryseedoil,J. Agr. Food Chem.,45,2533,1997.

200. Esquivel,M.M.,Bernardo-Gil,M.G.andKing,M.B.,Mathematicalmodelsforsuper-criticalextractionofolivehuskoil,J. Supercrit. Fluids, 16,43,1999.

201. Gomez-Prieto,M.S.,Caja,M.M.andSanta-Maria,G.,Solubilityinsupercriticalcarbondioxideofthepredominantcarotenesintomatoskin,J. Am. Oil Chem. Soc., 79,897,2002.

202. Ollanketo, M. et al., Supercritical carbon dioxide extraction of lycopene in tomatoskins,Eur. Food Res. Technol.,212,561,2001.

203. Shi,J.,Separation of carotenoids from fruits and vegetables, EuropeanPatentAppl.WO0179355,2001.

204. Rozzi,N.L.etal.,Supercriticalfluidextractionof lycopene fromtomatoprocessingbyproducts,J. Agric. Food Chem., 50,2638,2002.

205. Gomez-Prieto, M.S. et al., Supercritical fluid extraction of all trans-lycopene fromtomato,J. Agric. Food Chem., 51,3,2003.

206. Ruiz del Castillo, M.L. et al., Lipid composition in tomato skin supercritical fluidextractswithhighlycopenecontent,J. Am. Oil Chem. Soc., 80,271,2003.

207. Vasapollo,G.etal.,InnovativesupercriticalCO2extractionoflycopenefromtomatointhepresenceofvegetableoilasco-solvent,J. Supercrit. Fluids, 29,87,2004.

7089_C003.indd 100 10/15/07 5:29:57 PM


208. deLucas,A.,Rincon,J.andGracia,I.,Influenceofoperatingvariablesonyieldandqualityparametersofolivehuskoilextractedwithsupercriticalcarbondioxide,J. Am. Oil Chem. Soc., 79,237,2002.

209. Stavroulias,S.andPanayiotou,C.,Determinationofoptimumconditionsfortheextrac-tionofsqualenefromolivepomacewithsupercriticalCO2,Chem. Biochem. Eng. Q., 19,373,2005.

7089_C003.indd 101 10/15/07 5:29:58 PM

7089_C003.indd 102 10/15/07 5:29:58 PM

103

4 Extraction and Purification of Natural Tocopherols by Supercritical CO2

Tao Fang, Motonobu Goto, Mitsuru Sasaki, and Dalang Yang

Contents

4.1 Background................................................................................................. 1044.1.1 ProblemswithConcentratingTocopherolsUsingMolecular

Distillation....................................................................................... 1044.1.2 PretreatmentbeforeConcentratingTocopherol............................... 1054.1.3 FundamentalResearchonConcentratingTocopherols................... 106

4.2 MainExperimentalMaterials..................................................................... 1074.3 AnalyticalMethods..................................................................................... 1074.4 CorrelationforExperimentalData............................................................. 1074.5 BinaryPhaseEquilibria.............................................................................. 107

4.5.1 ApparatusandProcedure................................................................. 1074.5.2 HighPressureViewCell.................................................................. 1104.5.3 PhaseEquilibriumProperties.......................................................... 1104.5.4 Solubility.......................................................................................... 1134.5.5 DistributionCoefficient................................................................... 113

4.6 TernaryPhaseEquilibria............................................................................ 1184.6.1 ApparatusandProcedure................................................................. 1184.6.2 InfluencesofPressureandTemperatureonPhaseEquilibrium...... 1204.6.3 SeparationFactorbetweenTocopherolandMethylOleate............. 1224.6.4 EquilibriumLines............................................................................ 1234.6.5 PhaseBehaviorofME-DOD...........................................................124

4.7 SeparationwithSupercriticalCO2Fractionation........................................ 1264.7.1 FractionationApparatusandProcedure.......................................... 1274.7.2 PretreatmentResultandCompositionofME-DOD........................ 1294.7.3 EffectoftheInitialPressure............................................................ 1314.7.4 EffectoftheFinalPressure............................................................. 1324.7.5 CompositionofTocopherolConcentrate......................................... 134

7089_C004.indd 103 10/8/07 11:50:31 AM


4.7.6 ViscosityComparison...................................................................... 1344.7.7 ApplicationinCommercialProduction........................................... 136

4.8 Conclusions................................................................................................. 136References.............................................................................................................. 138

4.1 BaCkground

Tocopherols,commonlyknownasvitaminE,areknownforantioxidativeactivitywhich has been widely applied in the fields of food, medicine, and cosmetics.Figure4.1 illustrates the molecular structures of tocopherols. The main source ofnaturaltocopherolsisdeodorizerdistillate(DOD),abyproductoftheedibleoilrefin-ingprocessthatisrichintocopherolsandsterols[1].

4.1.1 Problems with ConCentrating toCoPherols Using moleCUlar Distillation

Molecular distillation, also called short-path distillation, has been applied to thecommercialproductionoftocopherolsfromDOD[2–5].Itischaracterizedbyhighvacuuminthedistillationspace,shortexposureofthedistilledliquidtotheoper-ating temperatures, and short distancebetween the evaporator and the condenser(20to70mm)[3].

However,onthebasisofprojectinvestigation,wefoundsomeproblemsinthecommercialproductionoftocopherolsusingmoleculardistillation.First,theprocessof molecular distillation is generally performed with multistage distillators (3 to5units)athighvacuum(0.1to10Pa)andhightemperature(433to503K).Notice-ably,ahighqualityvacuumpumpisabsolutelyindispensableforensuringenoughvacuumconditionforeachdistillator.Also,itisobviousthatthehighqualitypumpusedforcommercialproduction,thedistillator,withfinestructureandstrictopera-tionconditions,leadstorelativelyhighequipmentinvestmentandoperationcost.

O CH3

CH3

CH3 CH3CH3

CH3

HO

R1

R23'

4'2

7'8'

11'

Substituents R1 R2 Notation

CH3 CH3 α-tocopherolCH3 H β-tocopherol

H CH3 γ-tocopherolH H δ-tocopherol

Figure 4.1 Molecularstructureoftocopherols.(FromBrunner,G.,Gas Extraction: An Introduction to Fundamentals of Supercritical Fluids and the Application to Separation Processes, Darmstadt:Sternkopff,Springer,NewYork,1994.Withpermission.)

7089_C004.indd 104 10/8/07 11:50:32 AM

Extraction and Purification of Natural Tocopherols by Supercritical CO2 105

Second, according to our communication with some companies employingmoleculardistillation,itisgenerallydifficulttokeepallpressuresinthedistillatorconstantat low levels (0.1 to10Pa)during the longoperation.Consequently, theoperationtemperaturehastobeincreasedtocompensateforthedecreaseinvacuumdegreewithaviewtostabilizingtheconcentrationofthefractions.Asaresult,suchfluctuationsintemperatureandpressureleadtoanunstablequalityofthetocoph-erolproduct. Inaddition, thedefinitionofmoleculardistillation isnotveryaccu-ratebecausethereisnoeffectofrectificationcausedbyfractioncondensationandrefluence. As we know, the effect of rectification is the main difference betweendistillationandvaporization.Furthermore,thephenomenonofentrainmentprobablyoccurswithoutrectificationandinfluencesontheseparationselectivity.

Third,thegeneralopinionisthattheshortcontactorresidencetimeoftheproductathightemperaturesduringmoleculardistillationdoesnotcauseanydegradationoftheproductanddoesnotaffectthequalityoftheproduct.However,Mau[5]researchedonconcentratingtocopherolsbymoleculardistillationandreportedtheexistenceoftocopheroldimmersandotherdegradationproductsat433to493K,eventhoughpres-surewaslowerthan0.133Pa,andthetotalrecoveryoftocopherolsinallfractionsandresiduewasonlyabout75.16%oftheinitialamountoftocopherolsinfeed.

Finally, in the commercial operation of molecular distillation with three tofivedistillationunits,theresidencetimeoftocopherols,themaincompoundinthesecondorthirdfraction,isnolessthan1hour,whichisnotashorttimeforasepara-tionoperation.

Becausethermaldegradationoftocopherolsiscommonlycausedbyhighprocess-ingtemperature[6],developmentofnewalternativeisolationtechniques,includingsupercriticalfluidextraction(SFE),hasbeendesired.

4.1.2 Pretreatment before ConCentrating toCoPherol

Tomodifythecompositionofrawmaterial,aprocessofpretreatmentisgenerallynecessary. Pretreatment involves two steps (esterification and methanolysis) thatconvert free fatty acids (FFAs) and glycerides (Gly.), respectively, into fatty acidmethylesters(FAMEs).ThemainobjectiveistomodifythecompositionofDODandtoincreasethesolubilityofsoybeanDODinsupercriticalcarbondioxide(CO2)extraction.InthepublishedliteratureonchemicalmodificationofDOD,esterifica-tionwascarriedoutwiththecatalystssulfuricacid(H2SO4)[5,7–9,12],hydrochloricacid(HCL)[10],orNa[11].Additionally,someresearchersaddedasecondreactionofmethanolysiswiththecatalystssodiummethoxide(NaOCH3)[8,12]andsodiumhydroxide(NaOH)[9].Inourlabwork,H2SO4andNaOCH3wereselectedasthecatalystsofmethylesterificationandmethanolysis, respectively.Figure4.2 showsthepretreatmentprocessofDOD.Aftereachreaction,themixturewaswashedwithhot water until it became neutral. Finally, the mixture was held at low tempera-tureand,asaresult,mostofthesterolscrystallizedandwereremovedbyfiltration.Afterpretreatment,theoil,methylesterifiedDOD(ME-DOD),wasobtained,whichcontainsmainlyFAMEs(70%to80%),tocopherols(10%to15%),andimpurities(suchasresidualsterols,glycerides,squalene,pigments,andlongchainparaffins,comprisingintotalabout10%to15%).

7089_C004.indd 105 10/8/07 11:50:33 AM


4.1.3 fUnDamental researCh on ConCentrating toCoPherols

SupercriticalCO2isrelativelysuitableasaseparatingsolventforextractingsomefat-solublecomponents.AlthoughsomeresearcherstriedtoconcentratetocopherolsfromDODbysupercriticalCO2[6–16],theoperationparameters,especiallypressure,vary.Forexample,Zhaoetal.[7]concentrated75%tocopherolsat12MPa,whereasLee et al. [10] reported that 40 MPa could be used to obtain 40% extract. Kingetal. [14] combined SFE with supercritical fluid chromatography (SFC) for con-centratingtocopherols,andtheoptimizedconditionswere25MPa/353KforSFEand 25MPa/313 K for SFC. Generally, if pressure is low, satisfactory selectivitycanbeobtainedbutproductivityislow.Contrarily,higherpressureleadstolowerselectivityandhigherproductivity.Tofindmore reasonableoperatingconditions,phaseequilibriaofME-DOD+CO2mustbeclarified.

WhenconcentratingnaturaltocopherolsfromME-DOD,theimportantstepistoremoveFAMEs,whichcontributemorethan70%ofME-DOD.Toexplorethereasonableoperation conditions for this step, the complex systemofME-DOD+CO2wasinitiallyregardedasapseudo-ternary(methyloleate+tocopherol+CO2)system.ThereasonforchoosingmethyloleateisthattheME-DODappliedinour

Methanolysis (Reflux at 343 K for 120 min )

H2SO4(3 wt.% oil), methanol (65 wt.% oil)

DOD(FFA, glycerides, sterols, sterol esters, tocopherols and squalene)

Methyl Esterification (Reflux at 333 K for 120 min)

Oil Phase, MEDOD (FAMEs, Tocopherols, Squalene, Residual Sterols and Glycerides)

Solid Phase (Crude Sterols)

Standing Separation and Washing with Hot Water (353 K) Until Neutral (pH=7)

Upper, Oil Phase (partly methyl esterified DOD)

Lower, Water Phase

NaOCH3 (1 wt.% oil), Methanol (55 wt.% oil)

Standing Separation, Neutralization with HCL and Washing with Hot Water (353 K)

Lower, Water Phase

Chilling at 277 K, Crystallization and Filtration

Upper, Oil Phase

Figure 4.2 Pretreatment process for preparing ME-DOD from DOD. (From Fang, T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)

7089_C004.indd 106 10/8/07 11:50:34 AM


researchisfromthesoybeanoilindustryandthemainFAMEsaremethyloleateandmethyllinoleate(about75%to80%ofallFAMEs);thelatterissimilartomethyloleateinphysicochemicalproperties[17].

Asfarasphaseequilibriumisconcerned,twobinarysystemsofα-tocopherol+CO2andmethyloleate+CO2weremeasuredandcorrelated.Thentheternarysystem(methyloleate+tocopherol+CO2)andtherealisticsystem(ME-DOD+CO2)wereinvestigated.Finally,thephaseequilibriumdatawereanalyzedandaccordinglytheseparationofnaturaltocopherolsfromME-DODwascarriedoutwithsupercriticalCO2fractionation.

4.2 Main experiMental Materials

CO2wassuppliedbytheUchimuraSansoCo.,Ltd.(Osaka,Japan),withapurityof99.97%.MethyloleateandDl-α-tocopherolwereobtainedfromWakoPureChemicalIndustries,Inc.(Tokyo,Japan)withpuritiesof≥ 98%.DOD(9.23%tocopherols)wassuppliedbyKaidiFineChemicalIndustrialCo.,Ltd.(Wuhan,HubeiProvince,P.R.China),anditscompositionisillustratedinTable4.1.ME-DOD(10.19%tocopherols)waspreparedbyDODaccordingtotheprocedureshowninFigure4.2.

4.3 analytiCal Methods

TheapproximatecontentsofFFAsandglycerides,includingmonoglycerides,diglyc-erides,andtriglycerides)werecalculatedfromacidandsaponificationvalues(A.V.andS.V.,respectively)andexpressedasthecontentsofoleicacidandtriolein[18].

Analysesoftocopherols,sterols,andFAMEswereperformedwithhighperfor-manceliquidchromatography(HPLC)andgaschromatographwithflameionizationdetector(GC-FID),respectively[19–21].ThecompositionofME-DODandtocopherolconcentrate was determined with gas chromatograph-mass spectrometry (GC-MS).Additionally,theviscosityofthesamplesobtainedintheseparationexperimentweremeasuredwithanAR1000rheologymeter(TAInstrumentsCo.,Ltd,England)[21].

4.4 Correlation For experiMental data

TheSoave-Redlich-Kwong(SRK)EOS[22]withtheAdachi-Sugie(AS)mixingrule[23] was used to correlate the experimental data. The SRK EOS is the modifica-tionofthesimpleRedlich-KwongEOS,withwhichthevaporpressurecurvecanbereproducedwell.ThisprocedureofcorrelationwascompletedbyPE2000,whichwasdevelopedbyPfohl,Petkov,andBrunnerandcontainssomecommonEOSandprovedcapabilitytoobtainaconvergencesimilartothatfromASPENsoftware[24].

4.5 Binary phase equiliBria

4.5.1 aPParatUs anD ProCeDUre

Theexperimentalapparatususedinthisworkiscalled“gas-liquidalternatingcir-culationsystem,”whichisusedtosimultaneouslymeasurethecompositionsinboth

7089_C004.indd 107 10/8/07 11:50:34 AM


taB

le 4

.1C

hara

cter

isti

cs o

f do

d

a.V

.*

(mg

koh

/g)

s.V.

* (m

g ko

h/g

)FF

a*

(%)

gly

.* (

%)

toco

pher

ols

(%)

isom

er p

erce

ntag

e (%

)

ster

ols

(%)

isom

er p

erce

ntag

e (%

)

α-β-

+γ-

δ-C

ampe

ster

olst

igm

aste

rol

β-si

tost

erol

95.4

147.

148

.127

.29.

2311

.73

60.0

328

.24

9.45

33.5

23.5

43.0

*A

ccor

ding

to A

OC

Sm

etho

ds [1

8],t

he a

ppro

xim

ate

cont

ents

of f

ree

fatty

aci

d(F

FA) a

ndg

lyce

ride

s(G

ly. i

nclu

ding

mon

ogly

ceri

des,

dig

lyce

ride

s,a

nd tr

igly

ceri

des)

wer

eca

lcul

ated

fro

ma

cid

and

sapo

nific

atio

nva

lues

(A

.V.a

ndS

.V.,

resp

ectiv

ely)

and

exp

ress

eda

sth

eco

nten

tso

fol

eic

acid

and

trio

lein

.So

urce

:Fa

ng,T

.,G

oto,

M.,

Wan

g,X

.,D

ing,

X.,

Gen

g,J

.,Sa

saki

,M.a

ndH

iros

e,T

.,J.

Sup

ercr

itic

al F

luid

s, 4

0,5

0, 2

007.

With

per

mis

sion

.

7089_C004.indd 108 10/8/07 11:50:35 AM


liquidandgasphases.AsshowninFigure4.3,theequilibriumsystemisimmersedinawaterbath,withtwostirrersforkeepingthesystem’stemperatureuniformwiththeprecisiontemperaturecontrolof+0.5K.

About70mLofpurecomponentisinitiallychargedintoequilibriumvessel6(170mL,max.pressure30MPa),andthenCO2flowsintotheapparatusfromcylinder1,passing throughvalve2andfiltratingpipe3, thecoolerandsyringepump4(Isco260D,max.pressure57.71MPa,TeledyneIscoInc.,Lincoln,NE,USA),valve5,andthenintoequilibriumvessel6.Afterthepressureandtemperaturereachtherequiredvalues,valve5isclosedandthenmagneticpump8ispoweredon,whichcankeepthefluidflowingupwardatabout4mLperminute.Byrotatingfour-wayvalve7,gas(lightphase)orliquid(heavyphase)ischosenasthecirculatingfluidinthesystem.Forexample,thecirculatingfluidinFigure4.3isthegasphase.Afterthegasphaseiskeptcirculatingfor1.5hours,six-wayvalve9isturnedandthefluidinsampler11(20mL)istakenasthegassample.Aftervalve15isopenedandadjusted,theCO2inthegasslowlypassesthroughsamplingbottle13andflowmeter17(precision0.01L),whichrecordstheamountofCO2.Thepipesbetweenvalve9and15andbottle13areallheatedbyanelectronicheater.Thecompoundsprecipitatedinsampler11arewashedintobottle13withn-hexaneandrinsedwithabout10mLn-hexanetoavoidthesampleloss,sincethesolutesmayformaerosolparticlesandthenpassthroughthecollectingbottlewithCO2fluid.Aftereveryrun,theresidualn-hexaneinthesampleisremovedfromcylinder20withCO2andthenmergedintothesamplebattle.Then-hexanesolutionisquantifiedbyelectronicbalance(precision0.1mg)andanalyzedbyGC (formethyloleate)orHPLC(forα-tocopherol).From theamountsofCO2andchromatographicdata,thecompositioninthegasphaseiscalculated.Asforthe

1, 20: CO2 Cylinder 2, 5, 15, 16: Valve 3, 19: Filtrating Pipe4: Cooler and Syringe Pump 6: Equilibrium Vessel 7: Four-way Valve8: Electromagnetic Pump 9, 10: Six-way Valve 11: Gas Sampler12: Liquid Sampler 13, 14: Sampling Bottle 17, 18: Gas Flowmeter

2

3

41

5

79

86

Thermostated Water Bath

(170 ml)

11

10

1317

15

19

20

1814

1612

(5 m

l)(2

0 m

l)

PI TIR

Figure 4.3 Schematic diagram of experimental apparatus. (From Fang, T., Goto, M.,Yun,Z.,Ding,X.andHirose,T.,J.Supercritical Fluids, 30,1,2004.Withpermission.)

7089_C004.indd 109 10/8/07 11:50:36 AM


sample from the liquidphase, four-wayvalve7 is turned to the liquidcirculation.Aftercirculatingfor1.5hours,six-wayvalve10isturnedforsampling.Byasimilarmethodtothegasphases,theliquidcompositionisalsoobtained.

Thisstructureofourapparatuspreventspressurefluctuationduringsampling.Toobtainmoreaccurateresults,alldatarepresentmeanvaluesofthreesamplingsatauniformcondition,andtherelativestandarddeviationsarewithin0.005mol%forgascomposition(Y1)and0.5mol%forliquidcomposition(X1).

4.5.2 high PressUre View Cell

Forobservingtheequilibriumsystems,aviewcell(30mL,max.pressure30MPa,AkicoCo.,Tokyo,Japan)wasemployedinourexperiment,asshowninFigure4.4.Amagnetic stirrer is coupled with the cell and the temperature is controlledby electric heaters embedded inside the cell’s wall. The Isco pump was used forpressurizingthesystem.

4.5.3 Phase eqUilibriUm ProPerties

Thebinaryphaseequilibriumdataformethyloleate+CO2andα-tocopherol+CO2weremeasuredandcorrelatedat313.15to353.15Kinthepressurerangesof5to23MPaformethyloleateand5to30MPaforα-tocopherol[19].Theisothermsof313.15,333.15,and353.15Kformethyloleate+CO2andα-tocopherol+CO2weremeasuredoverthepressurerangesof5to23MPaand5to30MPa,respectively.TheexperimentalresultsandtheircorrelateddataareshowninFigure4.5andFigure4.6,inwhichthedatameasuredbyotherresearchers[25–37]arealsoillustrated.

Asforthephaseequilibriumofmethyloleate+CO2,at313.15Kand333.15K,our experimental data in Figure4.5 agree well with the data reported by otherresearchers, except thedata reportedbyChenget al. [25].Chenget al. foundanunusualwaist-shapecurveatthevicinityofthecriticalpressureofCO2,aninteresting

Filtrating PipePI

TIR

View Cell

Magnetic StirrerIsco Syringe Pump

CO2 Tank

Figure 4.4 Schematic diagram of high-pressure cell for visual observation. (FromFang,T.,Goto,M.,Yun,Z.,Ding,X.andHirose,T.,J.Supercritical Fluids, 30,1,2004.Withpermission.)

7089_C004.indd 110 10/8/07 11:50:37 AM


20(a

) T =

313

.15

K

(c) T

= 3

53.1

5 K

(b) T

= 3

33.1

5 K

Cram

pon

et al

., 199

9In

omat

a et a

l., 1

989

Zou

et al

., 199

0Yu

et al

., 199

2Ch

eng

et al

., 198

9Th

is W

ork

Cram

pon

et al

., 199

9In

omat

a et a

l., 1

989

Zou

et al

., 199

0Yu

et al

., 199

2N

ilsso

n et

al., 1

991

This

Wor

k

16 12 P (MPa) P (MPa)

8 4 24 20 16 12 8 4

24 20 16 12 8 4

0.5

0.6

Mol

e Fra

ctio

n of

CO

2 (%)

Mol

e Fra

ctio

n of

CO

2 (%)

0.7

0.8

0.9

0.97

0.98

0.99

1.00

0.5

0.6

0.7

0.8

0.9

1.0

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.98

1.00

20 16 12 8 4 0.2

0.3

0.4

0.5

0.6

0.7

(d) Th

is W

ork

and

Corr

elat

ion

Resu

lt (S

RK-E

OS

and

AS

mix

ing

rule

)Th

is W

ork,

313

.15

KCa

lcul

ated

at 3

13.1

5 K

This

Wor

k, 3

33.1

5 K

Calc

ulat

ed at

333

.15

KTh

is W

ork,

353

.15

KCa

lcul

ated

at 3

53.1

5 K0.

80.

960.

90.

981.

00

This

Wor

k

Fig

ur

e 4.

5 B

inar

yph

ase

equi

libr

iaf

or m

ethy

lol

eate

+ C

O2.

(Fr

omF

ang,

T.,

Got

o,M

.,Y

un,

Z.,

Din

g, X

.an

dH

iros

e, T

.,J.

Sup

ercr

itic

al F

luid

s, 3

0,1

,200

4.W

ith

perm

issi

on.)

7089_C004.indd 111 10/8/07 11:50:39 AM


(a) T

= 3

13.1

5 K

(c) T

= 3

53.1

5 K

(d) Th

is W

ork

and

Calc

ulat

ed D

ata

This

Wor

k, 3

13.1

5 K

Calc

ulat

ed at

313

.15

KTh

is W

ork,

333

.15

KCa

lcul

ated

at 3

33.1

5 K

This

Wor

k, 3

53.1

5 K

Calc

ulat

ed at

353

.15

K

(b) T

= 3

33.1

5 K

Joha

nnse

n et

al., 1

997

Chen

et al

., 200

0Sk

erge

t et a

l., 2

002

Mei

er et

al., 1

994

Pere

ira et

al., 1

993

Chra

stil,

198

2O

hgak

i et a

l., 1

987

This

Wor

k

Joha

nnse

n et

al., 1

997

Chen

et al

., 200

0Sk

erge

t et a

l., 2

002

Pere

ira et

al., 1

993

Chra

stil,

198

2Th

is W

ork

Joha

nnse

n et

al., 1

997

Sker

get e

t al.,

200

2M

eier

et al

., 199

4Ch

rast

il, 1

982

This

Wor

k

3236 28 24 20 16 12 8 4 0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.99

60.

998

1.00

00.

20.

30.

40.

50.

60.

70.

80.

90.

996

0.99

81.

000

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.99

60.

998

1.00

00.

50.

60.

70.

80.

90.

998

1.00

0

36 32 28 24 20 16 12 8 4 32 28 24 20 16 12 8 4

32

Mol

e Fra

ctio

n of

CO

2 (%)

P (MPa)P (MPa)

Mol

e Fra

ctio

n of

CO

2 (%)

36 28 24 20 16 12 8 4

Fig

ur

e 4.

6 B

inar

yph

ase

equi

libr

iaf

or α

-toc

ophe

rol

+ C

O2.

(Fr

omF

ang,

T.,

Got

o,M

.,Y

un,

Z.,

Din

g, X

.an

dH

iros

e, T

.,J.

Sup

ercr

itic

al F

luid

s, 3

0,1

,200

4.W

ith

perm

issi

on.)

7089_C004.indd 112 10/8/07 11:50:40 AM


phenomenonthatwasnotverifiedbyotherresearchers’andourdata.Additionally,inthecaseof353.15K,nodataarereportedintheliterature.

Forthesystemofα-tocopherol+CO2,Figure4.6a–cillustratesthatourmeasureddatamatchwiththeliteraturedataatthreetemperatures.Theresults,especiallythecompositiondataingasphase,areremarkablydifferentfromauthortoauthor.Thepossible reasonsfor theerrorswere thought tobehighviscosityofα-tocopherol,pressurechange,andaerosolscausedbysuddendepressurization[34].Additionally,thedataforliquidphasearenotasabundantasthoseforgasphase.

Our data illustrate that both methyl oleate and α-tocopherol mole fractionsin gas phase increase as pressure increases at constant temperature. Meanwhile,the CO2 fraction in liquid phase rises with increasing pressure. The equilibriumconcentrationofmethyloleateinCO2isalwaysmuchhigherthantheequilibriumconcentrationofα-tocopherolinCO2.Moreover,theinfluenceoftemperatureongascompositioniscontrarytothatofpressure.Inaddition,formethyloleate+CO2,withtheincreaseoftemperature,theCO2fractioninliquidobviouslydecreases,whereasforα-tocopherol+CO2at313.15Kand333.15K,theliquidcompositionchangesslightlywithtemperature.Ontheotherhand,at353.15K,theCO2molefractioninliquidisslightlylargerthanthoseatothertemperatureswithpressureshigherthan12MPa.

In addition, an ideal correlation for our experimental data, as shown inFigure4.5dand4.6d,canbeobtainedbytheSRKEOSwiththeASmixingrule,wheretheaveragedeviationobtainedislowerthan0.12%forgasand7%forliquid.

4.5.4 solUbility

The solubilities of methyl oleate and α-tocopherol in supercritical CO2 werecalculatedfromthegasphasecomposition.TheresultsareshowninFigure4.7.ThesolubilitiesofthetwocompoundsarepresentedasafunctionoftheCO2density.

Two effects are observed. At constant temperature, solubility increases withincreasingdensity.ThisisprobablyduetotheincreasingsolventpowerofCO2athigherdensity.Atconstantdensity,ariseof temperatureresults inanincreaseofsolubilityduetotheincreaseinvaporpressureofthesolutes.SimilarphenomenawerereportedbyJohannsenandBrunner[35].

4.5.5 DistribUtion CoeffiCient

Equilibriumdataoftwophasescanbeusedtocalculatethedistributioncoefficient,whichwasdefinedby:

Kyxi

i

i

= (4.1)

whereyi and xiaremolefractionsofcomponentiingasandliquidphase,respectively.Calculation results are shown in Figure4.8. As density increases at constant

temperature,thedistributioncoefficientofmethyloleateincreasessignificantly.Inthecaseofmethyloleate+CO2,whenpressurewasmorethan12.5MPaat313.15K,

7089_C004.indd 113 10/8/07 11:50:41 AM


16

2.5

2.0

1.5

1.0

0.5

0.0

12 8

Met

hyl O

leat

e Den

sity o

f CO

2(g/

ml)

Den

sity o

f CO

2(g/

ml)

Solubility of Alph-Tocopherol (g/100g CO2)

Solubility of Methyl Oleate (g/100g CO2)

T =

313.

15 K

Calc

ulat

ed at

313

.15

KT

= 33

3.15

KCa

lcul

ated

at 3

33.1

5 K

T =

353.

15 K

Calc

ulat

ed at

353

.15

K

Alp

ha-T

ocop

hero

l

T =

313.

15 K

Calc

ulat

ed at

313

.15

KT

= 33

3.15

KCa

lcul

ated

at 3

33.1

5 K

T =

353.

15 K

Calc

ulat

ed at

353

.15

K

4 0 0.0

0.2

0.4

0.6

0.8

0.0

0.2

0.4

0.6

0.8

(a)

(b)

Fig

ur

e 4.

7 T

hes

olub

iliti

eso

f m

ethy

lol

eate

and

α-t

ocop

hero

lin

CO

2.(

From

Fan

g,T

., G

oto,

M.,

Yun

, Z

.,D

ing,

X.

and

Hir

ose,

T.,

J.S

uper

crit

ical

Flu

ids,

30,

1,2

004 .

With

per

mis

sion

.)

7089_C004.indd 114 10/8/07 11:50:42 AM


Met

hyl O

leat

e

1.0

0.8

0.01

2

0.00

8

0.00

4

0.00

0

0.6

0.4

0.2

0.0

Den

sity o

f CO

2(g/

ml)

Den

sity o

f CO

2(g/

ml)

Distribution Coefficient of Methyl Oleate

Distribution Coefficient of Methyl Oleate

T =

313.

15 K

Ca

lcul

ated

at 3

13.1

5 K

T =

333.

15 K

Ca

lcul

ated

at 3

33.1

5 K

T =

353.

15 K

Ca

lcul

ated

at 3

53.1

5 K

Alp

ha-T

ocop

hero

l T

= 31

3.15

K

Calc

ulat

ed at

313

.15

K T

= 33

3.15

K

Calc

ulat

ed at

333

.15

K T

= 35

3.15

K

Calc

ulat

ed at

353

.15

K

0.0

0.2

0.4

0.6

0.8

0.0

0.2

0.4

0.6

0.8

(a)

(b)

Fig

ur

e 4.

8 D

istr

ibut

ion

coef

ficie

nts

of m

ethy

lol

eate

and

α-t

ocop

hero

lin

CO

2.(

From

Fan

g, T

.,G

oto,

M.,

Yun

, Z.,

Din

g, X

. and

H

iros

e,T

.,J.

Sup

ercr

itic

al F

luid

s, 3

0,1

,200

4 .W

ith

perm

issi

on.)

7089_C004.indd 115 10/8/07 11:50:43 AM


18.5MPaat 333.15K, and22.5MPaat 353.15K, there is no remarkablediffer-enceinequilibriumcompositionsbetweenthetwophases.Thismeansthatintheexperimental range investigated, critical points for the binary mixture probablyexist.Ifthedistributioncoefficientequalstheunity,thetwophases’compositionsanddensitiesareentirelyidentical,asaresultthebiphasicsystemchangingintoamonophasicsystem,whichmeansmethyloleateandCO2arecompletelymiscibleatsuchconditions.Thecorrespondingpressureiscalledacritical pressure forthebinarymixtureunderacertaintemperature,andallthecriticalpointsatdifferenttemperatureslineuptoacritical curve, whichischaracteristicforthismixture[1]inaP-T-xdiagramforabinarysystem.Becauseaccuratemeasurementofcriticalpoints is relativelydifficult,weadopted an approximatemethodby extrapolatingthecorrelatedcurvesofmethyloleate+CO2,asshowninFigure4.5d.Theapproxi-materangesforcriticalpointswereestimatedtobe13to14MPaat313.15K,19to20MPaat333.15K,and23to24MPaat353.15K.

Forverifyingthisprediction,ahigh-pressurecellwithwindowswasusedforvisualobservation.Initially,about20mLmethyloleatewerechargedintothecell.At 313.15 K, CO2 was slowly compressed (0.5 mL/min) into the cell. When thepressurewas8 to12MPa, the interfacebetweengas and liquidwasvery clear,as shown in Figure4.9a. When the pressure reached about 12.6 MPa, the inter-facebetweengasandliquidbecamethickerandtheliquidlevelincreasedalittlebecausemoreCO2dissolvedin liquid,asshowninFigure4.9b.Thenthesysteminsidethecellbecamemoreobscureandturbidwithpressureincrease,asshownin Figure4.9c. Finally, the interface became completely unidentifiable at about13.7MPa,andthewholesystemrecoveredclearasauniformphaseat13.9MPa,as shown in Figure4.9d. At 333.15 K and 353.15 K, a similar phenomenon wasrespectivelyobservedat19.7to20.1MPaand23.5to23.8MPa.Actually,itshouldbenoticedthatthedisappearanceoftheinterfacedoesnotsuddenlyhappen.Thechangeisaprogressiveprocessandtheendpoint isdifficult tobedeterminedbyvisualobservation.However,byvisualobservation,approximatepressure rangesareclosetothepredictionfromourcorrelation.

Inthecaseofα-tocopherol+CO2,thedistributioncoefficientindicatesthatthetwocomponentsarepoorlydissolvedineachother.Thedistributioncoefficientsofα-tocopherolareonetotwoordersofmagnitudelowerthanthoseofmethyloleate.

The discussion above indicates that there are considerable differences inthe solubilities and distribution coefficients of methyl oleate andα-tocopherol inCO2. Therefore, supercritical CO2 extraction can be thought feasible to separateα-tocopherol frommethyloleate.According tophaseequilibriumdataona fattyacid-CO2system[27,28,31,36],thesolubilitydifferencebetweenfattyacidsandα-tocopherolissmallerthanthatofmethyloleateandα-tocopherol.Thus,inordertoseparatetocopherolsfromDOD,thepretreatmentshowninFigure4.2isofgreatsignificance. The pretreatment converts fatty acids and glycerides, which consistof70%to80%ofDOD,intoFAMEs,resultinginenlargingthesolubilitybetweencomponentsofsupercriticalCO2.Therefore,thesupercriticalCO2processseemstobemorefeasibleforseparatingtocopherolsfromesterifiedDODthandirectlyfromDOD.Inaddition,pretreatmentissimultaneouslyadvantageousforremovingmoststerols and other solid impurities (wax, long-chain hydrocarbons) from esterified

7089_C004.indd 116 10/8/07 11:50:43 AM


(a) T

= 3

13.1

5 K

, P =

8 M

Pa(b

) T =

313

.15

K, P

= 1

2.6

MPa

(c) T

= 3

13.1

5 K

, P =

13.

7 M

Pa(d

) T =

313

.15

K, P

= 1

3.9

MP a

Fig

ur

e 4.

9 D

isap

pear

ance

of

the

inte

rfac

ebe

twee

nga

san

dli

quid

pha

ses

(met

hyl

olea

te+

CO

2).

(Fro

mF

ang,

T.,

Got

o,M

.,Y

un,Z

.,D

ing,

X.a

ndH

iros

e,T

.,J.

Sup

ercr

itic

al F

luid

s, 3

0,1

,200

4 .W

ith

perm

issi

on.)

7089_C004.indd 117 10/8/07 11:50:45 AM


DOD[5,7–10]becausethesolubilityofsterolsisfarlowerinfattyacidmethylestersthanthatinfattyacids.

Compared with methyl oleate, the solubility and distribution coefficient ofα-tocopherolaregenerallyonetotwoordersofmagnitudelower.Suchalargediffer-encebetweenthe twocomponents indicates that it ispossible toconcentratenaturaltocopherolsfromesterifiedDODintheexperimentalrangeinvestigated.Thisconclu-sionneedstobeprovedfurtherbystudyoftheternarysystemandtherealisticsystem.

4.6 ternary phase equiliBria

Aspreviouslydescribed,thecomplexsystemofME-DOD+CO2canberegardedas a pseudo-ternary (methyl oleate + tocopherol + CO2) system. Regretfully, nopublishedinformationwasfoundonphaseequilibriumofME-DODinsupercriticalCO2,eventhoughsomeliteraturereportedonternaryandmulticomponentsystemsinvolvingα-tocopherolorDOD[38–41].Thus,wemeasuredthephasebehaviorsfortheternaryandrealisticsystemswiththeviewofprovidingfundamentalinforma-tionforfurtherseparationexperimentsandprocessdesign.

Inthispart,weinvestigatedtheinfluencesofthreefactorsonphasebehavior:pressure (from10 to29MPa), temperature (from313.15 to353.15K), and initialfeedcomposition.Six initial feedcompositions(0,10.19,32.44,50.46,71.93,and100mass%)wereinvestigated.Amongthese,0%and100%stoodforthepurecom-positionsofmethyloleateandα-tocopherol,respectively.Theircorrespondingphaseequilibriumdatawerecitedfromthebinarydatainpart4.5.3.ThefeedcompositionofME-DODwas10.19%.Otherfeedcompositionswerepreparedbymixingmethyloleateandα-tocopherolaccordingtodifferentproportions.

4.6.1 aPParatUs anD ProCeDUre

Anexperimentalapparatuswasestablishedformeasuringthecompositionsofbothliquid and gas phases. As shown in Figure4.10, the apparatus consisted of feed,equilibrium, and sampling systems.Aviewcell (30mL,max. pressure30MPa,AkicoCo.,Tokyo,Japan)coupledwithamagneticstirrerwasemployedastheequi-libriumvessel,anditstemperaturewascontrolledwithanelectricheatercapableofmaintainingthetemperaturewithin±0.1K.

Initiallytheequilibriumcellwaschargedwithabout15to20mLfeed.CO2thenflowedintotheapparatusfromtheCO2tankviathefilteringpipeandsyringepump(ISCO260D,max.pressure57.71MPa,Teledyne Isco Inc.,Lincoln,NE,USA),whichisoperatedinthemodeofconstantpressure,andintotheequilibriumcell.Afterthepressureandtemperaturereachtherequiredvalues,themagneticstirrerwasturnedonandthesysteminsidetheequilibriumcellwasstirredforatleast2hours.Byrotating thesix-portvalve, thesampleswereconverted toandfromgas (lightphase)andliquid(heavyphase).Forexample,thesituationshowninFigure4.10isasampletakenfromthegasphase.Duringsampling,byadjustingthemicroswitchof thedigitalbackpressureregulator(BPR,JASCO880-81,JASCOInternationalCo.Ltd.,Tokyo,Japan),gaseousCO2slowlypassedthroughthesamplingbottleandflowmeter,whichrecordedtheamountofCO2(definedascompound3)consumed.

7089_C004.indd 118 10/8/07 11:50:45 AM


The pipes connected to the six-port valve, cell, and digital BPR were heated byelectronicheaters.Theirtemperatureswerecontrolledinamannersimilartothatoftheequilibriumcell.Additionally,becauseoftheeffectofadiabaticexpansion,thefluid temperaturedecreasedgreatlyandsuddenlyduringsampling.Consequently,somesolutesorCO2mayhavecondensedattheoutletoftheBPR,contributingmoreorlesstomeasurementinaccuracy.Toavoidsuchphenomena,theBPRoutletwasheatedandmaintainedatatemperatureof371K.Inaddition,about10mLn-hexanewasinitiallyloadedinthesamplebottlebecausethesolutesmayhaveformedaerosolparticlesandpassthroughthecollectingbottlewiththeCO2fluid[34].Then-hexanesolutionwasquantifiedbyelectronicbalance(precision10–4g)andanalyzedbyGC(formethyloleate,compound1)andHPLC(fortocopherol,compound2).Accord-ingtotheamountofCO2consumedandchromatographicdata,thegascomposition(y1, y2, y3)wascalculated.Byasimilarmethod,theliquidcomposition(x1, x2, x3)wasobtained.Additionally,becausesimilarsystemlineswereusedforsamplingfromtwophases,whenthesix-portvalvewasswitchedforsamplingfromanotherphase,thefluidfromthecellwaskeptflowingwithoutsamplingforabout1to2minutes,inordertoavoidcarryoverofthesamples.Anotherkeypointwastoensurethatthesamplewastakenfromanequilibriumsystembysamplingonlywhentheliquid-gasinterfacewasclearlyvisible.

Thestructureofourapparatusavoidedpressurefluctuationduringtheequilib-riumandsamplingstepsbecausetheequilibriumcellpressurewasmaintainedatthesetvalues(therequiredvalues)bysettingtheISCOpumpinthemodeofconstantpressure.Moreimportantly,duringsampling,theCO2flowrateshouldbekeptrela-tivelylowbyadjustingtheBPRmicroswitch.Inourexperiment,theCO2flowrate

Filtering PipeSix-part Valve

Digital BPR

Window

Equilibrium CellISCO Syringe

Pump

Feed System EquilibriumSystem

Magnetic StirrerCO2 Tank

SamplingSystem

Feed Pump

TIR PI

One-wayValve

Figure 4.10 Schematic diagram of the phase equilibrium apparatus. (From Fang, T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)

7089_C004.indd 119 10/8/07 11:50:46 AM


wasmaintainedatlowerthan20mL·min–1and5mL·min–1(at0.1MPaandroomtemperature)duringsamplingfromgasandliquid,respectively.

To obtain more accurate results, all data represented mean values of threesamplingsatuniformconditionswithanuncertaintyof±0.001massfractionforgascompositionand±0.002forliquidcomposition,respectively.

Forgasphasesampling,theCO2amountwasadjustedtoabout2L(at0.1MPaandroomtemperature)atexperimentalpressureslowerthan20MPasincelowsolubilityatlowpressureswasamainreasonforexperimentalerror;however,forhigherpressures(≥20MPa),theamountofCO2wasabout1L(at0.1MPaandroomtemperature).Forliquidphase,theCO2amountwasadjustedtoabout0.01to0.02L(at0.1MPaandroomtemperature).Thecontentsofmethyloleateandtocopherolintheliquidsampleweregenerallyhighandrequireddilutionbeforechromatographicanalysis.

Thesamplesdissolvedinn-hexanewereanalyzedbyGCtodeterminetheconcen-trationofmethyloleate.AnalysisoftocopherolwasperformedbyHPLC[19,20].

4.6.2 inflUenCes of PressUre anD temPeratUre on Phase eqUilibriUm

Theisothermsat313.15,333.15,and353.15Kfortheternarysystemofmethyloleate(1)+tocopherol(2)+CO2(3)weremeasuredoverthepressurerangefrom10to29MPa.At313.15K,thecompositiondataat10,20,and29MPaweredrawninatriangulardiagram,as shown inFigure4.11.Obviously, the two-phase region,which is sur-roundedbytheequilibriumdata,shrinkswithincreasingpressure.Inotherwords,

Methyl Oleate

Tocopherol

Tocopherol

Carbon Dioxide

Carbon Dioxide

Methyl Oleate

(a) (b)

0.0 1.0

0.8

0.6

0.4

0.2

0.0

0.2

0.4

0.6

0.8

1.00.0 0.2 0.4

W3

W3

W2W1

W1W2

0.6 0.8 1.0

0.0

0.5

1.00.8 0.9 1.0

0.2

0.1

0.0

Figure 4.11 Influence of pressure on the phase equilibria of (w1 methyl oleate + w2tocopherol+ w3CO2)atT =313.15K:(a)Liquid-gasequilibria;(b)Theequilibriumcomposi-tionsingas;⦁,experimentaldataatP=10MPa;◾,experimentaldataatP=20MPa;▲,theexperimentaldataatP=29MPa;•,thecorrelatedtielinesatP=10MPa;•,thecorrelatedtielinesatP=20MPa;•,thecorrelatedtielinesatP=29MPa.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)

7089_C004.indd 120 10/8/07 11:50:47 AM


themutualsolubilityofthecomponentsincreased.Figure4.11ashowstheCO2massfractioninliquidriseswithpressureincrease,whiletheCO2massfractioningasis reduced, as shown in Figure4.11b, which means the solubilities of other com-ponents ingasincrease.At lowerpressures(10MPa),becausebothmethyloleateandtocopherolhavelimitedmiscibilityinCO2,theternaryphasebehaviorrevealsaphaseequilibriumofternarytypeII[1].Thus,thereisnocriticalpoint.Athigherpressures(20,29MPa),becausemethyloleateandCO2arecompletelymiscible,thetwo-phaseareaisternarytypeI,whichischaracterizedbyacriticalpointwherethetwophasesbecomeidentical.InthetypeIsystem,highersolubilityinthegascanbereachedthaninternarytypeIIsystem.

Inadditiontothemeasureddata,Figure4.11showsthetielinesconnectingwiththeequilibriumdataintheliquidandgasphases.ThetielineswerecorrelatedwiththeSRKEOSandtheASmixingrule.Characteristically,thegradientoftheequilib-riumtielinesgraduallychangesfromonesidelineofthetriangletotheother.Thismeansthatwiththeincreaseofmethyloleatemassfractioninfeed,phasebehaviortendstobeclosetothatofthebinarysystemofmethyloleate+CO2.

Figure4.12showstheinfluenceoftemperatureonphaseequilibriumat20MPa.Obviously,theinfluenceoftemperatureiscontrarytothatofpressure.Withincreas-ing temperature, the two-phase area expands, as shown in Figure4.12a. In addi-tion, the phase equilibria are of ternary type I at 313.15 and 333.15 K, and thenat353.15KthephaseequilibriumdevelopsintoternarytypeII,wherethebinarycriticalpressure forCO2+methyloleate isgreater than20MPa.Noticeably, the

Methyl Oleate

Tocopherol

Tocopherol

Carbon Dioxide

Carbon Dioxide

Methyl Oleate

0.0 1.0

0.8

0.6

0.4

0.2

0.0

0.2

0.4

0.6

0.8

1.00.0 0.2 0.4

W3

W3

W2 W1

W1W2

0.6 0.8 1.0

(b)(a)

0.0

0.5

1.00.8 0.9 1.0

0.2

0.1

0.0

Figure 4.12 Influenceof temperatureon thephaseequilibriaof (w1methyloleate+w2tocopherol+ w3CO2)atP =20MPa:(a)Liquid-gasequilibria;(b)Theequilibriumcomposi-tionsingas;⦁,theexperimentaldataatT=313.15K;◾,theexperimentaldataatT=333.15K;▲,theexperimentaldataatT=353.15K;•,thecorrelatedtielinesatT=313.15K;•,thecorrelatedtielinesatT=333.15K;•,thecorrelatedtielinesatT=353.15K.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)

7089_C004.indd 121 10/8/07 11:50:48 AM


influenceof temperatureonthegascompositionseemstobe lesssignificant thanthatoftheliquidcompositionat20MPa,asshowninFigure4.12b.

4.6.3 seParation faCtor between toCoPherol anD methyl oleate

Accordingtothemeasuredgasandliquidcompositiondata,theseparationfactor(S)betweentocopherol(compound2)andmethyloleate(compound1)wascalculatedby:

S=(y2/x2)/(y1/x1) (4.2)

whereyi and xiaremassfractionsofcomponentiingasandliquid,respectively.The separation factor represents the process selectivity for separating methyl

oleatefromtocopherol.Indetail,alowervalueindicateshigherselectivity,whereasa higher value indicates that it is more difficult to separate the two compoundsunder certain conditions. Furthermore, when the separation factor equals unity,thecompositioningasissimilartothatinliquidandthesupercriticalCO2processcannotseparatemethyloleatefromtocopherol.

Figure4.13 and Figure4.14 show the influences of pressure and temperatureon the separation factor, respectively. In Figure4.13, both the experimental dataandcorrelatedcurveillustratethat,ataconstanttemperature,theseparationfactorincreasesaspressureincreases,exceptforonepartat10MPa.However,asshowninFigure4.14,theinfluenceoftemperatureiscontrarytothatofpressure.Asmentionedabove, a lower separation factor indicates a higher selectivity; accordingly, thetendenciesshowninFigure4.13andFigure4.14indicatethatlowpressureandhightemperature lead to high selectivity, which is advantageous for separating methyloleate from tocopherol with supercritical CO2. In addition, at constant pressure

0.5

0.4

0.3

0.2

S

0.1

0.00.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

x20

Figure 4.13 Influenceofpressureontheseparationfactor(S)atT=313.15K:x20, the

initialtocopherolmassfractioninfeed;▲,experimentaldataatP=29MPa;◾,experimentaldataatP=20MPa;⦁,experimentaldataatP=10MPa;····,correlatedatP=29MPa;---,correlatedatP=20MPa;—,correlatedatP=10MPa.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)

7089_C004.indd 122 10/8/07 11:50:49 AM


and temperature, the separation factor increases as the initial tocopherol content(x2

0) decreases. Moreover, this tendency is more obvious as pressure increases. Alowercontentoftocopherolmeansahighercontentofmethyloleatesincethefeedmainlyconsistedofmethyloleateandtocopherol,amongwhichtheformerismoresolubleinsupercriticalCO2.Consequently,moremethyloleateinthefeedgeneratesmoretocopherolfordistributioninsupercriticalCO2,resultinginanincreaseintheseparationfactor.Inotherwords,methyloleateactsasthecosolventfortocopherol.AsimilarphenomenonwasalsoreportedbyBambergeretal.[42],whomeasuredthesolubilitiesoffattyacids,puretriglycerides,andtriglyceridemixturesinsupercriticalCO2. They found that the solubilities of the less soluble triglycerides in mixtureslike tripalmitinwere enhancedby thepresenceofmore soluble triglycerides, liketrilaurin. In this situation, the more soluble compounds were said to be acting asthecosolvents.

4.6.4 eqUilibriUm lines

Usingtheobtainedequilibriumdata,equilibriumlinesweredrawninthepressureandtemperaturerangesinvestigated.Figure4.15andFigure4.16illustratetheequi-libriumlinesat313Kand20MPa,respectively.Thedatawererepresentedasthemassfractionofmethyloleate(CO2freebasis).

AsshowninFigure4.15andFigure4.16,eitherapressureincreaseoratempera-turedecreasemovestheequilibriumlineclosertothediagonalline.Infractionationdesigning, this tendencymeans thatan increase in theoretical stages isaccompa-niedbyadecreaseinprocessselectivity.Suchatendencyagreeswellwiththatofillustratedbytheseparationfactor.

0.4

0.3

0.2

S

0.1

0.1 0.2 0.3 0.4 0.5 0.6 0.7x2

0

Figure 4.14 Influenceoftemperatureontheseparationfactor(S)atP=20MPa:x20,the

initialtocopherolmassfractioninfeed;⦁,experimentaldataatT=313.15K;◾,experimentaldataatT=333.15K;▲,experimentaldataatT=353.15K;—,correlatedatT=313.15K;---,correlatedatT=333.15K; ····,correlatedatT=353.15K. (FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)

7089_C004.indd 123 10/8/07 11:50:51 AM


4.6.5 Phase behaVior of me-DoD

Duringthemeasurementofphaseequilibrium,therealisticsystemofME-DODwastakenasthe10.19%(tocopherolmassfraction)feedcomposition.Also,theME-DODsystemwastherawmaterialsforconcentratingnaturaltocopherols;thus,itsphasebehavior is discussed separately. Figure4.17 shows the influence of pressure andtemperatureontheseparationfactor.

1.0

0.8

0.6

0.4

0.2

0.00.0 0.2 0.4

x1

y 1

0.6 0.8 1.0

Figure 4.16 EquilibriumlinesatP=20MPa:x1’,massfractionofmethyloleateinliquid

(CO2freebasis);y1’,massfractionofmethyloleateingas(CO2freebasis);⦁,experimental

dataatT=353.15K;◾,experimentaldataatT=333.15K;▲,experimentaldataT=313.15K;—,correlatedatT=353.15K;---,correlatedatT=333.15K;····,correlatedatT=313.15K.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)

1.0

0.8

0.6

0.4

0.2

0.00.0 0.2 0.4

x1

y 1

0.6 0.8 1.0

Figure 4.15 EquilibriumlinesatT=313.15K:x1’,massfractionofmethyloleateinliquid

(CO2freebasis);y1’,massfractionofmethyloleateingas(CO2freebasis);⦁,experimental

dataatP =10MPa;◾,experimentaldataatP =20MPa;▲,experimentaldataP =29MPa;—,correlatedatP =10MPa;---,correlatedatP =20MPa;····,correlatedatP =29MPa.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)

7089_C004.indd 124 10/8/07 11:50:52 AM


The trends illustrated in Figure4.17 are similar to those in Figure4.13 andFigure4.14.Noticeably,theseparationfactorsatpressureslowerthan20MPaarerelativelysmall.Forinstance,at313.15K,theseparationfactorremainedlowerthan0.2forallpressureslowerthan15MPa.Aspressureincreases,theseparationfactorgreatlyincreases,reaching0.35at20MPa.Theincreaseoftemperatureoffsetstheeffectofpressuretosomeextents.Onthebasisoftheproperty,aseparationstrategyseems tobe reasonable and feasible.A fractionation column is necessary for theME-DODliquidsystem.Firstofall,lowpressure(15to20MPa)wasusedincombi-nationwithatemperaturedistributioninthecolumntoseparateFAMEslikemethyloleate.Thenthepressurewasincreasedtoseparatetocopherolfromotherimpurities.Thisprocedureneedstobeverifiedbyafractionationoperationinwhichoperationparameterscanbedeterminedandoptimized.

Duringourexperiments,somephenomenaoftherealisticsystemofME-DOD+CO2wereobservedthroughthevisualequilibriumcell.Figure4.18showstheliquid-gas interfacechanges thatoccuraspressure increases.The interface increasinglychangesfromcleartoobscure,withtheinterfacefinallydisappearingathighpres-sureabout29MPa.Athighpressure,acriticalpointprobablyexiststhatcausesthewholesystemtobecomeentirelymiscible.Inthissituation,theliquidandgascom-positionsare identicalandtheseparationfactorequalsunity.Itshouldbenoticedthat the disappearance of the interface does not happen suddenly. The change isaprogressiveprocessandanaccurateendpoint isdifficult todeterminebyvisualobservation. The critical pressure at 313.15 K is approximately estimated in thepressurerangefrom27.8to29.0MPabyvisualobservation.

Figure4.19showsthechangesinthefeedsituationwhenME-DODwaschargedbypumpintotheequilibriumcellatdifferentpressuresandataconstant5mL/min.Atlowpressure(5MPa),ME-DODcouldsmoothlyflowintotheequilibriumcell,butathighpressures,thechargedME-DODresemblesdropsorfog.Moreovertherateforflowingdownwardatahighpressurewasslowerthanthatatlowpressure.

0.4

0.3

0.2

0.1

10 15 20 25p/MPa

S

Figure 4.17 Separationfactor(S)ofME-DODinsupercriticalCO2:⦁,experimentaldataatT=313.15K;◾, experimental data atT=333.15K;▲, experimental dataT=353.15K.(From Fang,T., Goto, M., Sasaki, M. and Hirose, T., J. Chem. Eng. Data, 50, 390, 2004.Withpermission.)

7089_C004.indd 125 10/8/07 11:50:53 AM


WehypothesizedthatthemainreasonforthiswasthesmallerdifferenceindensitybetweenME-DODandsupercriticalCO2athighpressure;forexample,at20MPaand313.15K,thedensityofCO2is0.840g/mLandthatofME-DODis0.865g/mL.Suchasmalldifferenceindensityislikelytocausethedroporfogphenomenon,eventhoughtheclearinterfacebetweenliquidandgasremained.Thisphenomenonshouldbeconsideredwhendesigningacontinuouslycountercurrentoperation.

4.7 separation with superCritiCal Co2 FraCtionation

Asdescribedinsection4.1,theimportantstepinconcentratingnaturaltocopherolsfrom ME-DOD is to remove the FAMEs, which contribute more than 70% ofME-DOD. FAMEs are important chemical materials in biofuel, metal-cuttingoil,andcleaningagentproduction,aswellas in thesynthesisofotherfattyacidproducts [17]. In section 4.6, the fundamental research on ternary and realisticphaseequilibriahasestablishedapreliminaryseparationstrategy,whichmustbetestedthroughasupercriticalCO2fractionationoperation.

AfractionationcolumnisnecessaryfortheME-DODliquidsystem.First,lowpressure (the initialpressure) isused in combinationwitha temperaturegradientalongthecolumntoseparatetheFAMEs.Then,thepressureisincreasedtoseparatethetocopherolsfromotherimpurities.

20 MPa 25 MPa 27 MPa 29 MPa

Figure 4.18 The interface between liquid and gas at T = 313.15 K. (From Fang, T.,GotoM.,Sasaki,M.,Hirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)

P = 5 MPa P = 20 MPa P = 25 MPa

Figure 4.19 FeedsituationofME-DODatdifferentpressures(feedrate=5mL/minandT =313.15K).(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)

7089_C004.indd 126 10/8/07 11:50:55 AM


4.7.1 fraCtionation aPParatUs anD ProCeDUre

A fractionation system was rebuilt from a supercritical CO2 apparatus [43]. Theexperimentalsetupconsistedofacountercurrentcontactcolumn(2.4m×20mmi.d.,750mL)andaseparator(600mL)forthetopproduct,asshowninFigure4.20.Thecolumnwaspackedwithstainlesssteel3mmDixonPacking(NaniwaSpecialWireNettingCo.,Ltd.,Tokyo)overalengthof1.8m.Theseparationexperimentwascarriedoutinsemicontinuouscountercurrentoperationwithamaximumfeedof200g.

Before each run, about 120 g ME-DOD was charged into the column at40±1 g/h so that an abundance of raw material had accumulated at the columnbottom.Consequently, thefreshCO2fluidcouldcomeintocontactwithsufficientME-DODatthestartofthefractionationoperation.Thisensuresthateachexperi-mentbeganatasteady-statecondition,whichmeansboththefeedandtopfractionflow in a continuous situation with relatively stable flow rates. Fresh CO2 waschargedintothecolumnthroughvalve14(V14), thepressuresofthecolumnandseparatorwereadjustedbyBPR1andBPR2,respectively.Thetemperaturegradientofthecolumnwasconcurrentlyadjustedbyeightproportionalintegraldifferentialcontrollers.Theseparatorconditionsweremaintainedat3.8to4.0MPaand333K.Whenpressureandtemperaturereachedtherequiredvalues,CO2wasintroducedfromthecolumnbottombyopeningV15andsimultaneouslyclosingV14,indicatingthestartingpointof thefractionationoperation.Duringcontinuousoperation, the

V1 V2

V3V4

V5

V6

BPR1V7

V8P1

V15

V10

V11

V12

V13

V14

MV1

V17

BPR2

Window

Windows

V9

Feed(Continuous)

P2

Windows

Gas Meter 1

V16 Raffinate

Dixon Packing

Extract

Extraction Column Heater

CoolerCO2CO2

Figure 4.20 Schematic diagram of fractionation apparatus. (From Fang, T., Goto, M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)

7089_C004.indd 127 10/8/07 11:50:56 AM


taB

le 4

.2r

esul

t of

the

sca

le-u

p pr

etre

atm

ent

proc

ess

Mat

eria

l and

pr

oduc

tsM

ass

(kg)

FFa

(%

)aFa

Mes

(%

)b

gly

ceri

des

(%)a

toco

pher

ols

(%)c

ster

ols

(%)b

Con

tent

rec

over

yC

onte

ntr

ecov

ery

Con

tent

rec

over

y

DO

D35

3.6

48.1

027

.210

09.

2310

09.

4510

0

PME

-DO

D*

351.

51.

253

.58

22.3

81.5

9.19

99.0

9.51

100.

2

Cru

des

tero

ls47

.6/

//

//

/54

.777

.9

ME

-DO

D31

0.8

0.7

71.2

86.

119

.710

.19

97.0

2.7

25.2

* :“P

artly

met

hyle

ster

ified

DO

D,”

whi

chis

obt

aine

dby

met

hyle

ster

ifica

tion

a :D

eter

min

eda

ndc

alcu

late

dby

the

AO

CS

met

hods

[18

]b :

D

eter

min

eda

ndc

alcu

late

dby

GC

ana

lysi

s[2

1]c :

Det

erm

ined

and

cal

cula

ted

byH

PLC

ana

lysi

s[2

1]

7089_C004.indd 128 10/8/07 11:50:57 AM


ME-DODfeedlocationwaschangedbyswitchingon/offV11,V12,andV13.Addi-tionally,toachievedifferentsolvent-to-feed(S/F)ratios,theflowrateofME-DODwasmaintainedataconstant40±1g/hwhile theflowrateofCO2wasvariedbyadjustingmicrometeringvalve1(MV1).Thetotalmaximumfeedusedforeachrunwasabout200g,includingtheinitialfeedof120gbeforefractionationoperation.Intheintervalbetweenconsecutiveruns,theresidualmaterialswereremovedoutofthecolumnbyopeningthebottomvalve(V16)andreleasingthecolumnpressure.

4.7.2 Pretreatment resUlt anD ComPosition of me-DoD

Beforethefractionationexperiment,ME-DODwaspreparedfromDODaccordingtotheprocedureshowninFigure4.2.Table4.2liststheresultofthescale-upexperi-mentwith350KgDOD.Firstofall,ourpretreatmentmethoddoesnotcauseobviousdamagetotocopherolsasthetocopherols’recoveryinME-DODwas97%andsimul-taneouslyabout74.8%ofsterolswereremoved.Noticeably,thetotalsterols’recoveryin crude sterols and ME-DOD was larger than 100% because some sterols werereleasedfromsterolestersduringpretreatment.Additionally,aftermethylesterifica-tion,mostFFAwasconvertedintoFAMEs,whiletheconversionrateofglycerideswasonly18.5%,indicatingthatmethylesterificationisnotenoughtosimplifythecomplicatedsystemofDODandmethanolysisisnecessaryforconvertingglyceridesintoFAMEs.Finally,asforthetwoproducts,ME-DODandcrudesterols,theirtotalamountswerealittlelargerthanthatofinitialfeedbecausealittlewaterwasmixedintotheME-DODafterthestepofwashing.

ThecompositionofME-DODanalyzedwithGC-MSisshowninFigure4.21andTable4.3.Atotalof18compoundswereidentified.Thecompositionwas83.16%FAMEs,3.55%squalene,11.18%tocopherols,and2.11%sterols.ThemainFAMEswere methyl palmitate (14.57%), methyl linoleate (39.23%), and methyl oleate

Retention Time (min)

Abu

ndan

ce

5.00 10.00

11.26

9.62

11.1710.40 16.21

16.93

16.06

15.87TIC : ME-DOD.D

28.54

21.17

26.21

29.4029.13

31.4730.51

27.4423.24

15.00 20.00 25.00 30.00

Figure 4.21 GC-MS TIC chromatography of ME-DOD. (From Fang, T., Goto, M.,Wang,X.,Ding,X.,Geng, J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)

7089_C004.indd 129 10/8/07 11:50:58 AM


(21.16%),andthethreecompoundsmadeup90.14%ofallFAMEs.Becausesomecompoundswithhighmolecularweight,suchasglycerides,sterolesters,pigments,andwax,couldnotbeidentifiedwiththecurrentanalyses,theareapercentagesofcompoundswerenotaccurateandcouldnotbeusedforquantification.Thus,HPLCandGC-FIDwereemployedfordeterminingthecontentsoftocopherols,sterols,andFAMEs,respectively.Table4.3alsoshowstheanalysisdataobtainedbyHPLCand

taBle 4.3Composition of Me-dod

Composition determined by gC-Ms

peak no.rt

(min)area (%) Compounds trivial name of FaMe

1 9.62 1.08 Dodecanoicacid,methylester Methyllaurate(C12:0)

2 10.40 0.72 Tetradecanoicacid,methylester Methylmyristate(C14:0)

3 11.18 0.40 Unidentified /

4 11.26 14.23 Hexadecanoicacid,methylester Methylpalmate(C16:0)

5 15.87 39.23 9,12-octadecadienoicacid,methylester Methyllinoleate(C18:2)

6 16.06 21.16 9-octadecenoicacid,methylester Methyloleate(C18:1)

7 16.21 1.67 7-octadecenoicacid,methylester Methyloleate(C18:1)

8 16.93 3.73 Octadecanoicacid,methylester Methylstearic(C18:0)

9 21.17 0.37 13-docosenoicacid,methylester Methylbrassidate(C22:1)

10 23.25 0.57 Docosanoicacid,methylester Methylbehenate(C22:0)

11 26.21 3.55 Squalene

12 27.44 2.28 δ-tocopherol

13 28.54 6.98 γ-tocopherol

14 29.13 0.74 β-tocopherol

15 29.40 1.18 α-tocopherol

16 30.51 0.69 Campesterol

17 30.82 0.33 Stigmasterol

18 31.47 1.09 β-sitosterol

tocopherols (%) determined by hplC and isomers’ percentage

Tocopherols(%) α-Tocopherol β and γ-Tocopherols δ-Tocopherol

10.19 12.05 61.57 26.38

sterols (%) determined by gC-Fid and isomers’ percentage

Sterols(%) Campesterol Stigmasterol β-Sitosterol

2.71 32.11 23.18 44.71

FaMes (%) determined by gC-Fid and Main FaMes’ percentage

FAMEs C16:0 C18:2 C18:1

71.28 17.66 45.52 28.28

Source: Fang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.

7089_C004.indd 130 10/8/07 11:50:59 AM


GC-FID, inwhich thecontentsof tocopherols,FAMEs,andsterolswere10.19%,71.28%, and 2.71%, respectively. Noticeably, most FFA and glycerides were con-vertedintoFAMEsandmoststerolsinDODwereremovedthroughthepretreatmentprocess.Asaresult,tocopherolswerepartlyconcentratedinME-DOD,similartotheresultsreportedbyLeeetal.[10].

Fromtheaboveanalysis,ME-DODcontainsFAMEs(about70%),whichmustberemovedinthefirststepattheinitialpressure.

4.7.3 effeCt of the initial PressUre

For the greatest degree of tocopherol enrichment inside the column, the initialpressure was investigated so that most of the FAMEs were extracted with littletocopherolcontent.Inthefirst2hours,about80gME-DODwaschargedintothecolumn and the operation was in continuous countercurrent fractionation mode,whereCO2wasthecontinuousphaseandthefeedoilwasthedispersedphase.Afterfeeding,theoperationwaschangedtobatchfractionationmode.Theextractedfrac-tionswerecollectedintheseparator.Every30minutesor1hour,thefractionswereremoved from the separator and weighed until the total yield from the separatorreachedabout70wt.%(140g)ofthetotalfeed(200g).Atthatpoint,theexperimentwasterminated.

Thephaseequilibriumdatainsection4.6.4indicatedthattheseparationfactorbetween FAMEs and tocopherols change markedly from 15 to 20 MPa. Conse-quently,pressuresof14,16,and18MPawereinvestigatedfor theseexperiments,whileotheroperationparameterswerekeptatsimilarvaluesthroughout.Thecol-umntemperaturegradientwassetinalineardistributionfrom313Katthebottomto348Katthetop,theS/Fratiowasadjustedto75(theflowratesofCO2andME-DODwere3±0.05Kg/hand40±1g/h,respectively),andthefeedlocationwasV13.

Figure4.22showsthattheextractionyieldofFAMEswasgreatlyinfluencedbytheinitialpressures,ashigherpressureledtohighersolubilityandfasterextraction.Forinstance,theextractionyieldat18MPareachedmorethan70%in2.5hourswithabout7.5KgCO2,whileat14MPa,ittookfarmoretime(10hours)andmoreCO2(30Kg)fortheextractionyieldtoreachthesamelevel.Ontheotherhand,higherpressureresultedinmoretocopherolsextractedtogetherwithFAMEs,withthehigh-esttocopherolcontentbeing3.2%at18MPa,whichwasaboutthreetimesofthatat14MPa.Thistrendagreedwiththecommonrulethatsolubilitygenerallyincreaseswithpressure.However,highpressurealsoresulted indecreaseof theselectivity,whichisadisadvantagefortheseparation[6,10].

Fordetaileddiscussion,averagetocopherols’content(ATC)ofallfractionsandthe averageoil loading (AOL)wereused for evaluating the separation efficiency,withtheformerstandingforthepurityoftheFAMEproductandthelatterrepre-sentingtheprocessvelocity.AOLisdefinedastheratiobetweenthetotaloilmasscollectedandtheCO2consumedoveragiventime.Practically,AOLcanbecalcu-latedfromtheslopesoftheyieldcurvesshowninFigure4.22,andtheresultsarelistedinTable4.4.Aspressureincreased,AOLincreasedfrom0.48g/100gCO2at14MPato1.97g/100gCO2at18MPa.Similarly,ATCalsoincreased,indicatingthatmoretocopherolsweresolvatedwiththeFAMEsinthesupercriticalCO2.This

7089_C004.indd 131 10/8/07 11:50:59 AM


influencedthequalityoftheFAMEproductandledtoalossoftocopherolsenrichedinthefollowingprocess.Anotherphenomenonobservedwasthattheproportionoftocopherolisomersinthefractionswasgreatlyinfluencedbytheinitialpressure.Thetocopherolsatlowpressures(14and16MPa)weremainlyα-tocopherol,whereasthosepresentat18MPawerecomposedofthefourisomersinproportionssimilartothatoftherawmaterial.Hence,16MPawasselectedastheinitialpressureforseparatingFAMEs.

4.7.4 effeCt of the final PressUre

Whenthetotalyieldreachedabout70%,itmeantthatmostFAMEswereseparatedfromnaturaltocopherols,whichenrichedinsidethecolumn.Thenthesecondstepwascommenced,whereintocopherolswereconcentratedbyincreasingthecolumn

4 8 12 16 20 24 28 32 36

0

15

30

45

60

75

Consumption of CO2 (kg)

Extr

actio

n Yi

eld

of F

AM

Es (%

)

0

5

10

15

20

25

Toco

pher

ols’

Cont

ent (

%)

Figure 4.22 EffectoftheinitialpressureontheseparationofFAMEs.ExtrationyieldofFAMEs:⦁,18MPa;◾,16MPa;▲,14MPa;Tocopherols%:•,18MPa;◽,16MPa; ,14MPa.(Otherparameters:thecolumntemperaturegradientof313to348K,S/Fratio=75,thefeedlocationatV13). (FromFang,T.,Goto,M.,Wang,X.,Ding,X.,Geng, J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)

taBle 4.4effect of the initial pressure on the separation of FaMes

P (Mpa)

total Feed (g)

total yield (%)

aol (g/100 g Co2) atC (%)

proportion of tocopherol isomers

α β+γ δ

14 200 71.97 0.48 0.19 97.98 2.02 N.D.*

16 200 73.34 1.22 0.21 94.11 5.89 N.D.

18 200 73.82 1.97 2.11 16.61 71.56 11.83*: N.D.meansnotdetermined.Source: Fang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical

Fluids, 40,50, 2007.Withpermission.

7089_C004.indd 132 10/8/07 11:51:00 AM


pressuretoahighervalue(thefinalpressure).Theexperimentwasterminatedwhenthetotalyieldreachedabout85%to90%feed,becauseitwasfoundtobedifficulttoobtainmorethan90%yieldoffeedintheexperimentalrangeandtheextractionvelocitywaspracticallyveryslow(<5g/30min).Thetotalandtocopherolyieldswerecalculatedfromthemassflowandtocopherolcontentinthefractions.Wemainlyinvestigatedthe influenceof thefinalpressureonATCandtocopherols’recovery(TR).Figure4.23showsthefractionchangeasafunctionoftimeatdifferentfinalpressures,andFigure4.24illustratestheeffectofpressuremodesontotalyieldandTR.Inthefirst4hours,therewasnodistinctchangeinthefirststepofpressureto16MPa.Themassflowwasabout15to20g/30min(Figure4.23)and,after4hours,theyieldreachedabout70wt.%ofthefeed(Figure4.24).Evidently,themassflowdecreasedtolessthan10g/30min.Inaddition,tocopherolyieldat16MPawasonly3%to5%,whichisthatfavoredconcentrationoftocopherolsinthesubsequentstepathigherpressure. In thesecondstep,athigherpressure, themassflowandtotalyieldincreasedwithanincreaseinthefinalpressure,butthechangeintocopherolcontentat22MPawasnotasprecipitousasthatat20or18MPa(Figure4.23).Thisindicatedthat,whileahigherfinalpressureleadstoanincreaseinthetotalsolubil-ity,itsimultaneouslyresultsinadecreaseinselectivity.TheATCofallfractionsat22MPawasonly44.1%,althoughthecorrespondingTRwas82.8%.Both18MPaand20MParesulted inATCsgreater than50%,but theTRat18MPawasonly46.8%andlowerthan81.3%at20MPa(Figure4.24).Moreover,18MParesultedinalongerfractionationprocessbecauseoflowsolubility;forinstance,theextractedfractionwasalwayslessthan5g/30minafter7hours,andtheexperimentat18MPawasterminatedat9hoursbecauseoftooslowextraction.Therefore,20MPawas

1 2 3 4 5 6 7 8 90

10

20

30

40

50

Time (hr)

Mas

s Flo

w (g

)

0

20

40

60

80

Toco

pher

ols (

%)

Figure 4.23 Effect of the final pressure on mass flow and tocopherols’ content. Massflow:▲,18MPa;⦁,20MPa;◾,22MPa;Tocopherols%: ,18MPa;•,20MPa;◽,22MPa.(Other parameters: the initial pressure = 16 MPa for 4 hours, S/F ratio = 75, the columntemperaturegradientof313to348K,thefeedlocationatV12).(FromFang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)

7089_C004.indd 133 10/8/07 11:51:01 AM


selectedastheoptimalfinalpressureespecially,at5.5hours,afractionwithpurityof80.5%wasobtained,whichwasthehighestcontentobtainedinourstudy.

4.7.5 ComPosition of toCoPherol ConCentrate

Accordingtothefractionationoperation,itprovedtobefeasibleforconcentratingnaturaltocopherolsfromME-DODwithsupercriticalCO2fractionation.Withtheoptimizedpressureparameters,about29.1ghaving57.1%tocopherolconcentrate(allfractionsat20MPa)wereobtainedfrom200gME-DOD.GC-MSwasusedtoanalyzethecompositionintheconcentrate.TheresultisshowninFigure4.25.

The area percentage of tocopherols was 68.8%, higher than the 57.1% deter-minedbyHPLC.Themainreasonwasthatthereweresomeimpuritieswithhighmolecularweight that couldnotbe identifiedwith the currentGC-MScondition.Additionally, among the determinable compounds, FAMEs (C16:0, C18:2, C18:1,C18:0),squalene,andsterolswerethemainimpurities,withtheareapercentagesof15.4%,5.8%,and10%,respectively.

4.7.6 VisCosity ComParison

Inthiswork,DODandME-DODwereusedasrawmaterialandobtaineddiffer-entfractionsbysupercriticalCO2fractionation.Besidesthecompositiondifference,thesematerials aredifferent inphysicalproperties. Inparticular, theirviscositiesattractedourinterestsinceviscosityisaveryimportantphysicalpropertycommonlyused in engineering design. For example,when designing the feeding system forME-DOD,itsviscositydatahelpdeterminewhetherpreheatingisnecessary.

1 2 3 4 5 6 7 8 9

0

20

40

60

80

Time (hr)

Tota

l Yie

ld (%

)

0

20

40

60

80

100

Toco

pher

ols’

Reco

very

%

Figure 4.24 Effectof thefinalpressureon totalyieldandtocopherols’recovery.Totalyield:▲, 18MPa;⦁, 20MPa;◾, 22MPa;Tocopherols’ recovery: , 18MPa;•, 20MPa;◽,22MPa.(Otherparameters:theinitialpressure=16MPafor4hours,S/Fratio=75,thecolumn temperature gradient of 313 to 348K, the feed location atV12). (From Fang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)

7089_C004.indd 134 10/8/07 11:51:03 AM


Figure4.26 illustrates theviscositychangesofDOD,ME-DOD,andwaterasa functionof temperature.When the temperaturewasvaried from293 to303K,theviscositiesof the three samplesdecreasedquicklyand then thedecrease ten-dencybecamerelativelystableandslowathighertemperatures.Suchcharacteris-ticsindicatedthatthethreesamplesweretypicallypseudoplasticfluids.Inaddition,theviscosityofME-DOD is far smaller than thatofDODand similar to thatofwater.Thus,thepretreatmentprocessshowninFigure4.2leadstotwoadvantageousresults for continuous fractionation process. One is that the converted ME-DODhaslargersolubilityinsupercriticalCO2thanDOD;theotheristhattheviscosity

Retention Time (min)

TIC: TOCOD.D

11.24

15.8316.03

16.93 21.1723.24

23.47

26.21

27.46

28.60

29.4131.49

33.1630.53

30.8529.14

28.4028.20

5.00 10.00 15.00 20.00 25.00 30.00

Abu

ndan

ce

Figure 4.25 GC-MS TIC chromatography of 57.1% tocopherols. (From Fang, T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)

290 295 300 305 310 315 320 325 330 3350.000

0.004

0.008

0.012

0.1

0.2

0.3

0.4

0.5

Temperature (K)

Visc

osity

(Pa.s

)

Figure 4.26 ComparisonbetweentheviscositiesofDOD,ME-DOD,andH2O.◾,DOD;▲,ME-DOD;•,H2O.(FromFang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)

7089_C004.indd 135 10/8/07 11:51:05 AM


isgreatlyreducedafterconvertingmoreviscouscompounds(FFAandglycerides)intolessviscousFAMEs.Particularlyafterpretreatment,theviscosityofME-DODisfurtherdecreasedbyremovingmostofthesterols,whichpracticallyactasakindofthickeningmaterial.

Figure4.27 shows a comparison of viscosities among the fractions obtained.Theviscosityorderwasasraffinate>57.1%tocopherols>FAMEs.Here,raffinatewastheresidualmaterialatthebottomofthecolumnafterthefractionationopera-tionanditappearedasaverystickyliquid.

4.7.7 aPPliCation in CommerCial ProDUCtion

Onthebasisofthewholeresearch,anindustrialapplicationwascarriedoutinpastyears.Scale-upexperimentswithan18Lcolumnweredoneandtheresultswerereportedinliterature[44].Inaddition,aworkshop(Figure4.28)andacommercial-scale fractionation system of 350 L × 2 (Figure4.29) were established in KaidiFineChemical IndustrialCo.Ltd. (Wuhan,HubeiProvince,P.R.China)and thetechnologywasindustrializedwithanannualprocesscapacityof750tME-DODintheyearof2000.Accordingtoourwork,threepatentswereappliedandfinallyreleasedforpublication[45–47].

4.8 ConClusions

As the first step of the whole research, binary phase equilibrium data of methyloleate + CO2 and α-tocopherol + CO2 were measured and correlated with theSoave-Redlich-Kwong EOS and Adachi-Sugie mixing rule. According to theobtaineddata,thesolubilityanddistributioncoefficientwerecalculated.Compared

290 295 300 305 310 315 320 325 330 3350.000

0.004

0.5

1.0

1.5

Temperature (K)

Visc

osity

(Pa.s

)

Figure 4.27 Comparisonbetweentheviscositiesofthefractionsobtainedatdifferentpressures.◾,Raffinateat20MPa;▲,57.1%tocopherols(fractionat20MPa);•,FAMEs(fraction16MPa). (FromFang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)

7089_C004.indd 136 10/8/07 11:51:06 AM


Figure 4.28 Tocopherol concentration workshop established in Wuhan (Kaidi FineChemicalIndustrialCo.,Ltd.,Hubei,P.R.China).

Figure 4.29 Supercritical CO2 fractionation system (350 L × 2) for concentratingnaturaltocopherols.

7089_C004.indd 137 10/8/07 11:51:07 AM


withmethyloleate,thesolubilityanddistributioncoefficientofα-tocopherolweregenerallyonetotwoordersofmagnitudelower.SuchalargedifferencebetweenthetwocomponentsindicatesthatitispossibletoconcentratenaturaltocopherolsfromME-DODintheexperimentalrangeinvestigated.

Second,ternaryphaseequilibriumdataofmethyloleate+tocopherol+CO2weremeasuredandcorrelated.Onthebasisoftheexperimentaldataandcorrelationresults,the separation factor and equilibrium line were investigated. The discussion indi-catedthatlowerpressuresandhighertemperaturesleadtoahigherselectivity.Also,highercontentofmethyloleateinthefeedimprovesthedistributionoftocopherolsingas,resultingindecreasedselectivity.Morenoticeably,theexperimentaldataontherealisticsystemofME-DOD+CO2ledtotheformationofaseparationstrategy.

Finally,supercriticalCO2fractionationwasemployedtoconcentratetocopherolsfromME-DOD.TheinitialpressurewasinvestigatedforseparatingFAMEs.Forthefollowingtocopherolconcentrationstep,afinalpressureof20MParesultedinrela-tivelyhighaveragetocopherolcontent(>50%)andtocopherolrecovery(about80%).On the basis of the fundamental and separation research, an application in com-mercialproductionwasalsoconductedinthepastyears.Accordingtotheobtainedresults,itcanbeconcludedthatsupercriticalCO2fractionationistechnicallyfeasibleforconcentratingnaturaltocopherolsfrommethylesterifiedDOD.

reFerenCes

1. Brunner,G.,Gas Extraction: An Introduction to Fundamentals of Supercritical Fluids and the Application to Separation Processes,Darmstadt:Sternkopff,Springer,NewYork,1994.

2. Ito,V.M.,Martins,P.F.,Batistella,C.B.,MacielFilho,R.andWolf-Macie,M.R.,Naturalcompounds obtained through centrifugal molecular distillation, Appl. Biochem. & Biotech.,129–132,716,2006.

3. Martins,P.F.,Batistella,C.B.,MacielFilho,R. andWolf-Macie,M.R.,Comparisonof two different strategies for tocopherols enrichment using amolecular distillationprocess,Ind. & Eng. Chem. Res.,45,753,2006.

4. Sumner,C.E.,Jr.,Barnicki,S.D.andDolfi,M.D.,Processfortheproductionofsterolandtocopherolconcentrates,U.S.PatentUS5424457,A199506131995,1995.

5. Mau,J.andTsen,H.,Investigationontheconditionsforthepreparationofhigh-purityvitaminEconcentratefromsoybeanoildeodorizerdistillate,J. Chin. Agri. Chem. Soc. (Taipei),33,686,1995.

6. Lucas,A.,Martinez,E.O.,RincónJ.,Blanco,M.A.andGracia,I.,Supercriticalfluidextractionoftocopherolconcentratesfromolivetreeleaves,J. Supercrit. Fluids,22,221,2002.

7. Zhao,Y.,Sheng,G.andWang,D.,Pilot-scaleisolationoftocopherolsandphytosterolsfromsoybeansludgeinapackedcolumnusingsupercriticalcarbondioxide,inProc. 5th Int. Symp. on Supercritical Fluids,Atlanta,Georgia,USA,April8–12,2000.

8. Zhou,Q.,Sheng,G., Jiang,H. andWu,M.,Concentrationof tocopherolsby super-criticalcarbondioxidewithcosolvents,Eur. Food Res. Tech.,219,398,2004.

9. Nagesha, G.K., Manohar, B. and Udaya Sankar, K., Enrichment of tocopherols inmodifiedsoydeodorizerdistillateusingsupercriticalcarbondioxideextraction,Eur. Food Res. Tech.,217,427,2003.

10. Lee,H.,Chung,B.H.andPark,Y.H.,Concentrationoftocopherolsfromsoybeansludgebysupercriticalcarbondioxide,J. Am. Oil Chem. Soc.,68,571,1991.

7089_C004.indd 138 10/8/07 11:51:07 AM


11. Shishikura, A., Fujimoto, K., Kaneda, T., Arai, K. and Saito, S., Concentration oftocopherolsfromsoybeansludgebysupercriticalfluidextraction, J. Jpn. Oil Chem. Soc.,37,8,1988.

12. Liu,Y.,Fang,T.andDing,X.,PhaseequilibriumforsupercriticalCO2andthemethylesterified product form soybean oil deodorizer distillate, J. Food Lipids, 13, 390,2006.

13. Mendes,M.F.,Pessoa,F.L.P.andUller,A.M.C.,AneconomicevaluationbasedonanexperimentalstudyofthevitaminEconcentrationpresentindeodorizerdistillateofsoybeanoilusingsupercriticalCO2,J. Supercrit. Fluids,23,257,2002.

14. King,J.W.,Favati,F.andTaylor,S.L.,Productionoftocopherolconcentratesbysuper-criticalfluidextractionandchromatography, Sep. Sci. Tech.,31,1843,1996.

15. Chang,C.J.,Chang,Y.F.,Lee,H.,Lin,J.andYang,P.W.,Supercriticalcarbondioxideextraction of high-value substances from soybean oil deodorizer distillate, in Proc. 5th Int. Symp. on Supercritical Fluids,Atlanta,Georgia,USA,April8–12,2000.

16. Brunner, G., Malchow, T., Stürken, K. and Gottschau, T., Separation of tocopher-ols from deodorizer condensates by countercurrent extraction with carbon dioxide,J. Supercrit. Fluids,4,72,1991.

17. Swern,S.,Bailey’s Industrial Oils and Fats, JohnWiley&Sons,NewYork,1986. 18. AmericanOilChemists’Society,OfficialMethodsandRecommendedPracticesofthe

AmericanOilChemists’Society,17thed.,2000,A.O.C.S.,Washington,DC. 19. Fang,T.,Goto,M.,Yun,Z.,Ding,X.andHirose,T.,Phaseequilibriaforbinarysystems

ofmethyloleate-supercriticalCO2 andα-tocopherol-supercriticalCO2,J. Supercrit. Fluids,30,1,2004.

20. Fang,T.,Goto,M.,Sasaki,M.andHirose,T.,Phaseequilibriafortheternarysystemmethyloleate+tocopherol+supercriticalCO2,J. Chem. Eng. Data,50,390,2004.

21. Fang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,Separationofnaturaltocopherolsfromsoybeanoilbyproductwithsupercriticalcarbondioxide,J. Supercrit. Fluids, 40,50, 2007.

22. Soave,G.,Equilibriumconstants fromamodifiedRedlich-Kwongequationofstate,Chem. Eng. Sci.,27,1197,1972.

23. Adachi,Y.andSugie,H.,Anewmixingrule-modifiedconventionalmixingrule,Fluid Phase Equilibrium,28,103,1986.

24. Weber,W.,Petkov,S.andBrunner,G.,Vapour-liquidequilibriaandcalculationsusingtheRedlich–Kwong-Aspenequationofstatefortristearin,tripalmitin,andtrioleininCO2andpropane, Fluid Phase Equilibrium, 158–160,695,1999.

25. Cheng,H.,Zollweg,J.A.andStreett,W.,Experimentalmeasurementofsupercriticalfluid–liquidphaseequilibrium,InSupercritical Fluid Science and Technology, ACS Symposium Series 406,Johnston,K.P.andPenninger,J.M.L.,Eds.,AmericanChemicalSociety,Washington,DC,1989,86.

26. Inomata,H.,Kondo,T.,Hirohama,S.,Arai,K.,Suzuki,Y.andKonno,M.,Vapour-liquidequilibria forbinarymixturesofcarbondioxideandfattyacidmethylesters,Fluid Phase Equilibrium,46,41,1989.

27. Zou,M.,Yu,Z.R.,Kashulines,P.,Rizvi,S.S.H.andZollweg,L.A.,Fluid-liquidphaseequilibriaof fattyacidsandfattyacidmethylesters insupercriticalcarbondioxide, J. Supercrit. Fluids,3,23,1990.

28. Nilsson, W.B., Seaborn, G.T. and Hudson, J.K., Partition coefficients for fatty acidestersinsupercriticalfluidCO2withandwithoutethanol,J. Am. Oil Chem. Soc.,69,305,1992.

29. Yu,Z-R.,Rizvi,S.S.H.andZollweg,J.A.,Phaseequilibriaofoleicacid,methyloleate,andanhydrousmilk fat in supercritical carbondioxide,J. Supercrit. Fluids, 5, 114,1992.

7089_C004.indd 139 10/8/07 11:51:07 AM


30. Crampon,C.,Charbit,G.andNeau,E.,High-pressureapparatusforphaseequilibriastudies:SolubilitiesoffattyacidestersinsupercriticalCO2, J. Supercrit. Fluids,16,11,1999.

31. Chrastil,J.,Solubilityofsolidsandliquidsinsupercriticalgases,J. Phys. Chem.,86,3016,1982.

32. Oghaki,K.,Tsukahara,I.,Semba,K.andKatayama,T.,Afundamentalstudyoftheextraction with a supercritical fluid. Solubilities ofα-tocopherol, palmitic acid, andtripalmitin incompressedcarbondioxideat25°Cand40°C,Int. J. Chem. Eng.,29,303,1989.

33. Pereira,P.J.,Goncalves,M.,Coto,B.,deAzevedo,E.G.anddaPonte,M.N.,PhaseequilibriaofCO2+DL-α-tocopherolattemperaturesfrom292to333Kandpressuresupto26MPa,Fluid Phase Equilibrium,91,133,1993.

34. Meier,U.,Gross,F. andTrepp,C.,Highpressurephase equilibrium studies for thecarbondioxide/α-tocopherol (vitaminE) system,Fluid Phase Equilibrium,92,289,1994.

35. Johannsen,M.andBrunner,G.,Solubilitiesoffat-solublevitaminsA,D,EandKinsupercriticalcarbondioxide,J. Chem. Eng. Data,42,106,1997.

36. Chen,C.C.,Chang,C.M.J.andYang,P.W.,Vapor–liquidequilibriaofcarbondioxidewith linoleic acid, α-tocopherol, and triolein at elevated pressures, Fluid Phase Equilibrium,175,107,2000.

37. Skerget, M., Kotnik, P. and Knez, Z., Phase equilibria in systems containingα-tocopherolanddensegas,J. Supercrit. Fluids,26,181,2003.

38. Brunner,G.,Malchow,T.,Stuerken,K.andGottschau,T.,Separationoftocopherolsfrom deodorizer condensates by countercurrent extraction with carbon dioxide,J. Supercrit. Fluids,4,72,1991.

39. Stoldt, J.,Saure,C.andBrunner,G.,Phaseequilibriaof fatcompoundswithsuper-criticalcarbondioxide,Fluid Phase Equilibrium,116,399,1996.

40. Stoldt,J.andBrunner,G.,Phaseequilibriummeasurementsincomplexsystemsoffats,fatcompounds,andsupercriticalcarbondioxide,Fluid Phase Equilibrium,146,269,1998.

41. Araujo, M. E., Machado, N. T. and Meireles, M.A., Modeling the phase equilib-rium of soybean oil deodorizer distillates + supercritical carbon dioxide using thePeng-RobinsonEOS,Ind. Eng. Chem. Res.,40,1239,2001.

42. Bamberger,T.,Erickson,J.C.,Cooney,C.L.andKumar,S.K.,Measurementandmodelpredictionofsolubilitiesofpurefattyacids,puretriglycerides,andmixturesoftriglycer-idesinsupercriticalcarbondioxide.J. Chem. Eng. Data,33,327,1988.

43. Sato,M.,Kondo,M.,Goto,M.,Kodama, A. andHirose,T.,Fractionationof citrusoilbysupercriticalcountercurrentextractorwithside-streamwithdrawal, J. Supercrit. Fluids,13,311,1998.

44. Fang,T.,Goto,M.,Liu,Q.,Ding,X.andHirose,T.,Countercurrentextractionforthefractionation of natural tocopherols with supercritical CO2, in Proc. of the 6th Int. Symp. on Supercritical Fluids,Tome1,425,Versailles,France,April28–30,2003.

45. Wang,X.,Geng,J.,Liu,C.,Ding,X.andFang,T.,ProcessforfractionallyextractingnaturalvitaminEbysupercriticalCO2fluid,ChinaPatentCN1369487A,2002.

46. Wang,X.,Fang,T.,He,F.,Liu,C.,Liu,S.,Cao,Y.andWu,X.,ProcessforextractingvitaminEfromplant-oildeodorizerdistillates,ChinaPatentCN1418877A,2003.

47. Wang,X.,Geng,J.,Liu,C.,Ding,X.,andFang,T.,Anovelcolumnusedforthecon-tinuously countercurrent operation of supercritical CO2 fractionation, China PatentCN2561484Y,2003.

7089_C004.indd 140 10/8/07 11:51:08 AM

141

5 Processing of Fish Oils by Supercritical Fluids

Wayne Eltringham and Owen Catchpole

Contents

5.1 Introduction................................................................................................. 1415.2 FishOilComponents:Sources,Properties,andCommercialUses............ 142

5.2.1 Omega-3FattyAcids:EicosapentaenoicAcidandDocosahexaenoicAcid..................................................................... 142

5.2.2 SqualeneandDiacylGlycerylEthers.............................................. 1445.2.3 VitaminA(Retinol)......................................................................... 1465.2.4 WaxEsters....................................................................................... 146

5.3 Separation/FractionationTechnologies....................................................... 1475.3.1 TraditionalProcessingMethods...................................................... 148

5.3.1.1 Distillation.......................................................................... 1495.3.1.2 Low-TemperatureCrystallization....................................... 1515.3.1.3 UreaCrystallization........................................................... 1525.3.1.4 ChromatographicMethods................................................. 1565.3.1.5 EnzymaticTransformation................................................. 156

5.3.2 SupercriticalFluidProcessingofFishOils..................................... 1585.3.2.1 PhaseEquilibria:SupercriticalCO2andFishOil

Components........................................................................ 1585.3.2.2 PolyunsaturatedFattyAcidProcessing.............................. 1685.3.2.3 SqualeneandDAGEProcessing........................................ 1765.3.2.4 VitaminAProcessing........................................................ 1785.3.2.5 ProcessingofOtherMarineOilComponents.................... 181

5.4 Summary..................................................................................................... 181References.............................................................................................................. 181

5.1 IntroduCtIon

Extractionandfractionationoffishoilshasbecomeamajorareaofresearchoverthelast30yearsbecauseofthepotentialapplicationoftheseextractsandfractionsin thepharmaceutical,nutraceutical,andcosmetic industries.Themajorconstitu-entsoffishoilsaretriacylglycerides(TAGs).Minorcomponentsincludefreefattyacids, squalene, tocopherols, cholesterol, wax esters, sterol esters, phospholipids,diacylglycerides,diacylglycerolethers,pigments,andvitaminsA,D,andE.Oilsofmarineoriginaresignificantlymorecomplexthanthosefromplantsandterrestrialanimals.ThefattyacidsthatconstituteTAGsinfishoilsvaryconsiderablyaccording

7089_C005.indd 141 10/8/07 11:53:46 AM


to degree of unsaturation, variety of chain length, and number of isomeric com-pounds.TAGsaretypicallymadeupofstraight-chainfattyacidscontainingfrom12to24ormorecarbons,withthedegreeofunsaturationvaryingfromzerotosixdoublebonds.Fishoilsoftencontainmorethan60differentfattyacids,includingiso-mers,whichdifferaccordingtothepositionofunsaturationwithinthecarbonchain.Thecomplexcompositionoffishoilsmakesthemdifficulttoprocesstoconcentratespecificfattyacids.Highlevelsofunsaturationmaketheuseofhigh-temperatureprocessingmethodsproblematicbecauseofthesusceptibilityofthesecompoundstooxidativeandthermaldegradation.Thevariability infattyacidcompositionoffishoilishighlydependentonfishspecies,season,feedinghabits,partofthefishused(e.g.,liverorflesh),andcatchlocation.SomefattyacidprofilesofselectedfishoilsareshowninTable5.1[1],whichshowsthatliveroilcompositionisusuallylesssaturatedthanthatoftheflesh.

This chapter discusses several important fish oil constituents, includingtheir physical properties and applications in thenutritional, pharmaceutical, andcosmeticindustries.Moreover,thechapterisintendedtogiveanoverviewofthevariouslaboratory-to-productionscaletechniquesthatcanbeusedtoextractandfractionatevariousfishoil components.Acomparisonof various extraction andfractionationtechniquesisdiscussed,includinghowsomeoftheproblemsassoci-ated with “traditional” processing methods can be overcome using supercriticalfluid(SCF)technologies.

5.2 FIsh oIl Components: sourCes, propertIes, and CommerCIal uses

5.2.1 Omega-3 Fatty acids: eicOsapentaenOic acid and dOcOsahexaenOic acid

Productionofomega-3(ω3)fattyacidconcentratescontinuestobeatopicofinter-estforboththepharmaceuticalandhealthfoodindustries.Sincetheearlystudieson long-chainω3-polyunsaturated fatty acids (PUFAs) by Burr and Burr [2], thehealthbenefitsofthesecompoundshavebeenstudiedextensively.Withthegrowingpublicawarenessofthehealthbenefitsofω3-PUFAs,themarketforsuchproductsis expected to grow, increasing the demand for efficient production and isolationmethods.Themostwidelystudiedω3fattyacidsareeicosapentaenoicacid(EPA)anddocosahexaenoicacid(DHA)(Figure5.1).Extensiveclinicalfindingsontheireffectsonhumanphysiologyandtheiruseinthepreventionandtreatmentofdiseasessuchasarteriosclerosis[3,4],thrombosis[5],arthritis[6],andseveraltypesofcancer[7,8]havebeenreported.

Omega-3fattyacidsofmarineorigincanreduceserumTAGlevels[9].ThisisofparticularimportancebecauseraisedlevelsofserumTAGsareconsideredtobeariskfactorforcoronaryheartdisease[10].Urakazeetal.[5]showedthatintravenousinjectionsofanEPA-containingemulsioncanincreasetheEPAcontentinplasmaandplateletphospholipids.TheirstudyshowedthatplateletaggregationissignificantlydepressedandthatEPA-containingemulsionsmaybeusefulforpatientsrequiringpreventativecareofthrombosis.Othermedicalstudiesreportusingω3fattyacids

7089_C005.indd 142 10/8/07 11:53:47 AM

Processing of Fish Oils by Supercritical Fluids 143

for the reductionofbloodpressure [11,12].AstudybyKremerandcoworkers [6]reported theeffectofEPA intake inpatients suffering from rheumatoidarthritis.Aftera12-weekdiethigh inpolyunsaturatedfat, lowinsaturatedfat,andwithadailysupplementofEPA(1.8g),patientsnotedadecreaseinmorningstiffnessandjointpain.Otherworkershavealsoreportedanti-inflammatoryactivityofω3fattyacids [13].Researchers at thePaterson Institute (Manchester,UK)have identifiedamechanismbywhichω3fattyacidsmayprevent thedevelopmentofmetastatic

table 5.1Fatty acid profiles of selected Fish oils

salt Water oils

Freshwater oils

atl

anti

c C

od

atl

anti

c C

od l

iver

spin

y d

ogfi

sh

spin

y d

ogfi

sh l

iver

paci

fic

hal

ibut

paci

fic

her

ring

mac

kere

l

men

hade

n

stri

ped

mul

let

pink

sal

mon

lake

her

ring

rai

nbow

tro

ut

Fatty acid Weight (%) of total Fatty acids

14:0 1.8 2.8 2.0 1.6 2.8 7.6 4.9 8.0 4.6 3.4 5.5 2.1

15:0 0.5 0.4 0.5 0.3 0.3 0.4 0.5 0.5 6.3 1.0 0.4 0.8

16:0 33.4 10.7 21.2 13.2 15.1 18.3 28.2 28.9 17.3 10.2 17.7 11.9

16:1 2.4 6.9 6.0 5.7 8.9 8.3 5.3 7.9 11.0 5.0 7.1 8.2

16:2 0.6 1.0 0.9 1.0 0.8 1.0 0.7 0.8 3.8 1.7 0.7 1.2

17:0 0.9 1.2 1.2 1.0 0.7 0.5 1.0 1.0 0.8 1.6 0.6 1.5

18:0 4.0 3.7 2.7 4.3 3.4 2.2 3.9 4.0 5.0 4.4 3.0 4.1

18:1 11.9 23.9 27.5 28.5 25.7 16.9 19.3 13.4 8.4 17.6 18.1 19.8

18:2ω6 1.2 1.5 1.3 0.7 0.9 1.6 1.1 1.1 3.2 1.6 4.3 4.6

18:3 0.8 0.9 0.6 0.6 0.3 0.6 1.3 0.9 .4 1.1 3.4 5.2

18:4ω3 1.2 2.6 0.7 0.8 3.6 2.8 3.4 1.9 3.0 2.9 1.8 1.5

19:0 0.6 0.7 0.9 1.5 1.6 0.8 0.9

20:1 1.6 8.8 5.8 1.5 8.0 9.4 3.1 0.9 0.7 4.0 1.2 3.0

20:4ω6 3.2 1.0 2.5 0.8 2.5 0.4 3.9 1.2 2.6 0.7 3.4 2.2

20:5ω3(EPA) 12.4 8.0 7.9 3.7 10.1 8.6 7.1 10.2 7.5 13.5 5.9 5.0

22:1 0.7 5.3 4.1 10.3 5.1 11.6 2.8 1.7 0.7 3.5 2.8 1.3

22:6ω3(DHA) 21.9 14.3 10.4 6.5 7.9 7.6 10.8 12.8 13.4 18.9 13.3 19.0

Totalsaturated 40.6 19.4 27.6 21.1 22.3 29.0 38.5 43.3 35.5 22.2 28.0 21.3

Totalmonounsaturated 16.6 44.9 43.4 46.0 47.7 46.2 30.5 23.9 20.8 30.1 29.2 32.3

Totalpolyunsaturated 41.3 29.3 24.3 14.1 26.1 22.6 28.3 28.9 33.9 40.4 32.8 38.7

Totalω3 34.3 22.3 18.3 10.2 18.0 16.2 17.9 23.0 20.9 32.4 19.2 24.0

Source: AdaptedwithpermissionfromJournal of the American Oil Chemists’ Society,41,662.©1964AmericanOilChemists’Society.

7089_C005.indd 143 10/8/07 11:53:48 AM


prostate cancer [14]. They showed that theω6 polyunsaturated fatty acid arachi-donicacid(20:4ω6)isapotentstimulatorofmalignantepithelialcellularinvasion,which can increase the risk of prostate cancer development. They stated that theobservedcellularinvasionisinhibitedbyEPAandDHAintheratiosofω31:2ω6.SeveralotherstudieshavealsofocusedontheanticarcinogenicpropertiesofEPA[15–17].Omega-3fattyacids,especiallyEPA,alsoshowpromiseinthetreatmentofneuropsychiatricdisorders,suchasdepression[18],schizophrenia[19],andanorexianervosa[20].

BothEPAandDHAoccurnaturallyinthebody,wheretheyhavebeenshowntobeimportantinmembranestructureandfunction[21].Theyarefoundinespeciallylargeamountsinbraincells,eyes,nerves,andadrenalglands.Inparticular,DHAisoneofthemostabundantconstituentsofbrainstructurallipids,whereithasimpor-tanteffectsonmembraneorder(fluidity),theactivityofmembrane-boundenzymes,andsignaltransduction.DHAisconsideredtobeessentialforthevisualandneuro-logicaldevelopmentofinfants.Thelipidfractionofhumanmother’smilkcontainsDHA-to-EPAratiosof4:1,withDHAcontentbeing30timesmorethantheamountofDHAobservedincows’milklipid.IntheU.S.,80%ofinfantformulascontainDHAsothatchildrenreceivethisnutrientduringtheimportantphasesofbrainandnervoussystemgrowthanddevelopment.

5.2.2 squalene and diacyl glyceryl ethers

Thenaturaloccurrenceofsqualenewasfirstreportedin1906byMitsumaruTsujimoto,anindustrialengineerwhopioneeredthechemistryoffatsandoilsinJapan.Thirtyyearslater,NobellaureatePaulKarrerdescribedthechemicalstructureofsqualeneforthefirsttime.Squaleneisa30-carbonisoprenoid(Figure5.2)thatisusedcom-mercially as an additive in pharmaceutical preparations, cosmetics, sunscreens,dyes,lubricants,andhealthfoods[22,23].Duringthe1950s,squalenewasfoundtooccurnaturallyinthehumanbody[24].Humansebum,anaturalproductexpressedby sebaceous glands, contains around 12% squalene. Sebum helps keep the skinsuppleandhelpstomaintainskinmoisturelevels.Squalenewaslaterfoundtobe

OH

O

OH

OEicosapentaenoic Acid (EPA), 20:5 ω-3

Docosahexaenoic Acid (DHA), 22:6 ω-3

FIgure 5.1 ThechemicalstructuresofEPAandDHA.

FIgure 5.2 Thechemicalstructureofsqualene(C30H50).

7089_C005.indd 144 10/8/07 11:53:49 AM


theprecursortocholesterol[25],whichinturnistheprecursortoarangeofsteroidsandvitaminD.

GloorandKarenfeld[26]foundthatthebody’sconsumptionofsqualeneincreaseswhenskinisexposedtoultraviolet(UV)radiation.Kohnoandcoworkers[27]showedthatthefirstmoleculetargetedbyUVradiationintheskinwassqualene.TheseresultsledtomoreintenseresearchontheinteractionofsqualenewithUVradiationand,in1995,Kohnoetal.[28]demonstratedthatsqualenecanpreventUV-inducedoxidationoflipidsintheskin.Radioprotectiveanddetoxifyingeffectsofsqualenehavealsobeenreported[29].Dietarysqualenemayhavethepotentialtolowerbloodcholesterollevels[30,31].Anumberofresearchpaperssuggestthatsqualeneshowspotentialasananticarcinogen[32–34].Foramorethoroughtreatiseonthepropertiesofsqualene,itsactioninmetabolicpathways,anditspossiblepreventativecapabilitiesforhumandisease,thereaderisdirectedtoaworkbyDas[35].

Thereportsonthebeneficialeffectsofsqualeneonhumanhealthhaveledtoacommercialdemandforthislong-chainunsaturatedhydrocarbon.Althoughsqualeneisnaturallyfoundinsmallquantitiesinoliveoilandby-productsfromtherefiningofoliveoil,wheatgermoil,ricebranoil,andyeast,themostabundantsourcebyfaristheliversofdeep-seasharks.Othermajorcomponentsfoundintheliversofdeep-seasharksincludediacylglycerylethers(DAGEs)andTAGs.Becausesharksdonotpossessswimbladders,thepresenceoflargequantitiesoflow-densityoils(squalenedensity0.86gcm–3;DAGEdensity0.89gcm–3)intheirliversallowsthemtoachieveandmaintainbuoyancy.Table5.2showssometypicalcompositionsofsharkliveroilforseveralspeciesofshark[36,37].DAGEsareconsideredtobeefficientinwoundhealingapplicationsandinpreventingthemultiplicationofbacteria.Someresearch-ershavealso suggested thatDAGEsmayaid in the reductionofcertain typesofcancer,promoteformationofbloodcells,andprovideprotectionagainstradiation

table 5.2shark liver oil Compositions for some selected species of shark

shark species Common name squalene dages tags otherd ref.

Carcharhinus plumbeusa Sandbarshark 0.0 0.0 83.0 17.0 36

Centrophorus scalpratusb Endeavourshark 81.6 9.9 8.5 0.0 37

Centrophorus squamosusc Leafscalegulpershark 70 11 18 1.0 36

Centroscymnus crepidaterb Long-nosevelvetshark 73.0 20.0 5.0 2.0 37

Centroscymnus plunketib Plunketshark 0.9 76.6 22.5 0.0 37

Dalatias lichac Kitefinshark 79 18 2 1.0 36

Deania calceab Platypusshark 69.6 1.6 10.8 18.0 37

Etmopterus granulosusb Lanternshark 50.3 32.1 9.3 8.3 37

Hexanchus griseusa Bluntnosesixgillshark 1.0 70.0 29.0 0.0 36

Somniosus pacificusb Pacificsleepershark — 49.5 49.1 1.4 37

Squalus acanthiasc Pikeddogfish 0.0 12.0 87.0 1.0 36a Hawaiianwaters,bSouthernAustralianwaters, cChathamRise,NewZealand, dIncludingfreefatty

acids(FFA),phospholipids,sterols,pristane,waxesters,andsterolesters.

7089_C005.indd 145 10/8/07 11:53:50 AM


injury[38].SharkliveroilswithdefinedlevelsofDAGEsandsqualenearecurrentlysoldasnutraceuticals.

5.2.3 Vitamin a (retinOl)

Vitamin A, a fat-soluble compound, is derived in the bodies of animals fromβ-carotene(alsoknownasprovitamin A).VitaminA,carriedinthebloodbyretinol-bindingprotein,hasbeenidentifiedasanessentialnutrientnecessaryforgrowth,reproduction,andvision.Inthebody,itisoxidizedtoretinal,akeycomponentofthevisualsystem,andtoretinoicacid,whicheffectsgeneexpressionviaspecificnuclearreceptors. The discovery of an anticarcinogenic action of vitamin A by Saffiottoetal.[39]ledtorapidgrowthinvitaminAresearch.Inanimalexperiments,vitaminAanditsmetabolite,retinoicacid,wereshowntohaveanticancerproperties.Retinoicacidisnowaningredientusedinanti-ageingcosmetics.VitaminA–richcreamsareusedforseverecasesofacne[40].Forreviewsdescribingthediscovery,structureelucidation,androlesofvitaminAinthehumandiet,thereaderisdirectedtothepublicationsofWolf[41]andUnderwood[42].

TheimportanceofvitaminAinhumanhealthhasleadtoarequirementfordietarysupplementsintheformofcapsulesandfoodfortification.Theliversofcartilaginousfish,suchassharksandrays,areapotentiallyvaluablesourceofvitaminA.VitaminAisfoundinfishliveroilsalmostentirely(96–100%)asesters[43],suchasvitaminApalmitate(Figure5.3).

5.2.4 Wax esters

Oilsofcertaindeep-seaspeciesoffisharecomposedalmostentirelyofwaxesters,which are esters of long-chain fatty acids and fatty alcohols. Fish species in theSouthPacificwhoseoilsarecomposedofwaxestersincludeorangeroughy,blackoreo,andsmall-spinedoreo (Table5.3) [44].The fattyacidportionsof theestershavecarbonchainlengthsofC14–C24andareeithersaturatedormonounsaturated.ThefattyalcoholshavecarbonchainlengthsofC16–C24andaremostlysaturated(Table5.4)[44].Waxesterswithunsaturationneartheesterbondaremorevolatilethanwaxesterswithunsaturationnear the centerof the aliphatic chainbuthavesimilaroxidativestability[45].Inteleostfish(thosewithabonyskeletalstructure),theoccurrenceofwaxestersinmuscletissuecorrelatesbetterwithseadepthandverticalmigrationpatternsthanwithtaxonomy[46].

Infish,waxestersappeartofunctionassourcesofenergy,insulation,andaidstobuoyancyandbiosonar[47].Inindustry,theyhaveapplicationsasspeciallubri-cants,cosmeceuticals,andchemicalrawmaterialsforthemanufactureofsoapsand

O

O CH2(CH2)13CH3

FIgure 5.3 ThechemicalstructureofvitaminApalmitate.

7089_C005.indd 146 10/8/07 11:53:51 AM


detergents[48].Oilfromtheheadof thespermwhale,whichcontainsmorethan65% wax esters (Table5.3), is particularly suitable for these applications. Owingtothepresentstatusofthespermwhaleasanendangeredspecies,however,theoilisno longeran itemofcommerceandalternativesourceshavebeen investigated.Astudyofthechemicalandphysicalpropertiesoforangeroughyoilshowedthatitcouldreadilyreplacespermwhaleoil[44].TheproductionoforangeroughyoilhasbeencommercializedinNewZealand,withseveralplantsproducingthousandsoftonnesperyear[49].

5.3 separatIon/FraCtIonatIon teChnologIes

Therearemanymethodsforthefractionationoffishoils,butonlyafewaresuitableforlarge-scaleproduction.Thesuitablemethods,whichusuallyrequireconversionofTAGstofattyacidsorethylesters,includeadsorptionchromatography,fractionalormoleculardistillation,enzymaticsplitting,low-temperaturecrystallization,ureacomplexation and SCF extraction/fractionation techniques. Standard oil refiningtechnologiesarenotconsideredhere.

table 5.3total lipid Composition and Composition of the Wax ester Fraction of orange roughy, black oreo, small spined oreo, and sperm Whale

orange roughy black oreosmall spined

oreo sperm Whale

Component Weight (%) of oil

Waxesters 94.9 91.5 95.6 65.8

TAGs 3.1 4.8 2.5 30.1

Cholesterol/alcohols 1.0 2.7 1.5 4.0

Phospholipids 1.0 1.0 0.4 0.1

total Carbon number Weight (%) of Wax ester Component

C26 4.7

C28 14.0

C30 0.2 0.5 0.5 21.1

C32 2.1 3.5 2.9 23.2

C34 11.4 11.6 9.3 19.9

C36 16.7 21.8 18.3 11.7

C38 24.8 21.3 26.2 4.4

C40 23.4 19.8 25.4

C42 14.8 10.8 12.8

C44 5.5 6.1 4.3

C46 1.1 4.6 0.3

Source: Adapted with permission from Journal of the American Oil Chemists’ Society, 59, 390.©1982AmericanOilChemists’Society.

7089_C005.indd 147 10/8/07 11:53:51 AM


5.3.1 traditiOnal prOcessing methOds

Separationoffishoilfattyacidsandestersusingtraditionalmethodsiscomplicatedbyseveralfactors.First,methodsrelyingondifferencesinmolecularweight,suchasdistillation,arehinderedbytherelativelysmallmolecularweightdifferencesinthese compounds, especially when attempting to separate saturated and unsatu-ratedfattyacidsofthesamechainlength.Second,PUFAsarereadilysusceptibletodegradation,oxidation,polymerization,andstereomutation,evenatmoderatelyelevated temperatures. Berdeaux et al. [50] reported the thermal degradation ofPUFAs during the deodorization of fish oils. The high processing temperatures

table 5.4percentages of Fatty acids and Fatty alcohols of the Whole Fish Wax esters of orange roughy, black oreo, small spined oreo, and sperm Whale

orange roughy

% Fatty

black oreo

% Fatty

small spined oreo

% Fatty

sperm Whale

% Fatty

Component acid alcohol acid alcohol acid alcohol acid alcohol

saturated

<14:0 21.6

14:0 1.2 4.1 1.9 6.8 9.4 8.0

15:0 <0.1 0.8 <0.1 0.7 0.9 1.4

16:0 1.0 7.3 15.5 20.8 8.1 9.4 5.1 39.5

17:0 0.7 1.1 0.8 3.8 0.4 1.1

18:0 0.3 8.1 3.2 2.3 3.7 0.9 1.5 7.7

19:0 0.6 0.2

20:0 <0.1 0.2 <0.1 <0.1

22:0 <0.1 <0.1 <0.1 <0.1

24:0 <0.1 <0.1 <0.1 <0.1

Unsaturated

14:1 0.5 0.3 0.4 19.6

15:1 <0.1 0.7 0.1 0.2 0.2

16:1 11.8 7.9 10.9 15.6 4.1

17:1 1.0 0.8 0.8 3.7 1.3 1.0

18:1 56.0 34.6 26.9 19.0 32.8 23.3 17.8 35.4

18:2 1.9 1.0 0.9 0.5

20:1 17.8 30.6 15.8 29.6 16.5 33.7 3.9 1.4

22:1 7.8 13.8 11.6 20.3 9.5 31.6 1.4

23:1 <0.1 1.8 2.1 0.8

24:1 <0.1 5.4 8.3 3.9 <0.1 1.1 <0.1


7089_C005.indd 148 10/8/07 11:53:52 AM


associatedwithvacuumdistillationcanbeproblematicbecauseω3fattyacidsaresusceptibletooxidativedeteriorationduetotheirhighdegreeofunsaturation.Theprimaryoxidationproducts, lipidhydroperoxides,areespeciallyunstableandcandegrade to yield volatile secondary oxidation products. The secondary oxidationproductscanimpartundesirable,unpleasantfishyodorsandflavorstoendproducts.ThemixtureofTAGsinfishoilsistoocomplexforefficientisolationofindividualcomponentsandoftenonlymodestenrichmentscanbeexpectedfromfractionation.Therefore,mosteffortshavebeendirectedtowardthefractionationofacidsandtheirmethyl/ethylesters.Dealingwiththesesingle-chaincompoundsallowsdifferencesinchainlengthordegreeofunsaturationtobeeffectivelyaddressed.TheacidsandestershavegreatervolatilitythanTAGs,allowingtheuseoftemperature-controlledseparationmethods,suchasmoleculardistillation.Afterseparation,specificTAGsorfreeacidscanbereconstituted.Mostseparationmethods,whenusedalone,canonlyseparatefishoilacidsintogroupfractions.Therefore,twoormoreproceduresareoftenrequiredtoproduceindividualcomponentsinhighpurity.

5.3.1.1 distillation

Distillation relies on differences in mixture component vapor pressures, whicharestronglyrelatedtomolecularweightsforahomologousfamilyofcompounds.Enrichment is achieved by exploiting the differences in vapor pressure throughcountercurrentcontactingofvaporandliquidphasesinstagesusingplatesorcontin-uouslyusingrandomorstructuredpacking.Ifweassumeabinary(orpseudobinary)mixturecontainingcomponentsAandB,thenthedegreeofattainableenrichmentisdependentontheratioof theindividualcomponentvaporpressures,pAandpB.Assuming A is the more volatile component, the separation factor,α = pA/pB, isgreaterthanunityandsomedegreeofseparationcanbeachieved.

Figure5.4showsanidealizedvapor-liquidcompositiondiagramforamixtureofAandBthatisbeingseparatedinastage-wisedistillationcolumn.Atthefeedpointofthedistillationprocess,theconcentrationofAintheliquidphaseisX0andthatinthevaporphaseisY0.Thevapor-liquidequilibriumcurvepredictsthattheinitialconcentrationofAinthevaporphaseisgreaterthanthatintheliquidphasebyvirtueofitsgreatervolatility.Ifthevaporiscondensed(condensationrepresentedbythehorizontallinesdrawnbetweenthevapor-liquidequilibriumcurveandtheauxiliarylineinthestrippingorenrichmentsectionofthecolumn),theresultingliquidwillhaveaconcentrationofAequaltoY0/KA =X1,whereKAisthepartitioncoefficient(K-value)ofcomponentA.TheK-valueofanycomponentiisdefinedas:

K

y

xii

i

= (5.1)

whereyiistheconcentrationofcomponentiinthevaporphaseandxiistheconcen-trationofcomponentiintheliquidphase.

Similarly,thevaporinequilibriumwiththeliquidphaseofconcentrationX1hasaconcentrationofAequaltoY1.Again,theconcentrationofAinthevaporphaseisenhancedduetoitsgreatervolatility.Ifthisvaporisthencondensed,theresulting

7089_C005.indd 149 10/8/07 11:53:53 AM


liquidhasamolepercentconcentrationofAaccordingtothesamerelationship—thatis,Y1/KA = X2.Similarrelationshipsholdintherectificationsection,wherethe“heavy”componentBisconcentrated.Whenthesestepsarecarriedoutcontinuouslyinamultistageorcontinuouslypackeddevice,itistheoreticallypossibletoobtainhighlypureAas theextractandhighlypureBas theraffinate.Alternatively, theevaporationandsubsequentcondensationstepscanbecarriedoutasabatch-wisefractionaldistillationprocessuntilthedesireddegreeofenrichmentwithrespecttoAhasbeenachieved.

Stoutetal. [51]highlighted thepracticaldifficultyofobtainingω3-PUFAsinhighconcentrations in thenaturalTAGform.Moleculardistillationofmenhadenoil in itsnaturalTAGformincreased theEPAcontent in theresiduefrom16.0%to19.5%and theDHAcontent from8.4% to17.3%.Carryingout thedistillationusingthemenhadenoilethylesters,however,almostdoubledtheEPAconcentrationfrom15.9%to28.4%,whereastheconcentrationofDHAshowedanalmostfivefoldincrease,from9%to43.9%.

Fractionaldistillationoffishoils ispreferentiallycarriedoutusing fattyacidestersunder reducedpressure (0.1 to10 torr) since these,unlike free fatty acids,approximateideality.Also,thegreatervolatilityoftheestersallowsseparationtobecarriedoutat lower temperatures,which is importantconsidering the thermalinstabilityoftheω3components.Boilingpointsofunsaturatedestersaremarginallylowerthanthoseofsaturatedestersofthesamechainlength.Therefore,foragivenchainlength,unsaturatedestersareenrichedduringtheearlystagesoffractionation

Raffinate

X0 X1 X2

Y2

Y1

Y0

Feed

0 20 40 60 80 100

100

80

60

40

20

0

Extract

Rectificat

ion Section

Stripping S

ection

FIgure 5.4 SeparationofahypotheticalmixtureofcomponentsAandBbydistillation.

7089_C005.indd 150 10/8/07 11:53:54 AM


andsaturatedonesareenrichedintheendfractions.Duringthetransitionfromonechainlengthtothenext,thedistillatecontainsamixtureofsaturatedlowerchainlengthandunsaturatedhigherchainlengthcomponents.

Althoughthegreatervolatilityof thefattyacidestersallowstheuseof lowerprocess temperatures (compared with temperatures required for free fatty acids),temperatures are still moderately high (typically 423 to 473 K), and exposure todistillation conditions over a prolonged period of time can be detrimental to thepolyunsaturatedconstituents,causinghydrolysis,polymerization,isomerization,andthermaloxidation[52].Privettandcoworkers[53,54]foundconsiderabledecompo-sitionofarachidonateduringslowdistillationinaspinningbandcolumn.Fraction-ationofmarineoilesterscontainingchainlengthsofC20ormoreisdifficultbecauseseparationfactorsdecreasewithincreasingmolecularweight[55].

5.3.1.2 low-temperature Crystallization

Crystallization separationsarebasedon thedifferences incompositionof equili-brated liquid and solid phases. The process can be carried out using the crudeliquidoilor inasolventsolution.Anoperationwith thecrude liquidoil requiresslowcoolingandslowagitation.Thisproducesaslurryofsolidandliquidcompo-nents, the latterbeingenrichedwithPUFAs.Whenusinga solution, theequilib-rium isdependentoncomponent solubilities.Commonsolventsofchoice includemethanol,acetone,petroleumether,acetonitrile,nitromethane,andliquidpropane.Theformationofsolidcrystalsinevitablyresultsinentrapmentofsomeliquid,andsoseparationfactorsarenothigh.Thesolubilityoffatsinorganicsolventsincreaseswithincreasingunsaturationanddecreaseswithincreasingmolecularweight[56].Singleton[57]andStoutetal.[51]havemeasuredthesolubilityofseveralfattyacidsinavarietyofsolvents.Theirfindingsledtothefollowingrules:

Long-chainsaturatedfattyacidsarelesssolublethanshort-chainsaturatedfattyacids.Saturatedacidsare less soluble thanmonounsaturatedanddiunsaturatedacidsofthesamechainlength.Transisomersarelesssolublethancisisomers.Straight-chainacidsarelesssolublethanbranched-chainones.Freefattyacidsarealwayslesssolublethantheirmethylestercounterparts.

Themeltingpointsof fattyacidsvaryconsiderablyaccording to theirdegreeofunsaturation.Thiswidevariationinmeltingpointcanbeexploitedtoenabletheseparationofsaturatedandunsaturatedfattyacidcompounds[58].Atlowtempera-tures,long-chainsaturatedfattyacids(havinghighermeltingpoints)crystallizeout,leavingthePUFAsinsolution.Thefreeacidsarepronetoassociationinsolution.Although this can be overcome by processing the methyl ester analogues, thepracticaladvantageislimitedbecauseofthegreatersolubilityoftheesters.Also,theformationofeutecticspreventsachievementofthedegreeoffractionationonewouldpredictfromsolubilitydataalone.Thechoiceofsolventandprocessingtemperaturemustbeconsideredcarefullybecausethesefactorscanhaveapronouncedeffecton

•

•

•••

7089_C005.indd 151 10/8/07 11:53:55 AM


theconcentrationofPUFAsobtained.Thereisalsothepossibilityofseparatingfattyacidsonthebasisofdegreeofunsaturation[59].DHA(orsaltsthereof)crystallizesat a lower temperature than EPA in solvents such as acetone. However, the verylowtemperaturesrequiredmaketheprocesssomewhatimpractical[60]. Until theintroductionofSCFfractionation techniques, low-temperaturecrystallizationwasconsideredtobethemethodleastdetrimentaltopolyunsaturatedfattyacids.

5.3.1.3 urea Crystallization

Ureacomplexationwithstraight-chaincompoundswasfirstreportedbyBengen[61].Whilepureureacrystallizesinatightlypackedtetragonalstructure(Figure5.5a),in the presence of long straight-chain molecules, it crystallizes into a hexagonalstructuretoformaninclusioncomplex(Figure5.5b)[62].Theureacomplexiscon-structedofaspiralarrangementofureamolecules,andthestraight-chainmoleculesare held in the hexagonal channels by van der Waals forces, London dispersionforces, or induced electrostatic interactions [63]. The hexagonal channel is wideenough to accept molecules with a diameter of around 5 Å, but molecules withgreaterdiametersarenoteasilyaccommodated.Theformationandstabilityofureacomplexesisthereforegovernedbyshape,size,andgeometry.

Theuseofureacrystallizationseparatesfattyacidsmainlyaccordingtotheirdegreeofunsaturation.Whenureacrystallizesfromasolutioncontainingamixtureof fatty acids, the saturated and monounsaturated fatty acids are preferentiallyincluded in thecomplexwhile thepolyunsaturatedfattyacidsremain insolution.Thisisbecausethepresenceofdoublebondsinfattyacidspreventsthechainfromorientingintothe“ideal”geometryforcomplexformation.Thiscausesanimbalanceof theoptimum intermolecular distances,whichdisrupts thenet attractive forcesanddestabilizestheinclusioncomplex.Thestabilityofureainclusioncomplexesislessenedbyshorterchainlengthsandahighernumberofdoublebonds[64].Transisomersformmorestablecomplexesthanthecorrespondingcisisomers,andcom-pounds with conjugated double bonds form more stable adducts than those withmethylene interrupted or isolated double bonds. The most influential processingvariablesaffectingtheextentofcrystallizationaretheurea–fattyacidratiosandtheprocesstemperature.Theurea–fattyacidratiocanbeusedtofractionatefattyacidsaccording to differing degrees of unsaturation. When low concentrations of urea

(b) Palmitic Acid (C16:0)/Urea Inclusion Complex(a) Pure Urea

FIgure 5.5 Crystalstructuresforpureureaandurea–fattyacidcomplexes.

7089_C005.indd 152 10/8/07 11:53:56 AM


areused,thedifferentfattyacidscompeteforcomplexformationaccordingtotheabilitytoformthemoststableinclusioncompound.

RoblesMedinaetal.[65]studiedtheeffectofurea–fattyacidratioonthefattyacid composition of urea filtrates (noncomplexed fatty acids) from cod liver oil.Table5.5showstheirresultsfromureacrystallizationat277KandFigure5.6showstheeffectoftheseratiosonvariousfattyacidgroupsofinterest.Withaurea–fattyacid ratio of 1:1, the saturated fatty acids were partially removed from solution,while the concentration of monounsaturates remained constant. Increasing theratioto2:1facilitatedmaximumremovalofthesaturatedacidsalongwithpartialremovalofthemonounsaturatedacids.Ataratioof3:1,theremovalofsaturatedand

table 5.5the effect of urea/Fatty acid ratio on the noncomplexed Fatty acid Composition resulting from urea Fractionation of Cod liver oil at 277 K

Fatty acid Cod liver oil

urea/Fatty acid ratio

1:1 2:1 3:1 4:1

14:0 4.2 2.7 0.7 0.5 0.7

16:0 10.6 2.0 0.2 0.5 0.0

16:1ω7 7.8 9.6 6.9 2.5 3.2

18:0 2.6 0.9 0.1 0.0 0.0

18:1ω9 17.0 17.6 3.2 2.9 0.7

18:1ω7 4.6 5.9 1.4 1.0 0.0

18:2ω6 1.5 2.0 1.6 0.7 0.7

18:3ω6 0.2 0.2 0.4 0.5 0.5

18:3ω3 0.8 1.1 1.0 0.6 0.6

18:4ω3(SA)a 2.4 3.3 6.3 8.0 8.5

20:0 0.2 0.2 0.2 0.0 0.1

20:1ω9 10.8 9.0 1.3 0.6 0.8

20:3ω6 0.1 0.1 0.1 0.2 0.2

20:4ω6 0.5 0.8 1.0 0.9 1.0

20:5ω3(EPA) 9.4 13.0 22.6 24.8 25.6

22:0 0.1 0.0 0.0 0.0 0.0

22:1ω11 8.3 3.8 0.4 0.0 0.0

22:1ω9 0.1 0.0 0.0 0.0 0.0

22:4ω6 0.5 0.7 1.5 1.7 1.8

22:5ω3 1.2 1.6 2.4 1.4 1.6

22:6ω3(DHA) 11.0 15.8 45.4 58.2 59.9

24:0 0.0 0.0 0.0 0.0 0.0

Source: Reprinted with permission from Journal of the American Oil Chemists’ Society, 72, 575.©1995AmericanOilChemists’Society.

a Stearidonicacid.

7089_C005.indd 153 10/8/07 11:53:56 AM


monounsaturatedfattyacidswereatamaximum.Increasingtheratioto4:1didnotgreatlyincreasetheremovalofthesaturatedandmonounsaturatedcomponentsfromthesolution.Increasingtheurea–fattyacidratiosalsoincreasedtheconcentrationofstearidonicacid(SA),EPA,andDHAinthefiltrate.ThemaximumenrichmentofPUFAswasalsoobservedatanoptimumurea–fattyacidratioof3:1,aphenom-enonnotedbyotherresearchgroups[66–68].Inthesamestudy,RoblesMedinaandcoworkers[65]investigatedtheeffectoftemperatureonthefattyacidcompositionofureaconcentratesfromcodliveroilusingaurea–fattyacidratioof4:1;Figure5.7iscompiledfromtheirdata.ThefiltrateconcentrationsofSAandDHAwerefoundtobegreatestat261K,thetotalω3fattyacidconcentrationswerefoundtobegreatestat 277 K, and the concentration of EPA in the filtrate was maximized at around293K.Althoughdifferenttemperaturevalueswerereported,thistrendontheinflu-enceoftemperaturechangeissimilartothatobservedbyWillesandcoworkers[66],whousedureafractionationandhigh-performanceliquidchromatography(HPLC)toproducefractionsrichinPUFAsfromfishoils.Duringureacrystallization,theyreportedthattheconcentrationsofSAandDHAinthefiltrateweregreatestat268K,theconcentrationoftotalω3inthefiltratewasgreatestat283K,andthatofEPAwasmaximizedat288K.

WanasundaraandShahidi[69]optimizedtheproductionofω3fattyacidconcen-tratesfromsealblubberoilusingureacomplexation.Theyinvestigatedtheeffectsof the urea–fatty acid ratio, crystallization time, and crystallization temperature.

4:11:1CodLiverOil

Urea/Fatty Acid Ratio

SA

EPA

DHA

Total SA, EPA, DHA

Total Saturated+ Monounsaturated

Monounsaturated

Saturated

Fatty

Aci

d Co

ncen

trat

ion/

% w

/w

2:1 3:1

100

90

80

70

60

50

40

30

20

10

0

FIgure 5.6 Theeffectofurea–fattyacidratioonthenoncomplexedfattyacidcompositionresultingfromureafractionationofcodliveroilat277K.

7089_C005.indd 154 10/8/07 11:53:57 AM


The study was carriedout using free fatty acids.Under optimumconditions, themaximumamountof totalω3 fatty acids (88.2%;70.1%ofwhichwasDHAand9.36%ofwhichwasEPA)fromsealblubberoilwasobtainedataurea–fattyacidratioof4.5:1,acrystallizationtimeof24hours,andacrystallizationtemperatureof263K.

Thereareseveraladvantagestousingureacomplexationforthefractionationoffattyacids[70]:

Largequantitiesofmaterialcanbehandledusingsimpleequipment.Theycanbecarriedoutundermildconditions(e.g.,roomtemperature).Biocompatiblesolventssuchasethanolcanbeused.Separationsareoftenrelativelyefficientwhencomparedwithmethodssuchassolventextractionandfractionalcrystallization.Itisarelativelyinexpensiveprocess.

Ureainclusioncomplexationisusuallycarriedoutinmethanolorethanol.Oneshouldbecautiouswhenusingmethanolbecauseitmaycausemethylationofsomefattyacidsduringcomplexformation,producingamixtureoffattyacidsandmethylesters[71].UreaconcentrationshouldbenearsaturationsincetheconcentrationofrecoveredPUFAsfromthefiltratedecreaseswithdecreasingureaconcentration[70].Usingacetone,orhigherhydrocarbons,assolventsforureacomplexationshouldbeavoidedbecauseitcancompetewiththefattyacidsforinclusion[72].Ratherthanbeingusedasastand-aloneprocess,ureacrystallizationisoftenusedtopreconcen-tratefishoil fattyacidmixturesprior to furtherprocessing.Foramore thorough

••••

•

Temperature/K

SA

EPA

DHA

Total ω3

Fatty

Aci

d Co

ncen

trat

ion/

% w

/w

244

110

100

90

80

70

60

50

40

30

20

10

0252 260 268 276 284 292 300 308

FIgure 5.7 Theeffectoftemperatureonthenoncomplexedfattyacidcompositionresultingfromureafractionationofcodliveroilusingaurea–fattyacidratioof4:1.

7089_C005.indd 155 10/8/07 11:53:58 AM


treatiseofthetheoryandpracticeoffractionationwithurea,thereaderisdirectedtotheworksofSwern[63],Schlenk[72],andSmith[73].

5.3.1.4 Chromatographic methods

Chromatographicseparationstakeadvantageofthedifferentratesofmigrationofmixturecomponentsthroughacolumninatwo-phasesystem,comprisingamobilephase (the phase containing the components of interest) and a stationary phase(animmobilephase,whichisinsolubleinthemobilephase).Themobilephasecanbealiquid,gas,orSCFandthestationaryphaseisusuallyasolid.Thephasesarechosensuchthatthemixturecomponentshavedifferingstrengthsofinteractionwiththestationaryphase.Componentsthathaveagreateraffinityforthestationaryphasehavelongerelutiontimesthancomponentswithaloweraffinity,whichenablessepa-rationtotakeplace.

Carefulselectionofthestationaryphase(adsorbent)canpermittheseparationoffattyacidsaccordingtocarbonchainlengthordegreeofunsaturation.Silverionsformaweakπ-bondwithsitesofunsaturation,andsosilverion–impregnatedcolumnscanbe used to fractionate polyunsaturates. High performance liquid chromatography[74]andsilverresinchromatography[75]havebeenusedforthepreparationofω3concentrates.Teshimaandcoworkers [76]haveuseda silvernitrate–impregnatedsilicacolumntoisolateEPAandDHAmethylestersfromsquidliveroil.Puritiesof85%to96%forEPAand95%to98%forDHAwerereportedwithyieldsof39%and48%,respectively.AdlofandEmiken[75]enrichedtheω3contentofcommercialω3-PUFAconcentrates from76.5%to99.8%using isocraticelutionfromasilverresincolumn.Guil-GuerreroandBelarbi[77]purifiedEPAandDHAfromcodliveroil using a silver nitrate–impregnated silica column. The oil was saponified andtreatedwithurea,andthenoncomplexedfattyacidswerethenconvertedtomethylestersbeforechromatographicprocessing.Thecolumnwaselutedwithasequenceofsolvents.Theymanagedtoobtaina64%yieldofDHAwith100%purity.TherecoveryofEPAwas29.6%,withafinalpurityof90.6%.

The purity of eluted fractions also depends on the choice of eluting solvent.Perrut[78]usedmethanol/water(90:10v/v)toseparatefishoilethylesters.Puritiesof96%EPAand85%DHAwereachieved.Willeandcolleagues[66]usedthesamemethanol/ethanol(90:10v/v)solventsystemforthefractionationoffishoilmethylestersandproducedEPA-richandDHA-rich fractionswith86%and83%purity,respectively. Despite the developments in chromatographic techniques to refineand concentrate fish oil components, the use of very large volumes of solvents,potential product solvent residues, loss of column resolution after repeated use,andthepresenceofpotentiallytoxicsilverresiduesarelikelytohinderscale-uptoproductionscalevolumes.

5.3.1.5 enzymatic transformation

The hydrolysis or esterification of fatty acids can be catalyzed by lipases. Thesereactions can be carriedout at low temperatures, which is beneficial consideringthedetrimental effectshigh temperaturescanhaveonPUFAs.Thedirectionandefficiencyofthereactiondependsontheconditionsemployed.Watercontentinthe

7089_C005.indd 156 10/8/07 11:53:59 AM


reactionmediumisacrucialfactorinfluencingthedirectionofthereaction.Highwatercontentshiftsthechemicalequilibriumtowardhydrolysis,whereaslowerwatercontent shifts the equilibrium toward esterification. For esterification, thewatercontentshouldbekepttoaminimumtopreventpartialhydrolysisofproducts,butatthesametime,thewatercontentshouldbesufficientlyhighinordertopreventenzymedeactivation[79].

The TAG form of PUFA is considered to be nutritionally more favorablethanfattyacidestersbecausestudieshaveshownthat theintestinalabsorptionofω3-PUFAestersisimpaired[80,81].Also,TAGsareoftenpromotedasbeingmore“natural”thanfattyacidesters,leadingtoacommercialdemandofPUFAsintheTAGform.HeandShahidi[82]studiedtheglycerolysisofω3-PUFAsobtainedfromseal blubber oil using Chromobacterium viscosum lipase. Up to 94% conversionwasachievedwith13.8%,43.1%,and37.4%ofmonoglycerides,diglycerides,andtriglycerides,respectively,intheproduct.Bottinoandcolleagues[83]reportedtheresistanceofcertainlong-chainPUFAsofmarineoilstolipase-catalyzedhydrolysis.Theciscarbon-carbondoublebondspresentinsomefattyacidsresultinbendingof the carbon chain, causing the fatty acid terminal methyl group to lie close totheester linkage.Themethylgroup in this instanceproducesastearichindrancetolipase-catalyzedhydrolysis.Anincreaseinthenumberofdoublebondsfurtherincreases thestearichindrance.However, therearesomereports inwhichlipasesfrom Chromobacterium viscosum and Pseudomonas sp. released EPA and DHAfromtriglycerides[84–86].

Gámez-Mezaetal.[87]studiedtheconcentrationofEPAandDHAfromsardineoilbyenzymatichydrolysis.TheyinvestigatedfivecommerciallipasesfromPseudomonas(threeimmobilizedandtwosoluble).Theyfoundthattheimmobilizedlipaseprepa-rationPS-CI(alipasefromPseudomonas sp.immobilizedonachemicallymodifiedceramic)providedthegreatestdegreeofhydrolysisforEPAandDHA(81.5%and72.3% from their initial content in the sardine oil after 24 hours) and attributedthisobservationtothehigherproteincontentofthislipase.Atthestartofhydro-lysis (3hours), theynoticed that the lipasesdisplayed a significantpreference forsaturatedfattyacidscontaining14to16carbonatoms.However,theresistancetoreleaseEPAandDHAdecreasedasthehydrolysisreactionprogressed.SubsequentureacrystallizationofthePS-CIhydrolyzedoilenrichedEPAfrom14.5%to46.2%andDHAfrom12.6%to40.3%,witha78.0%yield.

Schmitt-Rozieresetal.[88]studiedtherecoveryofEPAandDHAfromeffluentsofthesardinecanningindustry.Theoilyeffluentcomponentcontainedaround10%eachofEPAandDHA.Aftertheremovalofsolidparticles,proteins,andpeptidesfromthecrudeeffluent,theresultantoilwashydrolyzedandEPAandDHAwereenrichedbyselectiveenzymaticesterification.UsingLipozyme™,DHAwasenrichedup to 80% but no enrichment was observed for EPA. By immobilizing Candida rugosa lipaseonanAmberliteIRC50cation-exchangeresin,a30%enrichmentofEPAwasachieved.

Zuyi and Ward [89] studied the lipase-catalyzed alcoholysis of cod liver oilto concentrateω3-PUFAs. They studied the effect of water content on reaction,indicating that thewater content is influencedby thehydrophobicityof the reac-tionmedium; themorehydrophobic thealcohol, the lower thewater content that

7089_C005.indd 157 10/8/07 11:53:59 AM


is required.Using isopropanol, theyobserved that thealcoholysisof triglyceridesincreasedwithincreasingwatercontentintherange0%to7.5%v/v.Athigherwaterconcentrations(>10%)severalnegativeeffects, includingenzymedestabilization,purificationcomplications,andpromotionofundesirablehydrolysisreactions,wereobserved.Carryingouttheisopropanolysiswith5%water(v/v)at383Kyieldedahighconcentrationofmonoglyceridecontaining40%ω3-PUFAs.

Lipase-catalyzed transesterification for the concentration ofPUFAs fromfishoilshasbeenshowntobeausefulalternativetotraditionalesterificationanddistil-lationmethods.Withaconversionof52%,Breivikandcoworkers [90]wereableproduceaconcentratecontaining46%EPA+DHA(theinitialsardineoilcontained24.7%EPA+DHA)usingPseudomonas sp.lipaseandastoichiometricamountofethanolwithoutsolvent,atroomtemperature.TheresultingTAGswereisolatedandconverted to ethyl esters using either conventional chemical means or enzymaticconversion by immobilized Candida antarctica lipase. Urea fractionation of theresulting product increased the EPA + DHA content to around 85%. The use oforganic solvents as reaction media for enzymatic conversions can be disadvanta-geousbecausethiscanleadtoenvironmentalandresidualsolventissuesassociatedwithproduct purification.SCFshavebeen investigated as alternative solvents forthisprocess.

5.3.2 supercritical Fluid prOcessing OF Fish Oils

AlthoughvariousSCFshavebeenusedtoextract/fractionatefattyacidsandtheiresters,carbondioxide(CO2)isbyfarthemostcommonlyusedsolventbecauseofitsavailability,lowcost,andnonreactivity.UsingCO2limitstheoxidation,decom-position,andpolymerizationofthePUFAspresentinfishoilsbecauseseparationsoccurunderaninertatmosphereandprocessescanbecarriedoutatmoderatelylowtemperatures.Inaddition,CO2isnontoxic,isnonflammable,andproducessolvent-residue-freeextracts,whichisparticularlyimportantifthedesiredmaterialsareforhumanconsumption.Theability tomodifysolventpropertiesbymanipulationoftemperatureandpressureorbytheadditionofacosolventgivessupercriticalCO2processesauniqueadvantage.Solventpropertiescanbetunedforspecificseparationproblemsofferinggreaterversatilityandflexibilityovermoreconventionalfraction-ationprocesses.Moreover,theincreasingsocialawarenessofthehealthbenefitsofcertainfishoilcomponentsinthediethasincreasedcommercialdemandfortheseproductsinthefoodandnutraceuticalindustries.TheincreaseddemandhasledtoareevaluationofprocessingmethodsandsupercriticalCO2methodologiesprovidesolvent-free,“natural”products,whichhavewideconsumerappeal.TheremainderofthischapterfocusesonthephaseequilibriaofCO2withvariousfishoilcompo-nentsaswellasthevarioussupercriticaltechniquesemployedtoextract/fractionateandisolatetheseproducts.Phaseequilibriadataareoffundamentalimportanceforoptimaldesignofextractionandfractionationoperations.

5.3.2.1 phase equilibria: supercritical Co2 and Fish oil Components

ThissectionprovidesabriefsummaryofsolubilitymeasurementsandmodellingofsolubilityandphaseequilibriaforfishoilcomponentsinsupercriticalCO2.Fish

7089_C005.indd 158 10/8/07 11:54:00 AM


oilsareacomplexmixtureoflipidcomponentsbelongingtoseverallipidclasses,includingacylglycerols,fattyacids,fattyacidesters,sterols,tocopherols,andhydro-carbons.Successful isolationusing supercritical processes requires reliable infor-mationonthesolubilitybehaviorofthesolutesofinterestasaffectedbyoperatingconditionsandsolute/solventproperties.Becausecompletepredictivemodellingofmulticomponentphasebehaviorinsupercriticalsystemsisnotyetrealized,experi-mentaldatastillplayanessentialroleinprocessdesignandthedevelopmentofbothsimpleandrigorousthermodynamicmodels.

SimpleempiricalmodelsbasedontheChrastilmodel[91]havebeenwidelyusedforpredictionofsolubilityinCO2atagiventemperatureanddensity.TheChrastilcorrelationcanonlybeusedforthevapor-phaseconcentrationofsolutesandgivesnoinformationontheliquid-phasecomposition.Equationofstatemodels,suchasthePeng-Robinson[92,93],Soave-Redlich-Kwong[94],excessfunction(gE)[95],groupcontribution[96],andlatticemodelequationsofstate(EOS)[97],havebeenshowntoprovidethemostrigorousmethodforpredictingphaseequilibriumbehavior.

TheChrastil correlation [91] for estimating lipid solubilities inSCFs takestheform:

ln ln /c k a T b= + +ρ (5.2)

wherecisthesolutesolubility,kistheassociationnumberrepresentingthenumberofmoleculesinthesolute-solventcomplex,ρisthepuresolventdensity,andaandbareempiricalconstants.Parameteraisdependentonthetotalheatofreaction(heatofsolvation+heatofvaporization),andbisdependentontheassociationconstantandsoluteandsolventmolecularweights.Parameterkreflectsthedensitydependenceofsolubilityatconstanttemperature,andparameterareflectsthetemperaturedepen-denceatconstantdensity.Table5.6providessomeChrastilcorrelationparametersforthesolubilityofseveralfishoillipidcomponentsinCO2[98–101].

Although, binary lipid/CO2 systems have been studied extensively, multi-componentdata are relatively scarce. In suchmulticomponentmixtures, complexintermolecularinteractionsmayleadtosignificantdeviationsfrompurecomponentsolubilities.However,purecomponentsolubilityinformationisstillimportant,asitcanbeusedtogiveaguidetothedegreeofseparationpossiblebetweentwoormoreclassesoflipidatagiventemperatureandpressure.GeneralsolubilitytrendsinbinarysystemscontributetoourbasicunderstandingoftheprincipalsoflipidsolubilityinSCFs.Thisinformationprovidesasoundbasisonwhichtoevaluatethesolubilitybehaviorofmore complexmulticomponent systems.However, researchers shouldexercisecautionwhenmakingsolubilitymeasurements, interpretingthedataand,ultimately,designingseparationprocessesbasedon this information.TemelliandGüçlü-Üstündag[102]reportseveraldiscrepanciesbetweenreportedsolubilitydataforlipid/CO2systemsfromdifferentlaboratories.Anexampleofsuchdiscrepanciesis given inFigure5.8 for some selected fatty acids inCO2[103–107]. Impurities,sampledegradation,isomericpurity,andlimitationstoexperimentalmethodsareallcontributingfactorstothereporteddatavariations.

Solutevaporpressuresandsolute-solventandsolute-soluteintermolecularinter-actions govern solubility behavior. In binary systems of a particular homologous

7089_C005.indd 159 10/8/07 11:54:01 AM


table 5.6Chrastil parameters for some selected Fish oil Components (all solubilities and densities are in units of g·l–1 unless otherwise stated)

lipid Componentk ± standard

errora ± standard

errorb ± standard

errorrange t/K;

p/mpa ref.

Fatty acids

Myristicacid,C14:0 6.42±0.33 –9300±1727 –10.2±5.9 98

Palmiticacid,C16:0 7.00±0.39 –12029±1043 –7.0±4.1 98

Stearicacid,C18:0 5.81±0.54 –15890±741 12.0±3.7 98

Oleicacid,C18:1 7.92±0.37 –3982±691 –38.1±2.3 98

Linoleicacid,C18:2 9.71±0.90 –5211±1626 –46.3±5.3 98

triglycerides

Triolein 10.28±0.66 –2057±480 –61.5±4.6 98

ethyl esters

Stearicacid 5.80±0.50 –2446±857 –26.7±3.9 98

Oleicacid 7.78±0.34 –1947±503 –40.9±2.7 98

Linoleicacid 7.17±0.63 –2193±896 –36.2±4.4 98

EPA 8.62±0.17 2473±262 –45.2±1.2 98

DHA 7.76±0.32 –1784±529 –42.1±2.5 98

hydrocarbons

Squalenea 6.54±0.06 –3936.6±155 –28.24±0.7 313–333;10–30

99

minor Components

VitaminAa 5.07±0.44 –3072±339 –21.7±2.12 313–353;20–35

100b

VitaminApalmitatea 7.66 0 –49.2 333;12.5–30

101

β-carotenea 8.63±0.61 –11576±461 –23.3±3.04 313–353;20–35

100b

Fish oils

Codliveroila 10.91±0.18 –4078±122 –59.2±0.98 313–333;20–30

99b

Spinydogfishliveroila 9.97 0 –65.4 333;20–30 101

Orangeroughyoila 7.79 0 –50.6 333;20–30 101a Solubilitiesareing·kg-1anddensitiesareinkg·m–3;bDerivedfromdatapresentedinthisreference.

7089_C005.indd 160 10/8/07 11:54:01 AM


series,whereintermolecularinteractionsaresimilar,molecularweightandvaporpres-suresdeterminecomponentsolubilities.Forexample,inFigure5.8,thesolubilityoffattyacidsincreaseswithdecreasingmolecularweight(chainlength).Ofthesystemsreportedintheliterature,fattyacidestershavethehighestvaporpressures,followedby fattyacids.Vaporpressuresof theglyceride lipidclass follow the trendmono-glycerides>diglycerides>triglycerides[98].EsterificationwithaC1orC2alcoholsubstantiallyincreasesthesolubilityoffattyacidsinCO2becausethepolaracidgroupisconvertedtoalesspolarestergroup[108].Forfattyacidsofthesamechainlength,meltingpointsdecreasewithincreasingdegreeofunsaturation(Figure5.8).Inthisinstance,solubilityisaffectedbythephysicalstateofthecompound.

JohannsenandBrunner[100]measuredthesolubilitiesoffat-solublevitaminsinsupercriticalCO2in the temperaturerangeof313to353Kandpressurerangeof20 to35MPa.The solubility for bothβ-carotene (provitaminA) andvitaminAincreasedwithincreasingpressure.Overthetemperaturerangestudied,vitaminA shows retrograde condensation behavior (solubility decreases with increasingtemperature)uptoaround30MPa(Figure5.9).Athigherpressures,thesolubilitycurvesofvitaminAexhibitacrossoverpointandthesystemexhibitsnonretrogradebehavior.ThesolubilityofvitaminApalmitatehasbeenmeasuredbyCatchpoleetal.[99]at333Kand12.5to30.0MPa.Thesolubilityiscomparedtothatofthevitamin A free acid in Figure5.9. The decrease in polarity of the ester over thefreeacidiscounterbalancedbythelargeincreaseinmolecularweight,leadingtoamodestdecreaseinsolubility.

Mollerupetal.[109–111]carriedoutaseriesofphaseequilibriameasurementsforfishoilfattyacidethylesters(FAEEs)ofthesandeelwithCO2.MeasurementswereobtainedusingthecrudefishoilFAEEs(283to343K,2to22MPa),urea-fractionated fish oil FAEEs (313 to 343 K, 1.6 to 25 MPa), andω3-rich fish oilFAEEs(313to343K,8to26MPa).TheinitialFAEEoilcompositionsaregiven

Pressure/MPa

C16:0; 318 K; ref. 104C16:0; 318 K; ref. 103

C18:0; 318 K; ref. 103C18:0; 318 K; ref. 104

C18:1; 323 K; ref. 105C18:1; 323 K; ref. 106C18:2; 313 K; ref. 107C18:2; 313 K; ref. 103

Solu

bilit

y/g

kg–1

of C

O2

5 10 15 20

50

40

30

20

10

025 30 35 40 45

FIgure 5.8 ComparisonoffattyacidsolubilitiesinCO2fromvariousliteraturesources.(Lineshavebeendrawntoaidtheeye.)

7089_C005.indd 161 10/8/07 11:54:02 AM


inTable5.7.TheK-values,onaCO2 freebasis, for someselectedω3-FAEEsaregiven inFigure5.10 at 313Kand343 Kas a functionof pressure.TheK-valuesdependstronglyontemperature,pressure,andmixturecomposition.Highselectivi-ties(higherrelativedifferencesbetweenK-values)andlowsolubilitieswereobservedatlowpressures,whereasathighpressurestheselectivitywaslowbutthesolubilitywashigh.ThecrudeFAEEmixturewasmoresolublethantheurea-fractionatedandω3-richmixtures (whichhadsimilar solubilities)because thecrudemixturecon-tainedalargeamountofsaturatedandmonounsaturatedFAEEsofmediumchainlength(C14–C18).Forthecrudefishoilesters,theK-valueswerefoundtovarywithchainlengthbutnotspecificallythedegreeofunsaturationandpositionofdoublebonds.Theauthorsreportedthatsolubilitiesincreasewithincreasingtemperature,and Figure5.10 shows that the selectivities at 343 K are greater than those at313K. The selectivities are largest in theω3-rich system because the number ofcomponentsandtheamountofmedium-chain-lengthmaterialhasdecreased.Usingurea-fractionationasapreconcentrationstepundertheconditionsinvestigated,theauthorsdeterminedthatoptimumseparationconditionsintermsofselectivityandsolubilitywereintherangeof16to18MPaat343K(correspondingtoCO2densitiesof550to615kgm–3).

Catchpole and von Kamp [92] studied the phase equilibria of the systemsqualene/CO2andsharkliveroil/CO2overtherange313to333Kand10to25MPa.Thesharkliveroilconsistedofaround50%squaleneand50%ofamixtureofTAGsandDAGEs.Therewasalso0.5%bymasspristane.TheTAG/DAGEmixturehasbeenassumedtobeasinglepseudocomponent,withahypotheticalcarbonnumberof54.Thephaseequilibriadataforsqualene/CO2at313Kand333KareshowninFigure5.11.Theamountofoildissolvedinthevaporphaseincreaseswithincreasingpressureanddecreasingtemperature,asdoestheamountofCO2dissolvedintheoilphase.Theexperimentaldataforthebinarysystemssqualene/CO2andC54-TAG/CO2weremodelledusingthePeng-RobinsonEOSwiththeusualmixingrules[92].ThePeng-Robinson EOS was also used to predict phase equilibrium and separation

Pressure/MPa

Vitamin A: 313 KVitamin A: 333 KVitamin A: 353 KVitamin A palmitate; 333 K

Solu

bilit

y/g

kg–1

of C

O2

15

30

25

20

15

10

5

020 25 30 35

FIgure 5.9 SolubilityofvitaminAandvitaminApalmitateinCO2.

7089_C005.indd 162 10/8/07 11:54:03 AM


table 5.7the Initial Faee Fish oil Compositions used in the studies of mollerup et al. [109–111]

Crude Fish oil urea-fractionated ω3-rich

Component Weight (%) of Faee

10:0 0.4

12:0 0.2

14:0 7.5 0.8

14:1ω5 0.5 0.2

15:0 0.5

15:1ω5 0.2 0.1

16:0 18.5

16:1ω7 12.4 0.5

16:2 1.4 2.3

16:3ω3 0.6 1.6 0.3

16:4ω3 0.8 2.9 0.2

18:0 2.2 0.03 0.5

18:1ω9 10.2 0.6

18:1ω7 2.3

18:2ω6 2.9 1.2

18:3ω6 0.4 0.8

18:3ω3 1.3 0.9

18:4ω3 3.8 12.7 2.3

20:0 0.2

20:1ω9 4.2 0.6

20:2ω6 0.3 0.5

20:3ω3 0.2 0.1

20:4ω3 0.7 1.0 2.9

20:5ω3 10.0 35.9 52.5

22:1ω11 6.5 0.3

22:1ω9 1.0 0.1

21:5ω3 0.4 1.5

22:5ω3 0.5 1.1 2.5

22:6ω3 9.6 33.2 36.1

7089_C005.indd 163 10/8/07 11:54:04 AM


factors for the ternary system C54-TAG/squalene/CO2. The predicted vapor andliquidmolefractionsofsqualeneintheternarysystemareshownforselectedtem-peraturesandpressuresinFigure5.12.TheliquidmolefractionsequatetodiscretemassfractionsofsqualeneonaCO2freebasisrangingfrom0to1.Itisinterestingtonotethattheequilibriumrelationshipisalmostlinear,asshownbytheregressionlines in Figure5.12. The mass fraction of CO2 dissolved in the liquid phase at agiventemperatureandpressurestaysalmostconstantevenwhenthesqualenemassfractionvariesfrom0to1(onaCO2-freebasis).Thepredictedvaporandliquid-phasemassfractionsofsqualeneforthesamesystemaregivenonaCO2-freebasisin Figure5.13. The K-values for squalene are also shown. The selectivity towardsqualeneisbestatlowpressureandlowmassfractionofsqualene.ThesolubilityofTAGsdecreasesmoresharplywithdecreasingpressurethansqualene,andsotheincreaseinselectivityistobeexpected.TheK-valuedecreasesasthetemperatureandvapor-phasedensityincrease,althoughitisstillsufficientlyhighat333Kand25.0MPatoenableseparationofsqualeneandC54-TAG.

Ruivoetal. [112]measuredthephaseequilibriaof the ternarysystemmethyloleate/squalene/CO2overtherange313to343Kand11to21MPa.Fourdifferent

Original Fish Oil

OriginalFish Oil

Pressure/MPa Pressure/MPa Pressure/MPa

Pressure/MPaPressure/MPaPressure/MPa

K-va

lues

at 3

43 K

(CO

2 fre

e bas

is)K-

valu

es at

313

K(C

O2 f

ree b

asis)

12 14 16 18 20 22 10

1088 9 10 11 12 13 14 15 10 12 14 16 18 11 12 13 14 15 16

12 14 16 18 20 2212

0.00.20.40.60.81.01.21.41.61.82.02.2

3.5

4.0

3.0

2.5

2.0

1.5

1.0

0.5

0.014 16 18 20 22

Urea-fractionated Fish Oil

Urea-fractionated Fish Oil ω3-Rich Fish Oil

ω3-Rich Fish Oil

C16:3 ω3C16:4 ω3C18:4 ω3C20:4 ω3C20:5 ω3 (EPA)C22:5 ω3C22:6 ω3 (DHA)

FIgure 5.10 Partitioncoefficients(K-values)onaCO2-freebasisforwholesandeeloil,urea-fractionated sand eel oil, andω3-rich sand eel oil [109–111]. (Reprinted from Fluid Phase Equilibria,161,169,©1999.WithpermissionfromElsevier.)

7089_C005.indd 164 10/8/07 11:54:05 AM


Liquid

0.725

26

24

22

20

18

16

14

12

10

80.750 0.775 0.800 0.825 0.995 0.996 0.997 0.998 0.999 1.000

Vapour

313 K333 K

Mole Fraction of CO2

Pres

sure

/MPa

FIgure 5.11 Liquidandvapormolefractionsforthesqualene–CO2system[92].

Liquid Phase Mole Fraction Squalene0.00 0.05 0.10 0.15 0.20 0.25

0.0040

0.0035

0.0030

0.0025

0.0020

0.0015

0.0010

0.0005

0.0000

Vapo

r Pha

se M

ole F

ract

ion

Squa

lene

250 Bar, 333 K200 Bar, 313 K200 Bar, 333 K125 Bar, 313 K

FIgure 5.12 Liquidandvaporphasemolefractionofsqualeneatselectedtemperaturesandpressures[92].Points:Peng-Robinsonequationofstatepredictions;lines:linearregressions.(ReprintedwithpermissionfromIndustrial and Engineering Chemistry Research,36,3762.©1997AmericanChemicalSociety.)

7089_C005.indd 165 10/8/07 11:54:07 AM


feedcompositionswereusedcontaining0.1079,0.3350,0.6447, and0.8779molefractionsof squalene.The selectivityof afluid canbequantified in termsof theseparationfactor,α,whichinthisexampleisgivenby:

α = ⋅

⋅y x

x yM S

M S

(5.3)

whereyisthemolefractionconcentrationinthevaporphase,xisthemolefractionintheliquidphase,andthesubscriptsMandSpertaintomethyloleateandsqualene,respectively.TheselectivityofCO2towardmethyloleateoverarangeofpressuresisdemonstratedinFigure5.14aasafunctionoftemperature(initialsqualenefeedmolefractionof0.6447)andFigure5.14basafunctionofsqualenefeedconcentrationat313K.Figure5.14showsthatCO2ishighlyselectiveformethyloleate,withsepa-ration factors ranging from2 to8.The separation factordecreaseswithdecreas-ing temperature and with increasing pressure, which results in a higher loading,givinggreaterthroughputattheexpenseofselectivity.Anincreaseinsolubilitywithtemperatureatfixeddensityisadvantageousforpackedcolumnfractionation,whichrequires adensitydifferencebetween the supercritical andoilphases tobe largeenoughtopreventflooding.

Additionof cosolvents, suchas ethanol, canenhance the solubilityof solutesin supercritical CO2. Cosolvents have also been shown to act as entrainers. Theentrainereffecthasbeendefinedasaphenomenoninwhichthesolventpowerofafluidisincreasedbytheadditionofcosolvents,whilsttheselectivityofthatfluidismaintainedorenhanced[113].Inmanystudies,theenhancedsolubilitieshavebeenattributedtosolute-cosolventinteractions,suchashydrogenbondingordipole-dipole

Mass Fraction Squalene in the Liquid

VLE

K Factors125 Bar, 313 K250 Bar, 333 K

125 Bar, 313 K250 Bar, 333 K

Mas

s Fra

ctio

n Sq

uale

ne in

the V

apou

r

Equi

libriu

m C

oeffi

cien

t K

0.0 0.2 0.4 0.6 0.8 1.0

8 1.0

0.8

0.6

0.4

0.2

0.0

7

6

5

4

3

2

1

0

FIgure 5.13 MassfractionintheliquidandvaporphaseandK-valuesforsqualeneonaCO2-freebasis[92].(ReprintedwithpermissionfromIndustrial and Engineering Chemistry Research,36,3762.©1997AmericanChemicalSociety.)

7089_C005.indd 166 10/8/07 11:54:08 AM


interactions.Specificintermolecularinteractionsbetweencosolventsandsolutescanenhancethesolubilityofthosespecificcomponents,whichcanbeparticularlyben-eficialforimprovingseparationselectivities.

Catchpoleetal.[99]measuredthesolubilityofsqualene,orangeroughyoil,codliveroil,andspinydogfishliveroilinsupercriticalCO2andCO2/ethanolmixturesat313to333Kand20to30MPa.Ethanolmassconcentrationsup to12%(onasolute-freebasis)wereused.Catchpoleandcoworkersfoundthatethanolsubstan-tially increased the solubility of all fish oil components studied (Figure5.15). At333K,theauthorscorrelatedtheincreaseinsolubilityduetotheadditionofethanolusingthefollowingequation:

Temperature 313 K 323 K 343 K

0.8779 0.6447 0.3350 0.1079

Mole Fraction ofSqualene in Feed

Pressure/MPa (a) (b)

Pressure/MPa

Sepa

ratio

n Fa

ctor

, α

Sepa

ratio

n Fa

ctor

, α

10 12 14 16 18 20 22 10 12 14 16 18 20 22

8

7

6

5

4

3

2

8

7

6

5

4

3

2

FIgure 5.14 TheseparationfactorsformethyloleateandsqualeneinCO2[112].(ReprintedfromJournal of Supercritical Fluids,29,77,©2004.WithpermissionfromElsevier.)

0

100

10

12 4

Mass % Ethanol (solute free basis)

Squalene

Solu

bilit

y (et

hano

l fre

e bas

is)/g

kg–1

of C

O2

Orange Roughy OilCod Liver OilSpiny Dogfish Liver Oil

6 8 10 12 14

FIgure 5.15 Enhancement of fish oil component solubilities as a function of ethanolcosolventconcentrationat333Kand20MPa[99].(ReprintedwithpermissionfromJournal of Chemical and Engineering Data,43,1091.©1998AmericanChemicalSociety.)

7089_C005.indd 167 10/8/07 11:54:10 AM


ln( / ) ln( / )S g kg S g kg kX⋅ = ⋅ +− −10

1 (5.4)

whereSistheenhancedsolubility,S0isthesolubilityinpureCO2,kisaconstant,andXisthemasspercentofethanol.Thekconstantsforsqualene,orangeroughyoil,andspinydogfishliveroilare0.14,0.15,and0.21,respectively.

ThesolubilityenhancementofsqualeneinCO2hasalsobeeninvestigatedusingn-hexane,toluene,andethanolintherangesof303.2to313.2Kand9to10MPa[114].Thedependenceofsolubilityonentrainerconcentrationwasdescribedbyaparabolicfunction.Theauthorscharacterizedtheinitialslopeof thisfunctionforeachsolute-entrainerpairwithasinglenumber,termedtheentrainer efficiency, E.TheEvaluewasrelatedtothesimilaritiesofthemolecularstructureandpolarityof theentrainerand thesolute.TheEvalues followed the trendn-hexane (3.6)>toluene(2.4)>ethanol(1.5),indicatingtheorderofsolubilityenhancementatfixedtemperature,pressure,andentrainerconcentrations.

Nilssonetal.[115]investigatedtheeffectsofadding5%ethanolasacosolventontheK-valuesandselectivityofmenhadenoilfattyacidestersat333Kand12.5MPa.Itwasnoted thatK-values increasedwith increasingnumberofdoublebonds foragivenchainlength.Additionofethanol increasedtheK-valuesforallfattyacidesters,regardlessofchainlengthordegreeofunsaturation.TheratiooftheK-valuesforCO2-ethanolandpureCO2rangedfromaround1.5forC14estersto3.1forC22esters,demonstrating that the solubilityenhancementachievedby theadditionofethanolincreaseswithincreasingchainlength.Thefluidselectivities,definedhereastheratiooftheK-valuesfortheC14:0estertothoseoftheothermixturecompo-nents,decreasedforallfattyacidestersupontheadditionofethanol.K-valuesforthefattyacidestersinpureCO2werealsomeasuredat333Kand13.1MPa.ThemeasuredpartitioncoefficientsinpureCO2undertheseconditionsyieldedK-valuesvery similar to those obtained using CO2-ethanol at 333 K and 12.5 MPa. Theyconcluded that ethanol serves no useful purpose as a cosolvent with CO2 for theconcentrationofEPAandDHAfromfattyacidestermixtures.

Althoughtheuseofcosolventsmaybebeneficial in termsof increasingsolu-bility, the complex nature of fish oils means their application may be limited intermsofselectivityenhancement.Theiruseshouldbeconsideredcarefullybecauseitcanincreasethecomplexityofprocessdesign.Anincreaseinsolventloadingmayresult in the coextraction of undesirable components. Also, using cosolvents canaffectmass transfer,greatly increaseprocessingcosts,andcanpotentially inducedegradation of the desired extract. For a more detailed discussion of binary/CO2andmulticomponent/CO2lipidsystems,thereaderisdirectedtothecriticalandin-depthreviewsofTemelliandGüçlü-Üstündag[98,102].Severalarticlescontainingcomprehensivetabulateddataonlipid/CO2systemsstudiedarealsoavailableintheliterature[98,116–118].

5.3.2.2 polyunsaturated Fatty acid processing

ThemainfocusofstudiesinvolvingSCFprocessingoffishoilshasbeentheisola-tionofω3-PUFAs,andinparticular,theisolationofEPAandDHA.Itiswellknown

7089_C005.indd 168 10/8/07 11:54:11 AM


thatonlymodestenrichmentsusingatriglyceridefeedstockcanbeachievedunlesstheoilisalreadyrichinDHA.Thecomplextriglyceridestructure,containingdiffer-entchainlengthacidsanddegreeofunsaturation,makesseparationstoachievecon-centrationoflong-chainPUFAsimpractical.Forthisreason,triglyceridesareeithersaponifiedorconvertedtofattyacidalkylesterspriortofractionation.TheresultantfattyacidsorestershavegreatersolubilityinCO2byvirtueoftheirlowermolecularweightandgreatervolatility[119].Somegroupshaveinvestigatedthefractionationofthefreefattyacidsbutthereporteddegreeofseparationwaslow.

SCFextraction/fractionationprocessesinvolvingethylestersarecarriedoutincontactingdevicescomprising twofluidphases:1)amoredense,ester-richphasecontainingdissolvedCO2and2)alessdense,CO2-richphaseinwhichsomeestersare dissolved. Separation processes exploit solubility differences of various feedcomponentsinthefluid-richphase.Eisenbach[120]carriedoutthefractionationofcodliveroilfattyacidalkylestersusingCO2inabatch-continuousprocess.Here,aquantityoffeedmaterialisloadedintothebaseofthefractionationcolumn,andCO2 is continuously passed through the column until the feedstock is depleted.The low-molecular-weight componentswith short chain lengths arepreferentiallyextracted.Toenhanceseparation,atemperaturegradientalongthecolumnwasusedtogenerate internalrefluxinthecolumncausedbyareductioninsolubility.Thiscausedthehigher-molecularweightcompoundstoprecipitateandindividualchain-lengthfractionswerecollectedinaseparatorasafunctionoftime.InEisenbach’sstudy[120],therefluxwasgeneratedbythepresenceofa“hotfinger”at363Kinthetopofthefractionationcolumn.Extractionswerecarriedoutat15.0MPausingaCO2flowrateof25Lhour–1.Theextractorandcolumntemperatureswere298and323K,respectively.Theestersremaininginsolutionexitedthetopofthecolumnandwerecollectedinaseparationvesselbypressurereduction.Usingthismethod,Eisenbachwasabletosuccessfullyseparatethefattyacidestersaccordingtocarbonnumber. He reported that a fraction containing C20 esters with greater than 95%puritywasobtained,consistingofaround13%ofthefeedmaterial,whichhadaninitialEPAcontentof14.5%.TheC20fattyacidesterfractionhadanEPAcontentof48.2%.

InalaterstudybyNilssonandcoworkers[121],abatch-continuousprocessforthefractionationofmenhadenoilwascarriedoutat15.2MPausingatemperaturegradientalongtheheightofthecolumn.Temperaturesinthecolumnvariedacrossfourtemperaturezonesfrom293Katthebottomto373Katthetop.Fractionationwascarriedoutwithandwithoutureacomplexationasapreconcentrationstep.TwoofthefeedmaterialsinvestigatedareshowninTable5.8.Fractionationofthewholeesters(Feed1)demonstratedthatsuccessfulseparationaccordingtocarbonnumbercouldbeachieved.Fractionsinexcessof60%puritywithrespecttoC20and90%puritywithrespecttoC22esterswereachievedwithEPAandDHAcontentsof51.9%(oftheC20fraction)and59.5%(oftheC22fraction),respectively.ThelowpurityoftheC20fractionemphasizedthedifficultyinseparatingtheC18andC20esters.Separationusingaurea-treatedfeed(Feed2)producedfractionsinexcessof80%puritywithrespect toC20estersandgreater than95%puritywithrespect toC22esters.Morethan95%of theC20 fractioncouldbeattributed toEPAandDHAaccounted foraround90%ofthetotalC22fraction.TheenhancementoftheoverallC20puritywas

7089_C005.indd 169 10/8/07 11:54:12 AM


attributedtothefactthatmostoftheC18fattyacidestersintypicalmenhadenoilaresaturatesandmonounsaturates,whichwerepreferentiallyremovedduringureacomplexation.Thesemaximumenrichmentswereachievedusingasolvent-to-feedratio(S/F)ofaround475.TheS/Fhasasignificantimpactontheeconomicviabilityofaprocess;highS/Fvaluesleadtolengthyfractionationtimesand/orhighenergycosts.AreductioninS/Fcanbeachievedbyincreasingtheoperatingpressureordecreasingtheoperatingtemperature,attheexpenseofseparationperformance.An

table 5.8Composition of Feed materials used in study by nilsson et al. [121]

Feed 1a Feed 2b

Fatty acid Weight (%) of Fatty acid esters

14:0 7.8

16:0 15.6

16:1ω7 10.9 <1.0

16:3ω4 1.1 5.3

16:4ω1 1.5 5.8

18:0 3.1

18:1ω9 7.6

18:1ω7 3.1 <1.0

18:2ω6 1.3 <1.0

18:3ω3 1.6 <1.0

18:4ω3 2.9 7.6

20:1ω9 1.2

20:4ω6 1.0 1.4

20:4ω3 1.5 <1.0

20:5ω3(EPA) 16.5 48.6

21:5ω3 <1.0 1.3

22:5ω3 2.5 <1.0

22:6ω3(DHA) 10.9 22.2

total by Carbon number

C14 9.0 <1.0

C16 32.7 13.1

C18 21.3 9.7

C20(%EPA) 21.8(75.7) 50.5(96.2)

C22(%DHA) 14.5(75.2) 25.0(88.8)


a Wholeesters,bUreafractionatedesters.

7089_C005.indd 170 10/8/07 11:54:12 AM


appropriatebalancebetweenproductyieldandS/Fshouldbeinvestigatedduringtheoptimizationprocess.

Asanextensiontothiswork,Nilssonetal.[122]employedanincreasingpres-sureprograminconjunctionwithatemperaturegradientforfractionationofurea-crystallizedfishoilethylesters.ThefeedsamplehadacompositionverysimilartothatgivenforFeed2inTable5.8.Usingseventemperaturezonesalongtheheightofthecolumn,rangingfrom313Kinthesecondzoneto353Katthetop(thebottomzone was at ambient temperature), and pressures ranging from 13.1 to 15.7 MPa(~6.9MPaincrements), theywereabletorecover85%ofEPAand88%ofDHAfromthefeedwithapurityof90%.ThemaximumenrichmentsinthisstudywereachievedusingaS/Fof340.TheauthorsconcludedthatatagivenpressuretheS/Fisprimarilygovernedbythetemperatureoftheuppermostzoneandisinsensitivetothelowerzonetemperaturesandflowrate.Increasingthetotalnumberoftem-peraturezonesandreducingthetemperatureatthetopofthecolumncanmaintainselectivitywhileloweringtheS/F.

Batch-continuousprocessingisinappropriateforlarge-scaleproductionbecauseprocessing costs are adversely affected by operating parameters, such as highS/Fvalues.Continuousprocessesaremoreefficient forproductionof largequan-tities of PUFAs. Krukonis [123] described a continuous countercurrent processfor fractionationof fattyacidestersand large-scaleproductionofEPAandDHAconcentrates.Inthisprocess,thefeedstreamiscontinuouslysuppliedtothetopofacolumn,whereitiscontactedbytheextractingsolventthatiscontinuouslyflowingintheoppositedirection.Thedirectionofflowdependsonthedensityofthetwofluids.Generally,forCO2countercurrentextractionoffishoils,theheavierfeedoilstreamflowsdownwardandthelighterCO2phaseflowsupward.Usingthestageconcept,Krukoniscalculatedthatatotalof13stageswererequiredtoobtainEPAof90%purityinthetopproduct(extractphase)andDHAofsimilarpurityintheraffinate.TheS/Fwassignificantlydecreasedtoaround30,whichismuchlowerthantheS/Ffor thebatch-continuousprocessesdescribedabove.Thecostofcarryingout thisprocesswasestimatedtobecomparabletotheproductionofω3fattyacidconcen-tratesusingconventionalmethods.

Riha and Brunner [124] investigated the continuous countercurrent fraction-ationofsardineoilFAEEswithsupercriticalCO2inthetemperatureandpressureranges313to353Kand6.5to19.5MPa,respectively.Theexperimentsfocusedonseparating low-molecular-weight components,with carbonnumbers ranging fromC14–C18,andhigh-molecular-weightcomponents(HMCs),C20–C22.AneconomicaloperationalpointwasdeterminedandtheprocesswasdeemedtobeusefulfortheenrichmentofHMCs,whichcouldbefurtherprocessedtoobtainfractionsrichinEPAandDHA.UsingaS/Fof63at353Kand19.5MPa,ina12mcolumnwithreflux(numberoftheoreticalplates=40),theHMCfractionswereobtainedingreaterthan95%puritywithgreaterthan95%yield.

ThePUFA-richfiltrateobtainedfromaureapreconcentrationstepalsocontainsuncomplexedurea.Thefattyacidsareusuallyrecoveredfromthefiltratebysolventextractionwithamixtureofanonpolarorganicsolvent,suchashexaneorisooctane,in which the urea is insoluble, and water in which the urea is soluble. The useof nonfood-grade organic solvents such as hexane in the extraction of PUFAs is

7089_C005.indd 171 10/8/07 11:54:13 AM


undesirable,particularlyiftheproductisintendedfordietarysupplementation.Lossof PUFAs may occur during the reduction of organic solvent residues to regula-tory levels.ApatentbyCatchpoleet al. [125]describesacombinedsupercriticalextraction-supercriticalantisolventprocessforextractingawiderangeoflipophiliccompoundsfromurea-containingsolutionsusingnear-criticalfluids,suchaspoly-unsaturatedfattyacidsfromfishoils.Thenear-criticalfluidiscontinuouslycontactedwiththeureafiltratesolution.Thesolventpropertiesofthenear-criticalfluidenableextractionofthefattyacid/alkylestersfromthesolution(alongwithethanol)whileantisolventpropertiesresultinurea(andwater)beingprecipitated.Thefattyacidsand ethanol can then be recovered separately by consecutive pressure-reductionsteps. Figure5.16 [126] shows the results for a single-stage urea fractionation oftunaoil(initialEPAandDHAcontentof5%and22%,respectively),andadouble-stageureafractionationofhokiliveroil(initialEPAandDHAcontentof6%and9%,respectively).ThesupercriticalantisolventprocessiscarriedoutafterthefinalstageofureaprocessingandseparatesureaandoxidationproductsfromthedesiredPUFAs. Typical antisolvent fractionation conditions are 333 K and 30 MPa. Thefinalproductscontainaround40%to50%DHA,andanω3contentof60%(tunaoil)andgreaterthan90%(hokiliveroil).

Kulas and Breivik [127] describe a process in which PUFA ethyl esters areextractedfromsolidurea/ethylestercomplexes.Thecomplexeswereobtainedfromthepriorureafractionationoffishoilethylesters.TheauthorsnotethatthePUFAsareselectivelyextractedduetothelowstabilityofthecomplex,whilestronglyboundmonounsaturatesarenotextracted.Theyalsonotethatsomeureaisextracted.Itisalsopossibletousesolidureaasaselectiveadsorbentforfattyacidsoralkylestersand CO2 or CO2 + ethanol as the mobile phase [125, 128]. The same processingconsiderationsapplyasforureasolutions:saturatesaremoststronglybound,andthestrengthofthebondingistemperaturedependent.Figure5.17[129]showsthebatch-continuousfractionationofhydrolyzedlingliveroil.Afattyacid/ethanolmix-turewascontinuouslymixedwithCO2andthenpassedthroughtwobedspackedwith finely ground solid urea. The resultant extract was around 50% DHA and

60

50

40

30

20

Feed Oil 1st Stage Feed Oil 1st Stage

Hoki Liver OilTuna Oil

% of

Fat

ty A

cid

Type

2nd Stage

10

0

SaturatesMonounsaturatesEPA, C20:5DPA, C22:5DHA, C22:6

FIgure 5.16 Fattyacidextractcompositionforsingle-stageureafractionationoftunaoilanddouble-stageureafractionationofhokiliveroil.

7089_C005.indd 172 10/8/07 11:54:14 AM


greaterthan90%ω3-PUFAsuntilaratioofureatofattyacidofapproximately5:1wasreached.Theprocesswasdeemednotsuitableforcommercialscaleoperationbecausethesolidureacomplexcouldnotberegeneratedin-situandbecamerockhardandverydifficulttoremovefromextractionvesselbaskets.

Supercriticalfluidchromatography(SFC)canofferthesamedegreeoffattyacidseparationas thatofferedbySCFextractionofurea-concentrated feeds,butonlyif thestationaryphasecanspecifically interactwithdoublebonds.Higashidateetal.[130]enrichedEPAandDHAfromesterifiedsardineoil(initialEPAandDHAcontentsof12%and13%,respectively)byextractingtheoilwithsupercriticalCO2anddirectlyintroducingtheresultingsolutionontoasilicagelcolumncoatedwithsilvernitrate.Extractionswerecarriedoutat313Kand8MPawithaCO2flowrateof9gmin–1.Chromatographicseparationswerecarriedoutat313Kusingpressureprogramming.Fivefractionswerecollectedsequentially.Using thismethod, theyobtainedEPA-andDHA-richfractionswithpuritiesof93%and82%,respectively.ThechromatographicoperatingconditionsandfractioncompositionsaregiveninTable5.9.Theorderof elutionwas in accordancewith the interactionof carbon-carbondoublebondswithsilver ions; theinteractionincreaseswithanincreasingdegreeofunsaturation,resultinginlongerelutiontimes.

Pettinelloetal.[131]investigatedtheproductionofEPA-enrichedmixturesfromfishoilusingSFCatboth laboratoryandpilot scale.Their startingmixturecon-tained67.6%EPAethylesters(EE)and6.1%arachidonicacid(AA;20:4ω6)-EE,whichwerefractionatedusingasilicaadsorptioncolumnandsupercriticalCO2astheelutingsolvent.Sampleswereanalyzedusingcapillaryandpackedcolumngaschromatography(GC).Theyusedandcomparedseveraltypesofsilicagelandinvesti-gateddifferentprocessoperatingconditions.Itwasemphasizedthatsampleloadingshould be optimized because this can strongly influence the yield obtained. Twomodesofoperationwerecarriedoutatthelaboratoryscale:1)constanttemperature

100

80

60

40

20

00 50 100 150

Polyunsaturated

DHA

EPA

MonounsaturatedSaturated

g Oil/kg Urea

% Co

mpo

sitio

n of

Ext

ract

200 250 300

FIgure 5.17 Extractcompositionversusfattyacid–urearatioforbatch-continuousfrac-tionationofhydrolyzedlingliveroilusingCO2andpackedbedsoffinelygroundsolidureaat290Kand30MPa.

7089_C005.indd 173 10/8/07 11:54:15 AM


withstepwiseincreasesinpressure(pressureprogramming)and2)constanttemper-atureandpressure.Duringthepressureprogrammingexperiments,thepressurewasraisedduringoperationinordertochangethesolubilityandselectivityoftheFAEEinsupercriticalCO2.Thepressurewasraisedfrom18.0MPato22.0or24.0MPaat343Kduringthechromatographicrun.Thelightercomponentswereelutedduringtheearlystagesoftheprocess,andtheEPA-EEremovalwasenhancedatthehigherpressures.Thepressureprogrammodeofoperationgave largeryieldsof agivenpurity(EPA-EE+AA-EEpurityof90%,yield49.0%),eveniftheconstantpressureoperation was carried out using a lower feed loading (EPA-EE + AA-EE purityof 90%, yield 10.0%). They reported that the selectivity obtained using pressureprogrammingwasnotsatisfactoryandsotheeffectoftemperatureonselectivitywasalsoinvestigated.Thetemperaturewasraisedto353Kandanoperatingpressureof20.8MPawasusedtogivethesameCO2fluiddensityasthatat343Kand18.0MPa.AreductioninretentiontimeandasharpeningoftheGCpeakwasnoted,indicatingthatsilicagelhasaweakerinteractionwithFAEEmoleculesathighertemperatures.A95%EPApuritywasachieved,withayieldof11%.ThepurityofEPAdroppedto90%whentheyieldwasincreasedto43%.

In the pilot-scale operation, Pettinello and coworkers [131] used a system inwhichtheCO2wasrecycled.Quantitiesoffeedmaterialsoftheorderofhundredsofgramswereprocessed.Thechromatographicfractionationswerecarriedoutusingtwotypesofsilicagel(75to200µm,60Åporesizeand75to200µm,40Åporesize),and theresearchersfoundthat thebehaviorofbothwassimilar in termsofseparationperformance.Intermsofeconomics,fortheprocesstobeusefulonanindustrialscale,itisimportantthatthesilicaadsorbentcanbeusedseveraltimes.Theresearchersshowed,usingthreesuccessiverunswiththesamecolumnofsilica,thatan84%pureEPAfractioncouldbeobtainedwithyieldsof34.3%,29.8%,and28.2%forruns1,2,and3,respectively.Pettinelloetal.[131]statedthataftertheinitialdeactivationofsilicaduringthefirstrun,theadsorbentmaterialmaintainedanacceptablelevelofactivity.Theeffectoffeedmass,pressure,andpressurepro-grammingwerealsoinvestigatedduringpilot-scaleoperation.TheEPA-EEpuritywasgreatestat93%with24.6%yieldwhenusingafeedmassof275.0g,atempera-tureof343K,andapressureprogrammingmethodintherange15.0to24.0MPa.

ApatentbyPerrutetal.[132]describesusingSFCforfishoilprocessinginacontinuous mode of operation by applying simulated countercurrent moving bed

table 5.9Chromatographic operating Conditions and Fraction Compositions from the Work of higashidate et al. [130]Fraction pressure (mpa) elution time (mins) % epa % dha

1 8 0–110 0 0

2 8 110–180 57 0

3 12 180–250 93 0

4 20 250–310 46 18

5 20 310–370 0 82

7089_C005.indd 174 10/8/07 11:54:15 AM


(SCMB)technology.Theauthorsclaimedthata93%EPA-EE-richfractionandan85%DHA-EE-richfractionwereachievedusingthecombinedSFC-SCMBmethod.In the first step, a starting mixture containing 32.8% EPA and 20.9% DHA waspassedthroughaSFCcolumn(300×60mm)at323Kand16.0MPausingRPC-18(octadecyl silica gel, 12 to 45 µm) as a stationary phase and a CO2 flow rate of40kghour–1.Theyobtaineda73.6%EPA-EE-richfractionwithayieldof67.1%(thisEPA-richfractioncontained15.1%DHA).TheDHA-EE-richfractionwasobtainedwith56.3%puritywithyield76.1%(thisDHA-richfractioncontained35.4%EPA).In a second step, these fractions were reprocessed using eight RPC-18 columns(100×80mm)at323Kand13.0MPawithaCO2flowrateof55kghour–1.Inthesecondstep,thetotalyieldsforbothEPAandDHAwere99%andthefractionpuri-tiesincreasedto93%and85%forEPA-EEandDHA-EE,respectively.

Alkioandcoworkers[133]studiedtheeconomicfeasibilityofproducinglargequantitiesofEPAandDHAfromtunaoilusingSFC.UsingsupercriticalCO2asthe mobile phase, they carried out a systematic study to find optimum processparametersformaximumproductionrate.At338Kand14.5MPa,usingoctadecylsilane-typereversed-phasesilicaas thestationaryphase,DHAandEPAcouldbeproducedsimultaneouslyinonechromatographicstepwithpuritiesof80%to95%and50%,respectively.Aprocessforproducing1,000kgofDHAand410kgofEPAconcentrateperyearrequires160kgofstationaryphaseand2.6tonshour–1ofCO2recycle.Theystated that thestationaryphasewouldpreferablybepacked in fourparallel600mm i.d. columnsand theestimatedcostof suchaplantwasaroundU.S.$2million.Assumingthatthestationaryphasewouldhavetobereplacedonceayear,theprocessoperatingcostswerecalculatedtobeU.S.$550perkilogramofDHAandEPAconcentrate(calculationswerebasedonmaterialandoperatingcostsfortheyear2000).

Enzymaticmethodsofenrichmenthavealsobeeninvestigatedundersupercriticalconditions.Linetal.[134]studiedtheenrichmentofω3-PUFAcontentinTAGsofmenhadenoilby lipase-catalyzed transesterification in supercriticalCO2.Prior toreaction,menhadenoilwastreatedbytheureainclusionmethodtoproducean80.1%ω3-PUFAconcentrate,71.2%ofwhichwasEPA+DHA.Using the immobilized1,3-regiospecific lipase, IM60fromMucor miehei, theauthorsstudied theeffectsofseveraloperatingparametersonthereaction,includingcosolventconcentration,reactiontime,temperature,pressure,andsubstrateratio(freeω3-PUFA:TAG).Forallreactions,theenzymeconcentrationwaskeptat10%w/wofthetotalsubstrates.Bothwater and ethanolwere examined as cosolvents andbothfluids exhibited amaximumforω3-PUFAcontentinTAGsasafunctionofconcentration.Thereac-tionwasstudiedwithawatercontentrangingfrom0%to10%w/wandamaximumω3-PUFAcontentof46%wasobservedat4%watercontent.Asimilartrendwasobservedforethanol(studiedintherange0%to15%w/w)withamaximumω3-PUFAcontentof56%beingobtainedat10%w/w.Following thisobservation, allotherreactionswerecarriedoutusing10%w/wethanolasacosolvent.Foraprocesstobeeconomicallyviable,optimumresultsmustbeobtainedwithintheshortesttime-frame.At323Kandpressuresrangingfrom10.3to20.7MPa,thetotalcontentofω3-PUFAsinTAGsincreasedwithtimeupto5hours,irrespectiveofpressure.Thetemperatureeffectonreactionwasinvestigatedintherange313to333K,anditwas

7089_C005.indd 175 10/8/07 11:54:16 AM


found that theω3-PUFAcontent inTAGs increasedwith increasing temperature.However,itshouldbenotedthatelevatedtemperaturesshouldbeusedwithcautionbecausemostproteindenaturationoccursat318to323K.Also,Kametetal.[135]notedthatbelow313K,CO2reactsreadilywiththefreeaminogroupontheenzymesurfacetoformacarbamate-enzymecomplex,whichreducesenzymeactivity.Thetotalω3-PUFAcontentinTAGsdecreasedwithincreasingpressure.Twopossibili-tieswereofferedtoexplainthisphenomenon:1)Thesuppressionofthethree-dimen-sionalmolecularstructureoftheactivesiteontheenzymeleadstoareductionofenzymeactivity;2)Atelevatedpressures,ahigherdissolutionofCO2intothewateronthesurfaceoftheenzymecausesadecreaseinpHandinducesthereversereac-tion.Theω3-PUFAcontentinTAGsalsoincreasedasthefreeω3-PUFA:TAGratioincreased.Underoptimumconditions(10%ethanol,5hourreactiontime,10.3MPa,323K,andasubstrateratioof4:1),theauthorswereabletoproduceTAGscontain-ing56%w/wofω3-PUFAsusingthismethod.

5.3.2.3 squalene and dage processing

Catchpole andcoworkers [101,136,137] investigated the continuousextractionofsqualenefromsharkliveroilinlaboratoryscale(5mLmin–1oilprocessingcapability)pilotscale(30mLmin–1oil)andproductionscale(1Lmin–1oil)packedcolumnplantandalaboratoryandpilotscalestaticmixerapparatususingsupercriticalCO2.Sepa-rationperformancewasdeterminedasafunctionoftemperature,pressure,oil-to-CO2flowrateratio,packedheight,staticmixerdimensions,packingtype,andrefluxratio.The initial shark liver oil contained 50% by weight squalene and 0.1% by weightpristane(C19H40),withthebalancebeingnonvolatiletriglyceridesandglycerylethers.ThepilotscalepackedcolumnapparatusisshowninFigure5.18.ThebasicapparatusconsistedofaCO2compressor,a2.5m×56mmi.d.packedcolumn,highpressurepistonpumpsforsupplyoftheliquidsharkliveroilandliquidreflux,andtwojack-eted separationvessels for the recoveryof squalene,fishodors, andpristane.CO2waspassedupwardthroughthecolumnatoperatingpressure.Thesharkliveroilwaspumpedintothetopofthefirst(withnoreflux)orsecond(withreflux)sectionofthecolumn.Therefluxliquidwaspumpedintothetopofthefirstsection.Theraffinate,whichishighlyenrichedinDAGEsandTAGsandstrippedofsqualene,wascollectedat regular timeintervals fromthebottomof thecolumn.TheCO2solutionpassedthroughapressurereductionvalveintothefirstseparationvessel,wherethebulkofthesqualenewasrecovered.Thefishodorsandpristanewererecoveredinthesecondseparationvesselbyfurtherpressurereduction,andCO2wasrecycled.Theproduc-tionscaleplant(Figure5.19)wasoperatedinasimilarmanner[101].Thelaboratoryscaleexperimentsalsousedthesamemethodology,althoughCO2wasnotrecycled.Thetotaloilloadingwasinvestigatedasafunctionofoil-to-CO2ratio(theratioofthetotalmassoftopproductfromthecolumntotheCO2massthathaspassedthroughthecolumnoveragiventime)overthetemperaturesandpressures313to333Kand12.5to25.0MPa,respectively.Investigationswerecarriedoutwithoutrefluxatfixedtemperatureandwithinternalrefluxusingatemperaturegradientovertheheightofthecolumnintherange313to333K.Theloadingfollowedthepatternofsqualenesolubility,withthehighestvaluesobtainedat333Kand25.0MPa,andthelowestat

7089_C005.indd 176 10/8/07 11:54:16 AM


313Kand20.0MPa.Pristanewasthemostsolubleoilcomponentandwasvirtuallycompletelyextractedfromthefeedoil,evenathighoil-to-CO2massratios.Pristaneisaskinirritantandisthusundesirableinsqualenethatisdestinedforuseincosmeticapplications.Separatorconditionswereoptimizedtomaximizetherecoveryofsqua-leneinthefirstseparator(313Kand9.0MPa),andmaximizingpristanerecoveryinthesecondseparator(313Kand6.0MPa).

Theeffectofpackingtype,scaleofoperation,andcountercurrentversuscocur-rentcontactingonmass transferefficiencywas investigated.Stainless steelwool,Raschigrings,andFenskeheliceswereinvestigatedaspackingsacrossarangeofpackedheightsatalaboratoryscale.NoreliableresultsusingFenskeheliceswereachievedduetoexcessivehold-upofliquidinthecolumn.Raschigringsgavepoorermass transfer performance than stainless steel wool. Other researchers have alsofoundthatRaschigringscomparedpoorlywithwirewooltypepackingsforlipid/near-criticalfluidpackedcolumnseparations[138,139].Theperformanceofwirewool also diminished significantly when only one packed section was used. Theauthorsconcludedthatatleast0.8mofpackingwasrequiredtoachieveahighlevelofseparationofsqualenefromtriglyceridesatalaboratoryscale,and2.5matapilotscale.Thepackedheightavailableinthedemonstrationscaleplantwasinsufficienttoachievemass transfersignificantlybetter thanastaticmixer,whichgivesonlyoneequilibriumstage(Figure5.20).Underoptimumconditionsatpilotscale,a92%

P P

P

P

P

P

H1

Air

CO2 Supply Cylinders

Cooling Water

Raffinate Squalene

Separation Vessel 1

Separation Vessel 2

RefluxPumpPiston

Pump Liquid Feed

Tank

Pristane

Hot Water System

Hot Water System

H2

H3

FIgure 5.18 ThepilotscalepackedcolumnapparatususedbyCatchpoleetal.[101,136,137]forthefractionationoffishoils.(ReprintedwithpermissionfromIndustrial and Engi-neering Chemistry Research,36,4318.©1997AmericanChemicalSociety.)

7089_C005.indd 177 10/8/07 11:54:18 AM


squalenebymassproductwasobtainedinseparator1atcolumnconditionsof333Kand25.0MPa.Usingthe92%squalenefractionasarefluxfeedstock,experimentswereperformedwithafixedratiooffeedoiltoCO2usingarangeofrefluxpumprates.Increasingtherefluxtofeedoilmassflowratiocausedboththetopproductstreamloadingandconcentrationofsqualeneinthetopproducttoincrease,asshowninFigure5.21.TheoilloadingincreasedlinearlytowardtheequilibriumloadingofpuresqualeneinCO2,withincreasingreflux-to-feedratio.SincethemassflowofthefeedoilandCO2wasfixed,therewasalsoanincreaseofsqualeneconcentrationintheraffinatewithincreasingrefluxratio.Toachieveoptimumseparationperfor-manceintermsofproductpurityandlossinraffinate,itisdesirabletouserefluxofthetopproductwithaloweredfeedrateofsharkliveroilatfixedCO2flowrate.Pilot-scaleoperationunderoptimumprocessconditionswithrefluxyieldeda99%bymasssqualene-richfractioncontaininglessthan0.5%pristane,withalossoflessthan5%bymassofsqualeneintheraffinatestream.

5.3.2.4 Vitamin a processing

The processing of fish oils to recover fractions enriched in vitamin A was firstcarriedoutusingnear-criticalpropane [140].Amulticolumnprocesswasused to

FIgure 5.19 Production scaleplantused for fractionationof squalene fromshark liveroil[101].

7089_C005.indd 178 10/8/07 11:54:19 AM


100

80

60

40

20

00 1 2

Extract

L/GK, Ratio of Flow Rates and Equilibrium Coefficient

% Sq

uale

ne in

Ext

ract

and

Raffi

nate

Raffinate

‒ Lab Scale Static Mixer‒ Lab Scale Packed Column‒ Demo Scale Packed Column

3 4 5

FIgure 5.20 Extractandraffinateconcentrationsofsqualeneforstaticmixer,laboratoryscale,anddemonstrationscalecolumnsversusmodifiedoil-to-CO2flowrateratio.

100 28

26

24

22

20

18

16

80

60

Squa

lene

Con

cent

ratio

n, %

by M

ass

Oil

Load

ing,

g /k

g of

CO

2

40

20

00.00 0.05 0.10 0.15

Product Squalene ConcentrationRaffinate Squalene ConcentrationOil Loading

Reflux to Feed Oil Mass Ratio0.20 0.25 0.30 0.35 0.40

FIgure 5.21 Squalene concentration in the top product and oil loading in CO2 as afunctionofrefluxtofeedmassratio[136].(ReprintedwithpermissionfromIndustrial and Engineering Chemistry Research,36,4318.©1997AmericanChemicalSociety.)

7089_C005.indd 179 10/8/07 11:54:20 AM


fractionatemenhadenandcodliveroilsbyutilizingdifferencesinsolubilityofoilcomponents near to, but below, the critical point. In the first column, conditionsarechosensuch that theoil ismiscibleexcept forcolorcomponentsandnonlipidcontaminants.Insubsequentcolumns,thesolubilityisprogressivelyreducedtogivefractionsenriched inpolyunsaturatesuntil thefinal column,wherein thevitaminA-rich(mostsoluble)fractionisrecovered,isreached.ThisprocessfelloutoffavorwhensyntheticvitaminAataneconomicallycompetitivepricebecameavailable.

Catchpoleetal.[101]carriedoutthecountercurrentextractionofvitaminAfrommodelfishoilmixturesusingsupercriticalCO2.ThemodelmixturesweremadetosimulatetheliveroilsofsurfacedwellingsharksthathavehighvitaminAcontents[141].VitaminApalmitateisthepredominantformofvitaminAinfishoils[142].TheremainderofthesharkliveroilisaTAGformthathassimilarfattyacidcompo-sitionstocodliveroil[143,144].Therefore,separationswerecarriedoutusingcodliveroilandvitaminApalmitatemixtures,withvitaminAconcentrationsrangingfrom1%to20%bymass.Theseparationfactorwaslowduetosimilarsolubilitiesofthevitaminesterandthenon-esterifiedoil.ExtractionswerealsocarriedoutusingmixturesofcodliveroilethylestersandvitaminAasthefreealcohol.Thesolubil-ityofthefreealcoholismuchlowerthanFAEEsatlowtomoderatepressures(9to12MPa),andvitaminAwaspreferentiallyrecoveredintheraffinate.Theresultsofthefractionationexperimentsforcodliveroil/vitaminApalmitateandcodliveroilethyl esters/vitaminAare shown inFigure5.22.TheconcentrationofvitaminApalmitate(codliveroil)intheextractwasenhancedoverthatoftheraffinateandtheenhancementwasnotstronglypressuredependent.Highlossesofthevitaminester

50

Cod Liver Esters

45

40

35

Extr

act V

itam

in C

once

ntra

tion,

% b

y Mas

s

Extr

act V

itam

in C

once

ntra

tion,

% b

y Mas

s

30

25

20

15

10

5

0 5 10 15Raffinate Vitamin Concentration, % by Mass

20 25

1.00

0.80

0.60

0.40

0.20

0.00

2 %, 313 K, 9.5 MPa2 %, 313 K, 10.0 MPa1.8 %, 333 K, 13.0 MPa4.2 %, 333 K, 13.5 MPa

Cod Liver Oil5 %, 27.5 MPa5 %, 30.0 MPa20 %, 25.0 MPa20 %, 27.5 MPa20 %, 30.0 MPa

0

FIgure 5.22 FractionationofvitaminA/codliveroilandethylestermixtures[101].Filledsymbols:⦁,◾,▲,▼,♦ExtractionofvitaminApalmitatefromcodliveroil;hollowsymbols:•,◽, , ExtractionofvitaminAfromfattyacidethylesters.(ReprintedfromJournal of Supercritical Fluids,19,25,©2000.WithpermissionfromElsevier.)

7089_C005.indd 180 10/8/07 11:54:22 AM


occurred in the raffinate. Increasing the extract to raffinate ratio can reduce thisloss,butthisresultsinlowerextractconcentrations.Theseparationwassubstantiallyimprovedwhenusingcodliveroilethylesters.ThemajorityoffattyacidesterswereextractedfromthefeedtoleavearaffinateenrichedinvitaminA.Theconcentrationofthevitaminintheextractdidnotexceed0.5%.

5.3.2.5 processing of other marine oil Components

Waxesteroilswereprocessedusingthepilotscalesupercriticalpilotplantdescribedearlier for theprocessingof squalene from shark liveroil [101].Thefishoilwaspartially degraded due to extended storage at room temperature and had highperoxidelevels.CO2+ethanolwasusedasthesolvent,whichwascountercurrentlycontactedwiththewaxesteroil.Ahighoil-to-solventratiocouldbeusedduetothehighsolubilityoftheoilmixtureinthesolventphase.High-molecular-weightestersand astaxanthin were recovered in the raffinate, and medium-molecular-weightesterswererecoveredinthefirstseparator,alongwithpartoftheethanol.Theover-allextracthadveryhighperoxidevalues.Theextractmixtureseparatedinto twophases,atopwaxester-richphase,andabottomethanol-richphase,whichalsocon-tainedmostoftheperoxidesandmalodorouscompoundspresentintheoriginaloil.Thefinalseparatorcontainedlargelyethanol,withvolatileodorcompounds.

Theextractionofgreen-lippedmusseloil,soldunderthenameLyprinol™,usingsupercriticalCO2hasbeencarriedoutcommerciallyforseveralyears.ThemusselsareendemictoNewZealand.Theoilhasanti-inflammatorypropertiesandhasfoundappli-cationasanantiarthriticandantiasthmaticnaturalremedy.TheextractionprocessandpropertiesoftheextractaredescribedinapatentbyMacridesandKalafatis[145].Theoilisacomplexmixtureoffreefattyacids,TAGs,sterols,andsterolesters[146].

5.4 summary

Theconventionalmethods for isolationofhigh-valuefishoil components includevacuumdistillation,ureacrystallization,hexaneextraction,andconventionalcrys-tallization. These methods have the disadvantages of requiring high processingtemperatures,resultinginthethermaldegradationordecompositionofthethermallylabile compounds or employing flammable or toxic solvents, which have adversehealtheffects.Inthisinstance,separationsemployingSCFtechnologiesoffernewopportunitiesforresolvingtheseseparationproblems.

reFerenCes

1. Gruger,E.H.,Nelson,R.W.andStansby,M.E.,Fattyacidcompositionofoilsfrom21speciesofmarinefish,freshwaterfishandshellfish,J. Am. Oil Chem. Soc.,41,662,1964.

2. Burr,G.O.andBurr,M.M.,Anewdeficiencydiseaseproducedbyrigidexclusionoffatsfromthediet,J. Biol. Chem.,82,345,1929.

3. Dyerberg,J.,Linolenate-derivedpolyunsaturatedfattyacidsandpreventionofathero-sclerosis,Nutr.Rev.,44,125,1986.

7089_C005.indd 181 10/8/07 11:54:22 AM


4. Mehta, J., Lopez, L.M. and Wargovich, T., Eicosapentaenoic acid: Its relevance inatherosclerosisandcoronaryarterydisease,Am. J. Cardiol.,59,155,1987.

5. Urakaze,M.etal.,Infusionofemulsifiedtreicosapentaenoyl-glycerolintorabbits:Theeffectsonplateletaggregation,polymorphonuclearleukocyteadhesion,andfattyacidcompositioninplasmaandplateletphospholipids,Thromb. Res.,44,673,1986.

6. Kremer,J.M.etal.,Effectsofmanipulationofdietaryfattyacidsonclinicalmanifesta-tionsofrheumatoidarthritis,Lancet, 1,184,1985.

7. Braden,L.M.andCarroll,K.K.,Dietarypolyunsaturatedfatinrelationtomammarycarcinogenesisinrats,Lipids,21,285,1986.

8. Reddy,B.S.andMaruyama,H.,Effectofdietaryfishoilonazoxymethane-inducedcoloncarcinogenesisinmaleF344rats,Cancer Res.,46,3367,1986.

9. Svaneborg,N.etal.,Theacuteeffectsofasingleveryhighdoseofn-3fattyacidsonplasmalipidsandlipoproteinsinhealthysubjects,Lipids,29,145,1994.

10. Frontini, M.G. et al., Distribution and cardiovascular risk correlates of serumtriglyceridesinyoungadultsfromabiracialcommunity:TheBogalusaHeartStudy,Atherosclerosis,155,201,2001.

11. Morris,M.C.,Sack,F. andRosner,B.,Doesfishoil lowerbloodpressure?Ameta-analysisofcontrolledtrials,Circulation,88,523,1993.

12. Weisinger,H.S.etal.,Perinatalomega-3fattyaciddeficiencyaffectsbloodpressurelaterinlife,Nature Med.,7,258,2001.

13. Ziboh,V.A.,Miller,C.C.andCho,Y.,Metabolismofpolyunsaturatedfattyacidsbyskinepidermalenzymes:Generationofantiinflammatoryandantiproliferativemetab-olites,Am. J. Clin. Nutr.,71,S361,2000.

14. Brown,M.D.etal.,PromotionofprostaticmetastaticmigrationtowardshumanbonemarrowstomabyOmega6anditsinhibitionbyOmega3PUFAs,Br. J. Cancer,94,842,2006.

15. Shirota,T.etal.,Apoptosisinhumanpancreaticcancercellsinducedbyeicosapentaenoicacid,Nutrition,21,1010,2005.

16. Yonezawa,Y.etal.,Inhibitoryeffectofconjugatedeicosapentaenoicacidonmamma-lianDNApolymeraseandtopoisomeraseactivitiesandhumancancercellproliferation,Biochem.Pharmacol.,70,453,2005.

17. Jho, D.H. et al., Eicosapentaenoic acid supplementation reduces tumor volume andattenuates cachexia in a rat model of progressive non-metastasizing malignancy,J.Parenter.EnteralNutr.,5,291,2002.

18. Adams,P.B.etal.,Arachidonicacidtoeicosapentaenoicacidratioinbloodcorrelatespositivelywithclinicalsymptomsofdepression,Lipids,31,S157,1996.

19. Assies, J. et al., Significantly reduced docosahexaenoic and docosapentaenoic acidconcentrationsinerythrocytemembranesfromschizophrenicpatientscomparedwithacarefullymatchedcontrolgroup,Biol. Psychiatry,49,510,2001.

20. Ayton, A.K., Azaz, A. and Horrobin, D.F., A pilot open case series of ethyl-EPAsupplementationinthetreatmentofanorexianervosa,Prostaglandins, Leukot. Essent. Fat. Acids,71,205,2004.

21. Mitchell, D.C. et al., Why is docosahexaenoic acid essential for nervous systemfunction?Biochem. Soc. Trans.,26,365,1998.

22. Buranudeen,F.,Squalene,Infofish Market. Digest,1,42,1986. 23. Gopakumar, K. and Thankappan, T.K., Squalene. Its sources, uses, and industrial

applications,Seafood Export J.,17,1986. 24. McKenna,R.M.,Wheatley,U.R.andWarmall,A.,Thecompositionofthesurfaceskin

fat(subum),J. Invest. Dermatol.,15,33,1950. 25. Langdon,R.G.andBloch,K.,Theutilizationofsqualeneinthebiosynthesisofcholes-

terol,J.Biol.Chem.,200,135,1953.

7089_C005.indd 182 10/8/07 11:54:23 AM


26. Gloor,M.andKarenfeld,A.,Effectofultravioletlighttherapy,givenoveraperiodofseveralweeks,on theamountandcompositionof the skin surface lipids,Dermato-logica,154,5,1977.

27. Kohno, Y. et al., UV-B exposure to skin surface: Photo-oxidation of surface lipid,J. Jpn. Oil Chem. Soc. (Yukagaku),42,204,1993.

28. Kohno,Y.etal.,Kineticstudyofquenchingreactionofsingletoxygenandscavengingreactionoffreeradicalbysqualeneinn-butanol,Biochem. Biophys. Acta: Lipids and Lipid Metab.,1256,52,1995.

29. Storm,H.M.etal.,Radioprotectionofmicebydietarysqualene,Lipids,28,55,1993. 30. Strandberg, T.E., Tilvis, R.S. and Miettinen, T.A., Variations of hepatic cholesterol

precursors during altered flows of endogenous and exogenous squalene in the rat,Biochim. Biophys. Acta: Lipids and Lipid Metab.,1001,150,1989.

31. Strandberg,T.E.,Tilvis,R.S.andMiettinen,T.A.,Metabolicvariablesofcholesterolduring squalene feeding in humans: Comparison with cholestyramine treatment,J. Lipid Res.,31,1637,1990.

32. Reddy, B.S., Dietary fat and colon cancer: Animal model studies, Lipids, 27, 807,1992.

33. Desai,K.N.,Wei,H.andLamartiniere,C.A.,Thepreventiveandtherapeuticpotentialof the squalene-containing compound, Roidex, on tumor promotion and regression,Cancer Lett.,101,93,1996.

34. Rao,C.V.,Newmark,H.L. andReddy,B.S.,Chemopreventive effectof squaleneoncoloncancer,Carcinogenesis,19,287,1998.

35. Das,B.,The Science Behind Squalene, the Human Antioxidant,1sted.,UniversityofTorontoPress,Toronto,Canada,2000.

36. Wetherbee,B.M.andNichols,P.D.,Lipidcompositionoftheliverofdeep-seasharksfromtheChathamRise,NewZealand,Comp.Biochem.Physiol.B,125,511,2000.

37. Bakes,M.J.andNichols,P.D.,Lipid,fattyacid,andsqualenecompositionofliveroilfromsix speciesofdeep-sea sharks collected in southernAustralianwaters,Comp.Biochem.Physiol.B,110,267,1995.

38. Hallgren, B., Therapeutic effects of ether lipids, in Ether Lipids: Biochemical and Biomedical Aspects,Mangold,H.K.andPaltauf,F.,Eds.,AcademicPress,NewYork,1983,261.

39. Saffioti,U.etal.,Experimentalcancerofthelung,Cancer,20,857,1967. 40. Bershad,S.,Developments in topical retinoid therapy for acne,Semin.Cutan. Med.

Surg.,20,154,2001. 41. Wolf,G.,AhistoryofvitaminAandretinoids,FASEB J.,10,1102,1996. 42. Underwood,B.A.,VitaminAdeficiencydisorders: Internationalefforts tocontrol a

preventablePox,J.Nutr.,134,231,2004. 43. Swern,D.,Ed.,Baily’s Industrial Oil and Fat Products,Vol.1,4thEd.,JohnWileyand

Sons,NewYork,1979,81. 44. Buisson,D.H.etal.,Oilfromdeepwaterfishspeciesasasubstituteforspermwhale

andjojobaoils,J.Am.Oil Chem.Soc.,59,390,1982. 45. Hagemann,J.W.andRothfus,J.A.,Oxidativestabilityofwaxestersbythermogravi-

metricanalysis,J.Am.Oil Chem.Soc.,56,629,1979. 46. Nevenzel,J.C.,Occurrence,function,andbiosynthesisofwaxestersinmarineorganisms,

Lipids,5,308,1970. 47. Sargent,J.R.,Lee,R.F.andNevenzel,J.C.,Marinewaxes,inChemistry and Biochemistry

of Natural Waxes,Kolattukudy,P.E.,Ed.,ElsevierScientificPublishing,Amsterdam,1976,80.

48. Warth,A.H.,The Chemistry and Technology of Waxes,Reinhold,NewYork,1947,89.

7089_C005.indd 183 10/8/07 11:54:24 AM


49. Body, D.R., Johnson, C.B. and Shaw, G.J., The monounsaturated acyl- and alkyl-moietiesofwaxestersandtheirdistributionincommercialorangeroughy(Hoplostethus atlanticus)oil,Lipids,20,680,1985.

50. Fournier, V. et al., Thermal degradation of long-chain polyunsaturated fatty acidsduringdeodorizationoffishoil,Eur. J. Lipid Sci. Technol.,108,33,2006.

51. Stout,V.F.etal.,Fractionationoffishoilsandtheirfattyacids,inFish Oils in Nutrition,Stansby,M.E.,Ed.,VanNostrandReinhold,NewYork,1990,73.

52. Ackman,R.G.,Ke,P.J.andJangaard,P.M.,Fractionalvacuumdistillationofherringoilmethylesters,J.Am.Oil Chem.Soc.,50,1,1973.

53. Privett, O.S., Weber, R.P. and Nickell, E.C., Preparation and properties of methylarachidonatefromporkliver,J. Am. Oil Chem. Soc.,36,443,1959.

54. Privett,O.S.andNickell,E.C.,Preparationofhighlypurified fattyacidsvia liquid-liquidpartitionchromatography,J. Am. Oil Chem. Soc.,40,189,1963.

55. Weitkamp,A.W.,Distillation,J. Am. Oil Chem. Soc.,32,640,1955. 56. Chawla,P.anddeMan,J.M.,Measurementofthesizedistributionoffatcrystalsusing

alaserparticlecounter,J. Am. Oil Chem. Soc.,76,329,1990. 57. Singleton,W.S.,Solutionproperties,inFatty Acids,Markley,K.S.,Ed.,Interscience

Publishers,NewYork,1960,609. 58. Haraldsson,G.G.,Separationof saturated/unsaturated fattyacids,J. Am. Oil Chem.

Soc.,61,219,1984. 59. Rubin,D.andRubin,E.J.,USPatentnumber-US4792418,1989. 60. Brown, L.B. and Kolb, D.X., Application of low temperature crystallization in the

separationoffattyacidsandtheircompounds,Prog. Chem. Fats Lipids,3,57,1955. 61. Bengen,M.F.,GermanPatent869070,1953. 62. Domart,C.,Miyauchi,D.T.andSumerwell,W.N.,Thefractionationofmarineoilfatty

acidswithurea,J. Am. Oil Chem. Soc.,32,481,1955. 63. Swern,D.,Techniquesofseparation:E.ureacomplexes,inFatty Acids: Their Chemistry,

Properties, Production, and Uses, Part 3, 2nd Ed., Markley, K.S., Ed., IntersciencePublishers,NewYork,1964,2309.

64. Abu-Nasr,A.M.andHolman,R.T.,Highlyunsaturatedfattyacids.II.Fractionationbyureainclusioncompounds,J. Am. Oil Chem. Soc.,31,16,1954.

65. RoblesMedina,A.etal.,Concentrationandpurificationofstearidonic,eicosapentae-noic,anddocosahexaenoicacidsfromcodliveroilandthemarinemicroalgaIsochrysis galbana,J. Am. Oil Chem. Soc.,72,575,1995.

66. Wille,H.J.,Traitler,H.andKelly,M.,Productionofpolyenoicfishoilfattyacidsbycombinedureafractionationandindustrialscalepreparativehighperformanceliquidchromatography,Rev.Fr.Corps Gras,34,69,1987.

67. Ratnayake,W.M.N.etal.,Preparationofomega-3PUFAconcentratesfromfishoilsviaureacomplexation,Fat Sci. Technol.,90,381,1988.

68. Traitler,H.,Wille,H.J.andStuder,A.,Fractionationofblackcurrantseedoil,J. Am. Oil Chem. Soc.,65,755,1988.

69. Wanasundara,U.N.andShahidi,F.,Concentrationofomega-3polyunsaturatedfattyacidsofsealblubberoilbyureacomplexation:Optimizationof reactionconditions,Food Chem.,65,41,1999.

70. RoblesMedina,A.etal.,Downstreamprocessingofalgalpolyunsaturatedfattyacids,Biotechnol. Adv.,16,517,1998.

71. Haagsma,N.etal.,Preparationofanomega-3fattyacidconcentratefromcodliveroil,J. Am. Oil Chem. Soc.,59,117,1982.

72. Schlenk,H.,Progress in the Fats and Other Lipids,Vol.2,PergamonPress,London,1954,243.

73. Smith,A.E.,Thecrystalstructureoftheurea-hydrocarboncomplexes,Acta Cryst.,5,224,1952.

7089_C005.indd 184 10/8/07 11:54:24 AM


74. Beebe, L.M., Brown, P.R. and Turcotte, L.G., Preparative-scale-high-performanceliquidchromatographyofomega-3polyunsaturatedfattyacidestersderivedfromfishoil,J.Chromatogr.,495,369,1988.

75. Adlof,R.O.andEmiken,E.A.,Theisolationofomega-3polyunsaturatedfattyacidsandmethylestersoffishoilsbysilverresinchromatography,J.Am.Oil Chem.Soc.,62,1592,1985.

76. Teshima,S.,Kanazawa,A.andTokiwa,S.,Separationofpolyunsaturatedfattyacidsbycolumnchromatographyonsolvernitrate-impregnatedsilicagel,Bull.J.Soc.Sci.Fish,44,927,1978.

77. Guil-Guerrero,J.L.andBelarbi,E-H.,Purificationprocessforcodliveroilpolyunsatu-ratedfattyacids,J.Am.Oil Chem.Soc.,78,477,2001.

78. Perrut,M.,Purificationofpolyunsaturatedfattyacid(EPAandDHA)ethylestersbypreparativehighperformanceliquidchromatography,LC-GC,6,914,1988.

79. Shahidi, F. and Wanasundara, U.N., Omega-3 fatty acid concentrations: nutritionalaspectsandproductiontechnologies,Trends in Food Sci.Technol.,9,230,1998.

80. El-Boustani,S.etal.,Enteralabsorptioninmanofeicosapentaenoicacidindifferentchemicalforms,Lipids,22,711,1987.

81. Lawson,L.D.andHughes,B.G.,Humanabsorptionoffishoilfattyacidsastriacylglycerols,freeacids,orethylesters,Biochem. Biophys. Res. Commun.,152,328,1988.

82. He,Y.andShahidi,F.,Enzymaticesterificationofω3-fattyacidconcentratesfromsealblubberoilwithglycerol,J.Am.Oil Chem.Soc.,74,1133,1997.

83. Bottino,N.R.,Vandenberg,G.A.andReiser,R.,Resistanceofcertainlong-chainpoly-unsaturatedfattyacidsofmarineoils topancreaticlipasehydrolysis,Lipids,2,489,1967.

84. Garcia,H.S.etal.,Synthesisofglyceridescontainingn-3fattyacidsandconjugatedlinoleicacidbysolvent-freeacidolysisoffishoil,Biotechnol.Bioeng.,70,587,2000.

85. Hoshino,T.,Yamane,T.andShimizu,S.,Selectivehydrolysisoffishoilbylipasetoconcentraten-3polyunsaturatedfattyacids,Agri.Biol.Chem.,54,1459,1990.

86. Tanaka, Y., Hirano, J. and Funada, T., Concentration of docosahexaenoic acid inglyceridebyhydrolysisoffishoilwithCandida cylindracealipase,J.Am.Oil Chem.Soc.,69,1210,1992.

87. Gámez-Meza,N.etal.,Concentrationofeicosapentaenoicacidanddocosahexaenoicacidfromfishoilbyhydrolysisandureacomplexation,Food Res.Int.,36,721,2003.

88. Schmitt-Rozieres,M.,Deyris,V.andComeau,L-C.,Enrichmentofpolyunsaturatedfattyacidsfromsardinecanneryeffluentsbyenzymaticselectiveesterification,J.Am.Oil Chem.Soc.,77,329,2000.

89. Zuyi, L. and Ward, O.P., Lipase-catalyzed alcoholysis to concentrate the n-3 poly-unsaturatedfattyacidofcodliveroil,Enzyme Microb.Technol.,15,601,1993.

90. Breivik,H.,Haraldsson,G.G.andKristinsson,B.,Preparationofhighlypurifiedcon-centratesofeicosapentaenoicacidanddocosahexaenoicacid,J.Am.Oil Chem.Soc.,74,1425,1997.

91. Chrastil,J.,Solubilityofsolidsandliquidsinsupercriticalgases,J.Phys.Chem.,86,3016,1982.

92. Catchpole,O.J.andvonKamp, J-C.,Phaseequilibria for theextractionof squalenefromsharkliveroilusingsupercriticalcarbondioxide,Ind.Eng.Chem.Res.,36,3762,1997.

93. Jaubert,J-N.andConiglio,L.,Fromthecorrelationofbinarysystemsinvolvingsuper-criticalCO2andfattyacidesterstothepredictionof(CO2-fishoil)phasebehaviour,Ind.Eng.Chem.Res.,38,3162,1999.

94. Coniglio,L.,Knudsen,K.andGani,R.,Modelpredictionofsupercriticalfluid-liquidequilibriaforcarbondioxideandfishoilrelatedcompounds,Ind.Eng.Chem.Res.,34,2473,1995.

7089_C005.indd 185 10/8/07 11:54:25 AM


95. Coniglio,L.,Knudsen,K.andGani,R.,Predictionofsupercriticalfluid-liquidequi-libriaforcarbondioxideandfishoilrelatedcompoundsthroughtheequationofstate-excessfunction(EOS-gE)approach,Fluid Phase Equilib.,116,510,1996.

96. Espinosa, S., Diaz, S. and Brignole, E.A., Thermodynamic modelling and processoptimizationofsupercriticalfluidfractionationoffishoilfattyacidethylesters,Ind.Eng.Chem.Res.,41,1516,2002.

97. Bamberger,T.etal.,Measurementandmodelpredictionofsolubilitiesofpurefattyacids,puretriglycerides,andmixturesoftriglyceridesinsupercriticalcarbondioxide,J.Chem.Eng.Data,33,327,1998.

98. Güçlü-Üstündag,Ö.andTemelli,F.,Correlatingthesolubilitybehaviouroffattyacids,mono-,di-,andtriglycerides,andfattyacidestersinsupercriticalcarbondioxide,Ind.Eng.Chem.Res.,39,4756,2000.

99. Catchpole,O.J.,Grey,J.B.andNoermark,K.A.,SolubilityoffishoilcomponentsinsupercriticalCO2andCO2+ethanolmixtures,J.Chem.Eng.Data,43,1091,1998.

100. Johannsen,M.andBrunner,G.,Solubilitiesofthefat-solublevitaminsA,D,E,andKinsupercriticalcarbondioxide,J.Chem.Eng.Data,42,106,1997.

101. Catchpole,O.J.,Grey,J.B.andNoermark,K.A.,Fractionationoffishoilsusingsuper-criticalCO2andCO2+ethanolmixtures,J.Supercrit.Fluids,19,25,2000.

102. Temelli,F.andGüçlü-Üstündag,Ö.,Supercriticaltechnologiesforfurtherprocessingofedibleoils,inBailey’s Industrial Oil & Fat Products,Vol.5,6thEd.,Shahidi,F.,Ed.,JohnWiley&Sons,NewJersey,2005,397.

103. Maheshwari,P.etal.,Solubilityoffattyacidsinsupercriticalcarbondioxide,J.Am.OilChem.Soc.,69,1069,1992.

104. Kramer,A.andThodos,G.,Solubilityof1-octadecanolandstearicacidinsupercriticalcarbondioxide,J.Chem.Eng.Data,34,184,1989.

105. Nilsson,W.B.,Gauglitz,E.J.,Jr.andHudson,J.K.,Solubilitiesofmethyloleate,oleicacid,oleylglycerols,andoleylglycerolmixturesinsupercriticalcarbondioxide,J.Am.Oil Chem.Soc.,68,87,1991.

106. Škerget,M.,Knez,Ž.andHabulin,M.,Solubilityofα-caroteneandoleicacidindenseCO2anddatacorrelationbyadensitybasedmodel,Fluid Phase Equilib., 109,131,1995.

107. Chen,C-C.,Chang,C-M.J.andYang,P-W.,Vapor-liquidequilibriumofcarbondioxidewithlinoleicacid,a-tocopherolandtrioleinatelevatedpressures,Fluid Phase Equilib.175,107,2000.

108. Dandge,D.K.,Heller,J.P.andWilson,K.V.,Structuresolubilitycorrelations:Organiccompoundsanddensecarbondioxidebinarysystems,Ind.Eng.Chem. Prod.Res.Dev.,24,162,1985.

109. Staby,A.,Forskov,T.andMollerup,J.,Phaseequilibriaoffishoilfattyacidethylestersandsub-andsupercriticalCO2,Fluid Phase Equilib.,87,309,1993.

110. Borch-Jensen,C.,Staby,A.andMollerup,J.M.,Phaseequilibriaofurea-fractionatedfishoilfattyacidethylestersandsupercriticalcarbondioxide,Ind.Eng.Chem.Res.,33,1574,1994.

111. Borch-Jensen, C. and Mollerup, J., Phase equilibria of long-chain polyunsaturatedfishoilfattyacidethylestersandcarbondioxide,ethane,orethylenearereducedgastemperaturesof1.03and1.13,Fluid Phase Equilib.,161,169,1999.

112. Ruivo, R., Paiva, A. and Simoes, P., Phase equilibria of the ternary system methyloleate/squalene/carbondioxideathighpressureconditions,J.Supercrit.Fluids,29,77,2004.

113. Walsh,J.M.,Ikonomou,G.D.andDonohue,M.D.,Supercriticalphasebehaviour:Theentrainereffect,Fluid Phase Equilib.,33,295,1987.

114. Sovova,H.etal.,SolubilityofsqualeneanddinonylphthalateinCO2withentrainers,J.Supercrit.Fluids,14,145,1999.

7089_C005.indd 186 10/8/07 11:54:26 AM


115. Nilsson, W.B., Seaborn, G.T. and Hudson, J.K., Partition coefficients for fatty acidestersinsupercriticalfluidCO2withandwithoutethanol,J.Am.Oil Chem.Soc.,69,305,1992.

116. Staby, A. and Mollerup, J., Separation of constituents of fish oil using supercriticalfluids:Areviewofexperimentalsolubility,extraction,andchromatographicdata,Fluid Phase Equilib.,91,349,1993.

117. Brunner,G.andDohrn,R.,High-pressurefluidphaseequilibria:Experimentalmethodsandsystemsinvestigated(1988–1993),Fluid Phase Equilib.,106,213,1995.

118. Christov,M.andDohrn,R.,High-pressurefluidphaseequilibria:Experimentalmethodsandsystemsinvestigated(1994–1999),Fluid Phase Equilib.,202,153,2002.

119. Krukonis,V.J.,Supercriticalfluidfractionationoffishoils.Concentrationsofeicosapen-taenoicacid,J.Am.OilChem.Soc.,61,698,1984.

120. Eisenbach,W.,Supercriticalfluidextraction:Afilmdemonstration,Ber.Bunsenges.Phys.Chem.,88,882,1984.

121. Nilsson,W.B.etal.,FractionationofmenhadenoilethylestersusingsupercriticalfluidCO2,J.Am.Oil Chem.Soc.,65,109,1988.

122. Nilsson,W.B.,Gauglitz,E.J.,Jr.andHudson,J.K.,Supercriticalfluidfractionationoffishoil estersusing incremental pressureprogramming anda temperaturegradient,J.Am.Oil Chem.Soc.,66,1596,1989.

123. Krukonis, V.J., Processing with supercritical fluids: Overview and applications, inSupercritical Fluid Extraction and Chromatography: Techniques and Applications,ACS Symposium Series number 366, Charpentier, B.A. and Sevenants, M.R., Eds.,AmericanChemicalSociety,Chicago,1988,26.

124. Riha,V.andBrunner,G.,Separationoffishoilethylesterswithsupercriticalcarbondioxide,J.Supercrit.Fluids,17,55,2000.

125. Catchpole,O.J.,MacKenzie,A.D.andGrey,J.B.,PatentWO03/089399,US2006035350,2003.

126. Catchpole,O.J.etal.,Unpublisheddata. 127. Kulas,E.andBreivik,H.,PatentWO01/10809,US6528669,2001.128. Hiroshi,U.andHiroshi,S.,PatentJP60214757,1985.129. Catchpole,O.J.etal.,Unpublisheddata.130. Higashidate,S.,Yamauchi,Y.andSaito,M.,Enrichmentofeicosapentaenoicacidand

docosahexaenoicacidestersfromesterifiedfishoilbyprogrammedextraction-elutionwithsupercriticalcarbondioxide,J.Chromatogr.,515,295,1990.

131. Pettinello,G.etal.,ProductionofEPAenrichedmixturesbysupercriticalfluidchroma-tography:fromthelaboratoryscaletothepilotplant,J.Supercrit.Fluids,19,51,2000.

132. Perrut,M.,Nicoud,R.M.andBreivik,H.,U.S.Patent5719302,1998.133. Alkio,M.etal.,Purificationofpolyunsaturated fattyacidesters fromtunaoilwith

supercriticalfluidchromatography,J.Am.Oil Chem.Soc.,77,315,2000.134. Lin,T-J.,Chen,S-W.andChang,A-C.,Enrichmentofn-3PUFAcontentsontriglycer-

idesoffishoilbylipase-catalyzedtrans-esterificationundersupercriticalconditions,Biochem.Eng.J.,29,27,2006.

135. Kamet,S.etal.,Biocatalyticsynthesisofacrylatesinorganicsolventsandsupercriticalfluids.III.Doescarbondioxidecovalentlymodifyenzymes?,Biotechnol.Bioeng.,46,610,1995.

136. Catchpole,O.J.,vonKamp,J-C.andGrey,J.B.,Extractionofsqualenefromsharkliveroilinapackedcolumnusingsupercriticalcarbondioxide,Ind.Eng.Chem.Res.,36,4318,1997.

137. Catchpole,O.J.etal.,Fractionationoflipidsinastaticmixerandpackedcolumnusingsupercriticalcarbondioxide,Ind.Eng.Chem.Res.,39,4820,2000.

7089_C005.indd 187 10/8/07 11:54:27 AM


138. Czech,B.andPeter,S.,Efficiencyofdifferentpackingsincounter-currentnear-criticalfluid extraction, in Pre-prints of High Pressure Chemical Engineering, 2nd Inter-nationalSymposium, DECHEMA-GVC,1990,419.

139. Böhm, F. et al., Design, construction, and operation of a multipurpose plant forcommercial supercritical gas extraction, in ACS Symposium Series, Vol. 406,Johnston,K.P.andPenninger,J.M.L.,Eds.,AmericanChemicalSociety,WashingtonD.C.,1989,502.

140. Passino,H.J.,Thesolexolprocess,Ind.Eng.Chem.,41,280,1949. 141. Shortland,F.B.,TheaquaticanimaloilresourcesofNewZealand,N.Z.J.Sci.Technol.,

2,30,1950.142. Winholz,M.,The Merck Index,Vol.9,Merck,NewYork,1976,1133.143. Hilditch, T.P. and Williams, P.N., The Chemical Composition of Natural Fats,

ChapmanHall,London,1964.144. Vlieg,P.andBody,D.R.,LipidcontentsandfattyacidcompositionofsomeNewZealand

freshwaterfinfishandmarinefinfish, shellfish,and roes,N.Z.J.Marine Freshwater Res.,22,151,1988.

145. Macrides,T.andKalafatis,N.,U.S.Patent6,083,536,2000.146. Wolyniak, C.J. et al., Gas chromatography-chemical ionization-mass spectrometric

fatty acid analysis of a commercial supercritical carbon dioxide lipid extract fromNewZealandgreen-lippedmussel(Perna canaliculus),Lipids,40,3552005.

7089_C005.indd 188 10/8/07 11:54:27 AM

189

6 Supercritical Fluid Extraction of Active Compounds from Algae

Rui L. Mendes

Contents

6.1 Introduction................................................................................................. 1896.2 SupercriticalFluidExtractionfromAlgae................................................. 191

6.2.1 Botryococcus Braunii...................................................................... 1916.2.2 Chlorella Vulgaris........................................................................... 1936.2.3 Dunaliella........................................................................................ 1966.2.4 Haematococcus pluvialis................................................................. 1986.2.5 Hypnea charoides............................................................................ 2016.2.6 Nannochloropsis..............................................................................2026.2.7 Spirulina (Arthrospira)....................................................................205

6.2.7.1 Spirulina maxima...............................................................2056.2.7.2 Spirulina platensis.............................................................207

6.3 Conclusion...................................................................................................209References..............................................................................................................209

6.1 IntroduCtIon

Microalgaearealltheeukaryoticphotosyntheticmicroorganisms,whichpresentagreatgeneticvariety.Someauthorsincludeinthisdefinitionprokaryoticphotosyn-theticorganisms[1],suchasthecyanobacteria.Phytoplankton,whichcomprisetheautotrophicprokaryoticandeukaryoticmicroorganismssuspendednear thewatersurface,arethebaseoftheaquaticlifefoodchain.

Morethan50,000speciesofmicroalgaearesupposedtoexist,butonlyabout50havebeenwellstudiedandamuchlowernumberhasbeencultivatedinlargescale[2].Microalgaeproduceagreatvarietyofsecondarymetabolites,whicharesynthesizedattheendofgrowthphaseandatthestationaryone.Theunlimitedstructuraldiver-sityofthesecompoundscanstillbeenlargedapplyingtechniquesofcombinatorialchemistry[3]. The bioactive molecules produced by these microorganisms can bebeneficialorharmful,butevenphycotoxinsandrelatedproductsmayserveasmaterialsforusefuldrugs[4].Ontheotherhand,untilnow,microalgaehadnotbeenusedmuchfortheproductionofchemicalsandbiochemicals,althoughsome,suchassomepoly-unsaturatedfattyacids(PUFAs),arethegreatestreserveofthebiosphere[5].

7089_C006.indd 189 10/8/07 11:55:47 AM


Infact,microalgaecanproduce, insignificantamounts, lipidssimilar toveg-etableoils,fuels,proteins,andessentialfattyacids,withdietaryapplications,suchaslinoleic,g-linolenic,eicosapentaenoic,docosahexaenoic,andarachidonicacids;vitamins(β-carotene,B12,andE);pigments(carotenoids,phycobiliproteins),waxes,biosurfactants, sterols, andother chemical specialties [6–8].Reviews focusingoncommercial aspects of microalgae biotechnology have recently appeared [9, 10].Figure6.1summarizesthemainapplicationsofmicroalgae[11].

Themarinemacroalgae,alsoknownasseaweed,havebeenusedashumanfoodandfertilizersandtoobtainagar,alginicacid,andcarrageenan,amongotheruses.In this field, the discovery of metabolites with biological activity has increasedsignificantly in the last threedecades [12].Thepharmaceutical industryhaspri-marilyfocuseditsattentiononthefollowingsubstances:sulphatedpolysaccharidesas antiviral substances, halogenated furanones as antifouling compounds, andkahalalideFasapossibletreatmentoflungcancer,tumors,andacquiredimmuno-deficiencysyndrome[12].

The lipid content of microalgae can reach 85% (dry weight basis), althoughvaluesbetween20%and40%aremoretypical[7].Theproductionyieldofthesecompoundsreliesontheconditionsofculture,withtheamountofnitrogenbeingoneofthemainfactorstocontrollipidcontent[5].Thefinalyieldalsodependsontheextractionmethod,degreeofcrushing,andtypeofsolventsused.

Themostimportantfeatureofalgaeistheirphotosyntheticcapacity.Therefore,thecultureofmicroalgaeisusuallymadeinopenponds,butthiscanleadtotheircontamination and to diseases, restricting this method to species that are more

Micro Algae

Food Nutraceutical, Functional

Food,Food Additives (Emulsifier,

Thickner), Sweets

Commercial Products

Hydrocarbons (Fuel) Adsorbents

Enzymes and Other Reserach Materials

Colorants Food (Ice creams, Jellies. Confectionaries, Juices)

Cosmetics (Lipsticks, Creams,

Lotions)

Pharmaceutical Products

Pharmaceutical Antibiotic, Antibacterial, Bulking Agent, Binder, Hard and Soft Capsule

Shell, Thickener Diagnostic Agents

FIgure 6.1 Microalgae applications in various fields. (Source: Adapted from Dufosséetal.,Trends in Food Science & Technology,16,389,2005.Withpermission.)

7089_C006.indd 190 10/8/07 11:55:50 AM

Supercritical Fluid Extraction of Active Compounds from Algae 191

resistant, such as Dunaliella, Spirulina, and Chlorella [13]. Dunaliella salina iscultivatedinpondswithhighlevelsofsalt,whereasSpirulina canbecultivatedinhighlyalkalinewater.Ontheotherhand,thecostsassociatedwithharvestingthealgae(whichcan includemicroscreen,centrifugation,orflocculation)canpreventtheeconomicsuccessofthisprocess.

Geneticengineeringoffersthepossibilityofconvertingphotosyntheticmicro-algaeabletoproduceautotrophicallychemicalspecialtiesintoonesabletoproducethemheterotrophically[14].Theuseoffermentersandphotobioreactors(whichcanbetanksprovidedwithalightsource,polyethylenebags,plasticorglasstubes,etc.)fortheselectiveproductionofparticularcompoundsisapromisingmethodforthecultureofmicroalgae.Thefutureofthistechnologyalsopassesbytheuseofmixo-trophiccultures[15].Geneticengineeringhasalsobeenusedtocreateoverproduc-ingstrains,aconditionthathelpssomespeciesbemorecompetitive,aswellastomodifytheminordertoobtainspecificcompounds[16,17].

Supercriticalfluidextraction(SFE)ofthistypeofcompoundhassomeadvan-tages over the conventional methods. These compounds can be obtained withoutthermaldegradationandwithoutsolvents,thereforethereareneitherissuesrelatedto the toxicityof theorganic solventsusednor the legal restrictionsof theiruse.Moreover,withSFEitispossibletoobtainahighefficiencyofextraction,ashort-ened extraction time, and, in some cases, a higher yield. On the other hand, theselectivityforcertaincompoundsismuchhigherwithSFEthanwithorganicextrac-tion.Generallyallalgaehasaremainingbiomassafterlipidextractionthatconsistsmainlyofprotein(about50%)andcarbohydrates.Theorganicsolventextractioncanleadtodenaturationoftheproteins,unliketheSFE,whatwouldbedetrimentaltoitsuseinfoodorfeedapplications[18,19].

Someofthemoreinterestingearlystudiesofsupercriticalcarbondioxide(CO2)extractionofcompoundsfromalgaewerecarriedoutusingScenedesmus obliquus [20],Dilophus ligulatus(abrownmacroalga)[21],Dunaliella salina[22],Skeleto-nema costatum [46], and Ochronomas danica [46], with the objective of obtain-ing fatty acids, several secondary metabolites, eicosapentaenoic acid (EPA), andβ-carotene, respectively. Also, some reviews on this field have appeared[23–25].Table6.1showsacollectionofresearchliteratureonseveralmacroalgaeandmicro-algae (including also other microorganisms, such as fungi and yeasts), and therespectivetargetcompounds,whichhavebeentheobjectofSFE.

TheaimofthischapteristoreviewinsomedetailthestudiesofSFEtoobtainseveralactivecompoundsfromsomeofthemostimportantmicroalgaeandalsosomeseaweedspecies,focusingonthetypesofcompoundsextracted,comparisonwiththeconventionalmethodstoobtainthem,andthemostrelevantaspectsoftheirSFE.

6.2 superCrItICal FluId extraCtIon From algae

6.2.1 Botryococcus Braunii

Botryococcus braunii is a species of green microalgae that live in large naturalcolonies, either in salt water or freshwater, in temperate and tropical regions. Itpresents a unique particularity among the known photosynthetic microorganisms

7089_C006.indd 191 10/8/07 11:55:52 AM


becauseitscontentinhydrocarbonscanreach85%ofthedrybiomass.Thesealgaehavealreadybeenproposedasafuturerenewablesourceoffuel[51].Thelocaliza-tionofthehydrocarbonsisextracellular.Eachcellissurroundedbyseveralexternalwalls, where most of the hydrocarbons accumulate in globular formations. Theremaininghydrocarbons(about5%)arelocatedinthecytoplasm[52].

Race A of these algae produces and accumulates linear alkadienes, C25–C31,withanoddnumberofcarbonatomsandthetrieneC29H54[53].Thesecompounds,afterhydrogenationorfunctionalization,canbeusedassubstitutesforparaffinicandnaturalwaxes,withcosmeticandpharmaceuticalapplications[54].

SupercriticalCO2studieswerecarriedoutat40ºCandpressuresof125,200,and300bar[33].Figure6.2showsthecumulativecurvesofthehydrocarbonextrac-tionyieldversusthehexaneextraction.Themaximumextractionyieldsofhydro-carbonsobtainedwere76g/kg(dryalgaebasis)and72g/kgforhexaneandSFEextractions,respectively.

About95%oftheextracellularhydrocarbonswererapidlyextractedat300bar.Moreover,thesupercriticalextractswerelimpidandgoldenduetothenonextractionofchlorophyll,andtheycontainedabout60%hydrocarbons,unlikewhathappenedwithhexaneextracts,whichcontainedonly37%ofthesecompounds.Whenhydro-carbonsweredepleted from thealgae, theextractsbecamemoreviscous.On the

table 6.1typical Compounds extracted from algae and other related organisms

organism target Compounds refs.

Arthrospira (Spirulina) Lipids,γ-linolenicacid,carotenoids,phycocyanin 26,27,28,29,30,31,32

Botryococcus braunii(raceA) Linearalkadienes(C25,C27,C29,C31),triene(C29) 33

Chlorella vulgaris Astaxanthin,canthaxanthin,lipids 34,35

Dilophus ligulatus Lipids,secondarymetabolites 21

Dunaliella bardawill Trans-β-carotene,cis-β-carotene 36

Dunaliella salina Trans-β-carotene,cis-β-carotene 37,38

Isochrisis galbana Lipids,EPA,PUFAs 39

Haematococus pluvialis Astaxanthin,astaxanthinesthers 30,40,41

Hypnea charoides ω3fattyacids(EPA,DHA) 42

Mortierella (fungus) Lipids,GLA 43

Nannochloropsis gaditana Chlorophylla,β-carotene,vaucheriaxanthine 44

Nannochloropsis sp. Lipids,EPA 45

Ochronomas danica Lipids,EPA,PUFAs 46

Phaffia rodozyma(yeast) Astaxanthin 47

Pilayella littoralis Nonhumiccompounds 48

Saprolegnia parasitica(fungus) Lipids,EPA 49

Scenedesmus obliquus Lipids,proteins 20

Skeletonema costatum Lipids,EPA,PUFAs 46

Torulaspora delbrueckii(yeast) Squalene 50

7089_C006.indd 192 10/8/07 11:55:54 AM


otherhand,C27andC29intheextractsdecreasedproportionatelyduringtheextrac-tionprogress,whileC31alkadieneproportionincreased[35].Hydrocarbonfractionincreasedwithpressure: the initialconcentrationofhydrocarbons increasedmoresteeplythanthatofintracellularlipids,leadingtoextractsmorerichinhydrocarbonsathigherpressures[55].

6.2.2 chlorella Vulgaris

Chlorella vulgarismicroalgaearecarotenoidproducers (mainlyofcanthaxanthinand astaxanthin) [56]. Carotenogenesis can be induced through saline, luminous,ornutritionalstress.Ontheotherhand,thecontentincarotenoidscanbetailoredthroughthedurationoftheprocessandtheintensityoftheimposedstresses.

Carotenoids belong to a hydrocarbon class (carotenes) and their oxygenatedderivatives(xanthophylls).Theirbasicstructure,reflectingitssynthesispath,con-sists of eight isoprenoidunits,which are assembled in suchway that twomethylgroupsnearthemoleculecenterareinposition1,6,whiletheothermethylgroupsstayinposition1,5[57].Thesetofconjugateddoublebonds(eleventothirteen)con-stitutesthechromophoreresponsibleforthecolorofthesecompounds.Thesecolorsrangefromyellowtoredandareinfluencedbythepresenceofmoredoublebonds,functionalgroups,andthetypeofmolecularconformation.

Canthaxanthin(β-β-carotene-4,4’-dione),C40H52O2,aredpigment,togetherwithastaxanthin,isoneofthemostimportantketocarotenoids[13].Itisusedascoloranttoimprovethecolorofpoultrymeatsandeggyolksaswellasinaquaculturetogiveapinktonalitytosalmonandtroutflesh.Althoughlackingpro-vitaminAactivity,canthaxanthinhasanticarcinogeniccapacity[58].

0

20

40

60

80

0 20 40 60 80 100CO2/Dry Alga (kg/kg)

Hyd

roca

rbon

s/D

ry A

lga (

g/kg

)

FIgure 6.2 Hydrocarbons supercritical extraction yield, as a function of solvent-algaeratio,fromthemicroalgaeBotryococcus brauniiinsupercriticalCO2at313.1K.♦12.5MPa,⦁20.0MPa,◾30.0MPa,×hexaneextraction.(Source:Mendesetal.,Inorganica Chimica Acta,356,328,2003.Withpermission.)

7089_C006.indd 193 10/8/07 11:55:57 AM


ThesemicroalgaeweresubmittedtosupercriticalCO2atpressuresbetween10.0and35.0MPaandtemperaturesof40ºCand55ºC.Theextractionswerecarriedouton5goffreeze-driedChlorellaatseveralphysicalconditionsofthemicroalgae:notcrushed(whole),partiallycrushed,andtotallycrushedcells[34,35].

The initialconcentrationof lipids(asdeterminedby theslopeof thecumula-tivecurveoftheextractionatorigin)insupercriticalfluidincreasedwithpressure,for both temperatures, either with whole or crushed cells, but with the latter theincrease was more significant (Figure6.3).Above 15.0 MPa, therewas an initialconcentrationincreasewithtemperatureforwholecells,butwiththecrushedalgae,thatvaluewasaround25.0MPa.Forcrushedcells,thehighestvalueforthiscon-centrationwasobtainedat35.0MPa/55ºC(19mg/LCO2)andthelowest(3mg/L)at20.0MPa/55ºC.Thispressurewasthelowestusedforcrushedcells.Forwholecells,thehighestconcentrationobtainedwas5mg/LCO2at35.0MPa/55ºC.ThisbehaviorcanberelatedtothedifferentamountandtypeoflipidsavailableinthesupercriticalCO2,accordingtothephysicalconditionofthealgae.

Fortheconditionsofpressureandtemperaturestudied,usingwholecells,theglobalyieldoflipidsobtainedincreasedeitherwiththepressureatconstanttemperatureorwithtemperatureatconstantpressure(20.0and35.0MPa)[34].Withcrushedcellsat20.0MPa,theyielddecreasedwithtemperature,whereasat35.0MPa,itincreased.

The highest yield of lipids obtained by SFE was 13.3% (dry weight, partialcrushedcells)at35.0MPa/55ºC;thisvaluedroppedto5%atthesameconditionsusingthewholealgae.Theyieldoforganicsolventextractionforcrushedcellsusingacetoneandhexanewere16.8%and18.5%,respectively.

Theextractionofcarotenoidsshowedasimilarbehaviortothatoflipidsforpres-sureandtemperaturevariations.However,theyieldofcarotenoidswashigherwithsupercriticalCO2at35.0MPa(50mg/100gdryweightalgae) than thatobtained

Pressure (MPa)

Conc

entr

atio

n (m

g/L)

205

5

10

15

20

035 50

FIgure 6.3 Initial concentration of lipids in CO2 at standard temperature and pres-sure(STP),asafunctionofpressure.Wholecells,◾40°C,⦁55ºC.Crushedcells,♦40°C,▲55°C.(Source:Mendes,R.L.andPalavra,A.F., inChemistry, Energy, and the Environment,Sequeira,C.A.C.andMoffat,J.B.,Eds.,RoyalSocietyofChemistry,Cambridge,51,1998.Withpermission.)

7089_C006.indd 194 10/8/07 11:56:00 AM


either with acetone (40 mg/100 g dry weight algae) or hexane (30 mg/100 g dryweight algae). When the degree of crushing increased, the yields of lipids andcarotenoidsalsoincreased[35].

The fraction of carotenoids in the extracted oils increased with the pressure.Forwhole cells, this fraction ranged from18mg/100goil (at 20.0MPa/55ºC) to171mg/100gofoil(at35.0MPa/55ºC)when100gofCO2on5galgaewereused[34]. For crushed cells, the fractions obtained were higher: 100 mg/100 g oil at20.0MPa/55ºCand275mg/100gofoilat35.0MPa/55ºC[59].

When a completely crushed Chlorella is used, with a carotenoids content of3mg/gofdryalgae,theyieldofcarotenoidsextractionincreasedsteeplywhenabout20%ofthesecompoundswereextracted(Figure6.4).Thisincreasecanbeattributedtoahigheraccessibilityofthesupercriticalfluidtothecarotenoidsboundtothecellfragmentsafterthelipids’outsideparticlesareextractedortoacompetitiveeffectbetweenthelipidsandcarotenoidsforthesupercriticalsolvent[35].

Astaxanthin and canthaxanthin account for about two-thirds of the carot-enoidsinC. vulgaris.Theratioofastaxanthintocanthaxanthininthesupercriticalextracts(0.8)isthesameasthatfoundinacetoneextractswhencompletelycrushedcellsareused.However,whenpartiallycrushedorwholecellsareused,theratioof astaxanthin to canthaxanthin in the supercritical extracts drops consider-ably,from0.6(withacetone)to0.2.Possibleexplanationsforthisbehaviorareastrongerbondbetweenastaxanthinandthecellularmatrixoradifferentlocaliza-tionofthetwocarotenoids.

An unsteady model to describe the SFE of lipids from the microalgaeC. vulgariswasbasedonthemodelusedtodescribetheextractionoflipidsfromfungi (Saprolegnia parasitica) using supercritical CO2 and supercritical CO2 +cosolvent(10%ethanol)[49].Themodelassumesthattheaxialandradialdisper-sionsarenegligible,thepropertiesofthefluidandalgalbedremainconstant,andthecomplexmixtureoflipidsisconsideredasasinglecomponent[60].Themass

0

10

20

30

40

50

0 10 20 30 40 50CO2/Dry Alga (kg/kg)

%Car

oten

oids

FIgure 6.4 Recovery of carotenoids from Chlorella vulgaris, as a function of solvent-algaeratio,at35.0MPaand328.1K.▲Wholecells,⦁Slightlycrushedcells,◾Wellcrushedcells.(Source:Mendesetal.,Inorganica Chimica Acta,356,328,2003.Withpermission.)

7089_C006.indd 195 10/8/07 11:56:02 AM


balanceinfluidandsolidphasecanthereforebedescribedbythefollowingsetofdifferentialequations:

ερ ρ∂∂

= − ∂∂

+ −( )y

tu

y

hApK y y* (6.1)

1−( ) ∂∂

= − −( )ε ρs

x

tApK y y* (6.2)

whereεistheporosityofthealgaebed,ρisthesupercriticalfluiddensity,ApKistheoverallmasstransfercoefficient(basedonvolume),uisthesuperficialvelocityofthefluid,xisthemassoflipidspermassoflipid-freealgae,ρsisthealgaedensity,his theaxialdistancefromthebottomof thealgaebed,y is the lipidconcentra-tioninsupercriticalfluid,tisthetime,andy*isthelipidsolubility.Theinitialandboundaryconditionsarex=xoatt=0foranyh,andy=0ath=0andt≥ 0.

Tohaveacompletedescriptionoftheextractioncurve,theoverallmasstransfercoefficient(whichaggregatestheexternalandtheintraparticleresistances)mustvarythroughoutthewholeextraction,accordingtoanempiricalexpression(3)[49,60],foundbyatrial-and-errormethod:

ApK ApKo x x x xo o= ( ) −( ) −( ) exp ln .0 01 shift (6.3)

where xshift is the concentration of lipids (in the algae) at which the diffusion-controlledregimestartsandxo is theinitialconcentrationof lipids.Masstransfercoefficientsweredeterminedusinga least squares regressionof theexperimentalextractioncurves.

ThemodelwasappliedtodifferentphysicalconditionsofC. vulgaris atapres-sure of 35.0 MPa and 55ºC, using a CO2 flow rate of 0.8 g/min. For algae withwholecells,theshiftforthediffusion-controlledregimeoccurredwhen4%ofthelipidswereobtained,inthecaseofaC. vulgariswithathickercellularwall,and10%whenanalgawithathinnercellularwallwasused.ThethicknessofthiswallseemsrelatedtothecarotenoidcontentofC. vulgaris[61].Withapartialcrushing,theshiftoccurredwhenabout25%ofthelipidswereextracted,whereaswithatotalcrushing(almostallthecellsdisrupted),theshiftoccurredwhen55%ofthelipidswereextracted.ThelastvalueissimilartothoseobtainedwithSFEoflipidsfromfungiandofoilfromrapeseed,althoughinthiscase,theshiftoccurredbecausetheremainingoilwasinotherformofbindingtothematrix[62].Thecalculatedmasstransfercoefficients(ApKo)rangedbetween0.171kg/m3sand0.531kg/m3sfortheextractionusingwholecellsand0.7kg/m3sand2.2kg/m3s usingcrushedcells[60,61].

6.2.3 Dunaliella

ThemicroalgaeDunaliella salinacanproduceβ-caroteneup to14%(dryweightbasis) [2],with this compound being mainly a mixture of two geometric isomers(all-trans and 9-cis). The synthetic compound is the crystalline all-trans isomer,

7089_C006.indd 196 10/8/07 11:56:05 AM


whichisnotreallyfat-soluble;however,naturalβ-carotene(fromalgae)isnotcrys-tallineandishighlyfat-soluble.

Separating the two isomers is advantageous because the cis is more easilyabsorbedinhumantissues[63]andhasmoreantioxidantactivity[63].Theconven-tionaltechniquesofisomerseparationinvolvelargeamountsoforganicsolventsandareverytime-consuming.Ontheotherhand,formedicalandfoodapplications,itisimportantthatthisseparationisdonewithouttheuseoftoxicsolvents.

TheseparationofisomerscanbecarriedoutthroughSFEifthereisasignifi-cantdifferenceinsolubilityofthecompounds.Thecisisomerisaboutthreetimesmoresolublethantheall-trans,andbothcarotenespresenthighersolubilitythanthesyntheticall-transisomer.

The concentration in supercritical CO2 of the cis-β-carotene and all-trans-β-carotene from a solid mixture of carotenoids from Dunaliella salina is shown inFigure6.5.ThismixturewasobtainedbyacetoneextractionfromwetDunaliella[37].Theorganicextractwassaponified inorder to remove the lipidsandchlorophyll,and the separation of phases was carried out with diethyl ether. The carotenoidscontainedin theetherphasewererecoveredbyevaporationof thissolventwithastreamofnitrogen.

Gamlieli-Bonshteinetal. [36]usedadifferentapproach tocompare thesolu-bilityoftheβ-caroteneisomers.Duetononavailabilityofthepure9-cisisomertocarryoutsolubilitymeasurements, thesolubilityof thecompoundwascalculatedindirectly.SupercriticalCO2extractionofβ-carotenefromDunaliella bardawil wasperformedat448barand40ºCfromaconcentrateextractobtainedwithamixtureethanol/hexane/water.Theseconditionsofpressureandtemperaturewerepreviouslyfoundtobetheoptimalonesforthesupercriticalextractionofβ-carotene[36].Enter-ingwiththeratiooftheinitialratesoftheextraction(fromtheconcentrate)ofthetwoisomersandknowingthesolubilityofthetrans isomer,whichwaspreviouslymeasured,theauthorscalculatedthesolubilityofthe9-cisβ-caroteneasbeing,at448bar/40ºC,7,64×10–5gisomer/gCO2,nearlyfourtimesthevalueoftheall-trans

0 10 15 20 25 30 35Pressure (MPa)

10–3

10–1

10–2

10–4

10–5

100

Solu

bilit

y (g/

dm3)

5

FIgure 6.5 Solubility of β-carotene isomers in supercritical CO2 (40°C), ⦁ all-trans-β-carotene (mixture), ◾ cis-β-carotene (mixture), • synthetic trans-β-carotene. (Source:AdaptedfromMendesetal.,Inorganica Chimica Acta,356,328,2003.Withpermission.)

7089_C006.indd 197 10/8/07 11:56:06 AM


isomer. Supercritical CO2 extraction was also performed on raw algae under thesameconditionsofpressure and temperature.At the initial stages, the extractionrateof thecis isomerwashigher thanthatof the trans isomer,but thedifferencebetweentherateswasnotaslargeasinthecaseoftheconcentrate.Thissuggestedtotheauthorsthat,inthefreeze-driedalgaepowder,theeffectofinternaldiffusionlimitationsonβ-caroteneextractionrateissignificant.Therefore,theresultsindicatethatdecreasingthealgaeparticlesizeshouldincreasetherecoveryandselectivityofisomerseparation.

SupercriticalCO2extractionofcompoundsfromfreeze-driedDunaliella salina wasalsocarriedoutbyMendesetal. [38]atpressuresof200and300barandatemperatureof40ºC,inasemicontinuousapparatusattwoflowrates,of18.9g/minand10.8g/min,on27gofalgaehavingacontentof0.5%(dryweight)ofβ-caroteneand10%(dryweight)lipids.Theyieldoftheextractionoflipidsincreasedwiththepressure,butnoincreaseintheextractionyieldofβ-carotenewasobservedwiththepressure.Whenthesupercriticalextractionoflipidsandβ-carotenewerecomparedforthetwodifferentflowratesat300bar,itwasverifiedthattheextractionyieldwashigherforthelowerflowrate.Recoveriesof25%and80%werereachedforlipidsandβ-carotene,respectively,attheflowrateof10.8g/min.Itwasalsoverifiedthatitwaspossibletoobtainvaluesofthecis-transratiowellaboveoftheinitialoneinthealgae(1.3).Forinstance,whentheextractionwascarriedoutat300barandataflowrateofabout18.9gCO2/min,thecis-transratioreachedthehighestvalue(3.6)atitsbeginninganddecreasedexponentiallyalongtheextraction.ForalowerflowrateofCO2(10.8g/min),theratiodecreasedto2.2.Theseresultsshowedthatthecis-β-carotenewasquicklydepletedatthesurfaceofthealgaeparticles.ThisbehaviorcanbeexplainedbythehighersolubilityofthecisisomerinCO2andbythepresenceofahigheramountofthiscompoundintheouterpartofthealgaeparticles(wherethetransisomerwasmoreeasilyisomerized),asthecapacityofDunaliella toproducethecis isomer is related to lightexposition [65].Thevaluesof thecis-trans ratiowere higher (about 4) whenβ-carotene concentrates of Dunaliella salina [37] orDunaliella bardawil[36]wereused.

Inanattempttoincreasetheselectivityoftheseparationofβ-caroteneisomers,Nobreetal.usedacombinationofsupercriticalCO2andsilicagel[66].Theexperi-ments were carried out at a pressure of 200 bar and a temperature of 40ºC in aflowapparatus.Theextractorwasa32-cm3vesselfilledwith20gofglassbeads,inwhichabout100mgβ-carotene(obtainedfromDunaliella)hadbeenpreviouslyprecipitated.Asecond5-cm3vesselinseriescontainedthesilicagel.Severalloadsofsilicawereused:1.5g,0.5g,and0.15g.Thecis-transratioincreasedwhentheamountofsilicageldecreased,beingalwayshigherthantheoneobtainedbysimpleSFE,reachingavalueof7.4forthelowestamountofsilicagelused.ThisvalueoftheratiowasaboutthreetimestheoneobtainedbySFEatthesameconditionsofpressureandtemperature.

6.2.4 haematococcus pluVialis

Haematococcus pluvialis is a freshwater flagellate that accumulates astaxan-thininitsaplanospores.Itcanproduceastaxanthininrelativelyhighyields(upto

7089_C006.indd 198 10/8/07 11:56:08 AM


45mg/gdryweight algae) in laboratory conditions, but due toheavy contamina-tion, theuseofopenpondsisnotsatisfactorytoobtainthehighyieldsnecessaryforalgalastaxanthintocompetewiththesyntheticone.OthermethodsforgrowingH. pluvialis havebeentriedtoovercomethatdrawbackandalsotoincreaseyields[13].Olaizola[67]exploredthestrategyusedbyapharmaceuticalcompanytomakeHaematococcus astaxanthin more acceptable to consumers and to make it morecompetitive.ThisstrategyincludestheuseofSFE.

Astaxanthin (3,3´-dihydroxy-β,β-carotene-4,4´-dione), C40H52O2, is one ofthe most important xanthophylls either from a commercial or a biotechnologicalpoint of view, being the most abundant carotenoid and pigment found in certainaquatic animals, such as salmon, trout, shrimp, and lobster. Several isomers arefound inastaxanthinpresent innature,with3S,3S’being themainonefound inH. pluvialis[68].ThepresenceofthehydroxylandketogroupsintheiononeringexplainswhymostoftheastaxanthinappearsinHaematococcus inesterifiedforms(monoanddiester)andalsowhyitisverypronetooxidation.Theactivityofthecom-poundisseveraltimeshigherthanthatofβ-caroteneandvitaminE.Guerinetal.[68]reviewedthemainapplicationsforhumanhealthandnutritionofHaematococcus astaxanthin,namelyitsusesagainstultraviolet-lightphotooxidation,inflammation,cancer,andagingandage-relateddiseasesandinthepromotionofimmuneresponse,liverfunction,andheart,eye,andprostatehealth.Ontheotherhand,asinglehighdose(100mg)wasadministeredtohumansinastudyofbioavailability,anditwasverifiedthatastaxanthinwasreadilyabsorbedandincorporatedinhumanplasmalipoproteinataconvenabledegree[69].

Valderramaetal.[30]andMachmudahetal.[41]carriedoutsupercriticalCO2extraction of astaxanthin from H. pluvialis.Another study of the same type thatfocusedonastaxanthin,aswellasontheothercarotenoidspresentinthesemicro-algae,wascarriedoutbyNobreetal.[40].Pressuredfluidextraction(PFE),whichusesconventionalsolventsatcontrolledtemperaturesandpressures,hasalsobeenperformedtoextractcarotenoidsfromH. pluvialis[70].

InthestudiesofValderramaetal.[30],thesemicontinuoussupercriticalappa-ratuswasprovidedwithanextractionvesselof450ml,andtheexperimentswerecarriedoutat300barand60ºC.Threetypesofrunswereperformed:run1,inwhichthedriedsamplesofmicroalgaewerecrushedbycuttingmillspriortoextraction;run2,inwhichthesampleswerecrushedlikeinrun1,followedbymanualgrindingusingdryice(solidCO2);andrun3,inwhichthesamplesweretreatedasinrun2,but theextractionwasperformedusingsupercriticalCO2containingethanolasacosolvent(9.4%weight).

Theastaxanthinyielddependedstronglyonsamplepreparationandontheuseofethanolasacosolvent.Amoreefficientgrindingledtohigheryield.WithpureCO2,astaxanthinisrecoveredonlypartially.WithH. pluvialis wellcrushedandusingthecosolvent,anastaxanthinrecoveryof97%wasreached.Thisrecoverywasdeter-minedfromtheinitialandfinalcontent(residueoftheextraction)ofthecompoundinthemicroalgae.Valderramaetal.[30]didnotprovidethemethodbywhichthosecontentsweredeterminedandnomentionofesterifiedastaxanthin,theusualformof astaxanthin in thesemicroalgae,wasmade.The supercritical extraction (yield

7089_C006.indd 199 10/8/07 11:56:09 AM


versussolvent-feedratio)wasmodeledwiththesameempiricalmodelusedinthesupercriticalextractionoflipidsfromSpirulina(seesection6.2.7.1).

Nobreetal.[40]carriedoutamoredetailedstudyofsupercriticalCO2extrac-tionofcarotenoidsfromH. pluvialisusingfreeze-driedsamplesgroundwithaballmill.Theeffectsofpressure(200and300bar),temperature(40and60ºC),degreeofcrushing(relatedtomillingtime),andtheuseofethanol(10%)ascosolventontheextractionyieldweredetermined.Theextractionwithacetonewascarriedoutusingglassbeadsmixedwiththealgaeuntiltotalabsenceofcolorwaspresentinthebiomass.

Besidesfreeastaxanthin(whichrepresentsabout2%ofthecarotenoids),theyieldofesterifiedastaxanthin(73%ofthecarotenoidspresentinH. pluvialis),β-carotene(7%of thecarotenoids),canthaxanthin,andluteinwereassessedandtherecover-iesobtainedbySFEwerecomparedwiththetotalcontentofcarotenoidsfromthemicroalgae.Figure6.6showsthetotalastaxanthinrecoveryasafunctionofsolvent-feed ratio, showing the beneficial effect of ethanol as a cosolvent as well as thedegreeofcrushingofthemicroalgaecells.Arecoveryofabout90%wasobtained.TheimprovementinyieldduetoethanolwasascribedtotheincreaseofastaxanthinsolubilityinsupercriticalCO2duetoitspolarcharacterand,ontheotherhand,totheswellingofthemicroalgaeparticlepores,whicheasedthereleaseofthecompounds.Therecoveryimprovementduetothecrushingofthecellscanbeattributedtotheincreaseinthenumberofdisruptedcellsandthedegreeofdisruption, increasingtheamountofextractablecarotenoids.Inthiswork,theresearchersalsoverifiedanincreaseintherecoveriesofcarotenoidswhenthepressureincreasedfrom200to300bar,butonlyaslightimprovementwasobtainedwhenthetemperature,atthesamepressures,increasedfrom40ºCto60ºC.

All the carotenoids identified in H. pluvialis (esterified and free astaxanthin,β-carotene,lutein,canthaxanthin)wererecoveredwithvaluesnearorhigherthan90%atapressureof300barandatemperatureof60ºC,usingethanolasacosolvent.

0

20

40

60

80

100

0 100 200 300CO2/Dry Alga (g/g)

Tota

l Ast

axan

thin

Rec

over

y (%)

FIgure 6.6 Recoveryoftotalastaxanthin(freeplusesters)asafunctionofCO2amountwith(◾)andwithout(▲)ethanolasacosolventusingslightlycrushedalgae,andCO2withethanolusingwell-crushedalgae(♦),at60°Cand300bar.(Source:Nobreetal.,Eur. Food Res. Tecnol., 223,787,2006.Withpermission.)

7089_C006.indd 200 10/8/07 11:56:10 AM


Machmudahetal. [41]studiedthesupercriticalCO2extractionofastaxanthinfromH. pluvialis usingaflowapparatusprovidedwitha50-cm3extractionvesselcontaining7gofalgaeineachexperiment.Thecontentofastaxanthinwas3.33%(drybasis)asdeterminedbySoxhletusingdichloromethane.Theconditionstestedwere temperature (313 to 353 K), pressure (20 to 55 MPa), CO2 flow rate (2to4cm3/min),andethanolconcentration(ethanol-solvent=1.67–7.5%).Thealgaewerenot crushed, unlike in the aforementioned studies.Without ethanol entrainer, theamountoftotalextract,theamountofastaxanthinextract,andtheastaxanthincon-tent in the extract increased with an increase in temperature. This behavior wasattributedtotheincreaseofvaporpressureofastaxanthinandalsotothedecom-positionofthecellwallwiththetemperature,whichcontributedtotheincreaseoftheextractablecompounds.Theincreaseofpressurealsoledtoanincreaseofthoseyields.Theamountofthetotalextractandtheamountofastaxanthinextract,butnottheastaxanthincontentintheextract,slightlyincreasedwithCO2flowrate.

The highest astaxanthin extracted (recovery) and astaxanthin content (in thetotalextract)were77.9%and12.3%,respectively,whichwereobtainedat55MPa,at343K,andataCO2flowrateof3cm3/min.

Usingethanolasanentrainer,ahigheramountofastaxanthin(80.6%)couldbeextractedatamoremoderatepressure(40MPa).Ontheotherhand,theincreaseinentrainerconcentration,upto5%(v/v)ethanol,increasedtheamountofastaxanthin.With theuseofentrainer, theCO2flowrateconsiderablyaffected theastaxanthinextractedandahigheramountofastaxanthinwasextractedasCO2flowratedecreased.Thislastpointsuggestedtotheauthorstheimportanceoftheinternalmassdiffusionin the extraction of astaxanthin fromH. pluvialis. This is in accordance with thepreviousstudiesofValderrama[30]andMendes[40],whichdemonstratedthatthecrushingofthealgaecontributedhighlytothesuccessoftheextraction.

InstudiesofPFEusingacetoneassolvent,higherorequalamountsofcarotenoidswereextractedthanwereextractedwiththetraditionalorganicsolventmethod[70].Thetimeofextractionwas20minuteswithPFE,whereas90minuteswereneces-sarytoperformthetraditionalacetoneextraction.

6.2.5 hypnea charoiDes

Hypnea charoidesisasubtropicalredseaweed.Amongtheseaweeds,theredonesareknownfortheirhighomega3(w3)fattyacidcontents[71].Amongthefattyacidconstituentsofalgallipids,themostimportantarethepolyunsaturatedfattyacids(PUFAs).TheyrangefromC12(twelvecarbonatoms)toC22andcancontainuptosixallylicbondsseparatedbyacarbonatom.Withinthisgroup,theessentialfattyacids(EFAs)forhumansarelinoleicacid,g-linolenicacid(GLA),dihomo-g-linoleicacid,arachidonicacid,andEPA.TherearetwotypesofPUFAs:w3andomega6(w6),knownofficiallybyn-3orn-6fattyacids,respectively[72].Thesenumberscorrespond to the position of the last double bond counted from the last methylgroup.Formosteukaryoticalgae,whichcontainpredominantlymonoandsaturatedfatty acids, the triglycerides constitute up to 80% of the lipid fraction [73]. In ageneralway,themarinealgaearerichinw3fattyacids,namelyEPA(C20:5,w3)anddocosahexaenoicacid(DHA,C22:6,w3).Themainsourceforhumanconsumption

7089_C006.indd 201 10/8/07 11:56:11 AM


of thesePUFAs ismarinefish,whose feedingbasis isconstitutedbymicroalgae.Withthedepletionoffishstocks,microalgaeandmacroalgae(seaweeds)couldbealternativesourcesofw3fattyacids[42].

Alargenumberofepidemiological,animal,andclinicalstudieshaveshownthatintakeofw3fattyacidsisbeneficialforpreventingacertainnumberofdiseases(e.g.,cardiovasculardiseases,someformsofcancer,anddiseaseswithimmunoinflamma-torycomponents)andcanalsoplayaroleinbrainandnervedevelopmentofgrowingfetusesandinfants[72].Thetherapeuticeffectsofthesefattyacidshavealsobeenshown[74].

Cheung[42]carriedoutsupercriticalCO2extractionofn-3fattyacidsfromthesemacroalgae,withemphasisontheeffectsoftemperature,pressureontheyield,andcompositionoftheextracts.Theexperimentswereperformedon2gofdriedandground(1mmsieve)algae,attemperaturesof40ºCand50ºCandpressuresof24.1,31.0,and37.9MPaataCO2flowrateof2mg/min.Theextractioncellwasprovidedwitha10-cm3stainlesscartridge.

Cheungfoundthatlipidextractionincreasedwiththepressureatconstanttem-perature.Athigherpressures(31.0and37.9MPa),anincreaseintemperatureledtoanincreaseintheyieldandalsointherateofextraction(intheinitialperiod).However,atthelowestpressureused,theyielddecreasedwhenthetemperaturepassedfrom40ºCto50ºC.TheseresultsaresimilartopublisheddataofsupercriticalCO2extrac-tionoflipidsfromChlorella vulgaris[34].Thisbehaviorisexplainedintermsofthecombinedeffectofthepressureandtemperatureonthedensityofthesolventandvaporpressureofthesolutes.

Themaximumyieldof lipids fromH. charoides at theconditions studiedbyCheung was 67.1 mg/g (dry weight algae) at 37.9 MPa/50ºC and the lowest was33.7mg/gat24.1MPa/50ºC.Sixw3fattyacidswerefoundintheextracts,represent-ingattheconditionsofmaximumyield(37.9MPa/50ºC),24%and39%ofthelipidsandtotalfattyacids,respectively.EPA(20:5w3)representedabout60%ofthetotalw3fattyacidsandα-linolenicacid(18:3w3)16%ofthesefattyacids.DPA(22:5w3)andDHA(22:6w3)werealsofoundtorepresent8%and12%,respectively,ofthetotalw3fattyacids.

WiththeexceptionofDPAandDHA,theyieldofallthew3fattyacidshadasimilarbehaviortothatofthetotallipids,withthevariationofpressureandtempera-ture.Evenathigherpressures,theC22w3fattyacidsshowedaloweryieldathighertemperatures.ThisbehaviorcouldbeattributedtothefactthatchainlengthisamoreimportantfactorinsupercriticalCO2solubilitythanthedegreeofsaturation.

Theauthorconcludes thatalgal lipidsofH. charoides couldbeanonconven-tionalalternativetow3fattyacids.

6.2.6 nannochloropsis

Nannochloropsisspeciesaremarinemicroalgaeabletoproduceahighcontentofbothlipidsandeicosapentaenoicacid(EPA).ThiscompoundisthemajorfattyacidofthosefoundinNannochloropsis(rangingfrom29%to33%),withthecontentofn-3PUFAsbeingabout40%ofthetotalfattyacids[45].Itisalsoasourceofvaluablepigments,suchaschlorophylla,astaxanthin,zeaxanthin,andcanthaxanthin[75].

7089_C006.indd 202 10/8/07 11:56:11 AM


Andrichetal.[45]verifiedthatinsupercriticalextractionstudiesofalgaelittleinformation about the kinetics of the process and the influence of the operatingconditionsonthecompositionofthelipidextractswereavailable.Therefore,theyfocusedtheirstudiesatexaminingthesepoints.

Supercritical CO2 extraction of bioactive lipids from the microalgae Nanno-chloropsis sp. wascarriedoutinapilotplantapparatusatpressuresof40,55,and70MPaandtemperaturesof40ºCand55ºC.Anamountof180gmicroalgae,mixedwith100gof3-mmglassspheres,wasusedforeachrun.ThesupercriticalCO2flowratewas10kg/h.Theauthorsalsoperformedextractionbypercolationwithhexane(Soxhlet).

Thefollowingequation isused todescribe theevolutionofextractedoilovertime(t),forbothsupercriticalextractionandhexaneone:

Oe H Os e kt= −( )−* 1 (6.4)

whereOeistheamount(g)ofoilextractedatatime(t)pergramofalgalbiomass,H*isaconstantrangingfrom0to1,[Os]istheamount(g)ofoilpresentin1gofstartingmaterial,andkisthekineticconstant.TheproductH*[Os]representstheasymptoticvalueoftheextractioncurve.Inahighlyefficientprocess,H*tendsto1,meaningthattheamountofextractableoilpergramofbiomasscoincideswiththeconcentrationofoilinthestartingmaterial.

Themaximumextractionrate(Rmax)isgivenby:

R kH Osmax = + (6.5)

Theauthors interpreted thecumulativeextractioncurvesasa functionof time intermsofthekineticparameters.Intermsofoilextractable,alltheprocesses(super-criticalCO2extractionsatalltheconditionsandhexaneextraction)aresubstantiallyequivalent(about250mglipids/gdryalgae).Thevalueofkincreasesatconstanttem-peraturewiththeincreaseofpressureandalsoincreasesforagivenpressurewhenthe temperature increases, although in this case more moderately. The combinedeffectofpressure-temperature(P-T)affectskmorethanPandTalone,increasingitsvaluethreetimesfromtheextractionat40ºCand400bartotheextractionat55ºCand700bar.SFEwasclearlyfasterthantheextractionbyhexane(Rmaxwasseveraltimeshigher).

Rmaxwasexpressedintermsof theconcentrationof lipidsinthesupercriticalfluidatthebeginningoftheextraction,R*max(g/l),andrelatedtothedensity,r(g/l),andtemperature,T(K),ofthesolventthroughtheequationduetoChrastill[76]:

R ea b T Cmax* = +( )ρ (6.6)

The values of the parameters, obtained from the several extraction conditionsstudied,werea=10.92±2.57;b=3506.57±1225.65;andc=62.68±16.18.Theauthorsclaimthatthiskineticmodelseemssuitabletoevaluatetheeconomicfeasibilityoftheprocess,althoughmoreinformationisneededforitsgeneralization.

7089_C006.indd 203 10/8/07 11:56:14 AM


ThepercentageofEPA in the total fatty acids ranged from29.4% to33%fortheconditionsofextractionstudied.However,thesevaluesseemlowerthanthe37%claimedforthecommercialproduct.TheauthorsattributedthistothefactthattheuseofsupercriticalCO2causedsomelossesinthemostpolarfractions,whereEPAispos-siblyprimarilylocated.Ontheotherhand,noparticulardifferenceswerefoundinthefattyacidprofilesofextracts.However,aslightincreaseinEPAandDPA(C22:5n-3)seemsperceivablewhenpassingfromthemildesttothehardestSFEconditions.

Macías-Sánchezetal.[44]carriedoutsupercriticalCO2extractionofpigmentsfrom Nannochloropsis gaditana in a microscale apparatus, which is providedwithtwoextractorsof10mleach.Inoneoftheextractorswasinsertedacartridgecontaining0.2goffreeze-driedmicroalgaeand,after15minutesofstaticextrac-tion, the runswereperformedat aflowrateof4.5mmol/min for3h.A totalof15experiments were carriedout in a random way, in order to fulfill amultilevelfactorialdesigntodeterminetheeffectoftemperatureandpressureontheextractionofthecarotenoidsandchlorophylla.

Organic solvent extraction, using methanol, was performed by sonication on0.2gofN. gaditana,with5mlof solvent foreachof the14cyclesnecessary toobtainthemethanolwithoutanycoloration.Thealgaepelletremainedgreenishafterthesolventextraction.Theyieldsobtainedwere0.8mg/mgand18.5mg/mg(dryweightbasis)forcarotenoidsandchlorophylla,respectively.

SFEwascarriedoutattemperaturesof40ºC,50ºC,and60ºCandpressuresof100,200,300,400,and500bar.Theanalysisoftheexperimentaldesignshowsthatthetemperature,pressure,andinteractionofbothvariablessignificantlyinfluencetheprocess(p-value<0.05).Theyieldincreaseswithpressureatagiventempera-turefrom100to400bar,wherethemaximumyieldisreachedat60ºC(400bar)forbothcarotenoidsandchlorophylla(0.343mg/mgand2.238mg/mg,respectively).At 100bar, no pigments were extracted and at each pressure the yield increasedwithtemperatureforpressuresabove200bar.Asusual,thisbehaviorisexplainedintermsofbalancebetweensolventdensityandvaporpressure.Ontheotherhand,at500bar,theyielddecreased.Thefactofthemaximumyieldbeobtainedatanintermediatepressure(400bar)isexplainedintermsofthedecreaseofthediffu-sioncoefficientduetotheincreaseoftheCO2density,whichreducesthepenetrationcapacityofthefluidandtheyieldatthehighestpressureused.ThisalsoalreadyhadbeensuggestedfortheextractionofcarotenoidsfromSpirulina[28].

Thefollowingempiricalcorrelationsforcarotenoids(6.7)andchlorophylla(6.8)wereobtained[44]:

R T P= − + + −0 233163 0 00492577 0 00121779 0 000. . . . 00923077

0 00002705 0 00000353013

2

2

T

PT P+ − −. . (6.7)

R P T= − − −3 43203 0 00140362 0 14499 0 00000725. . . . 9952

0 0001963 0 001144

2

2

P

PT T+ +. . (6.8)

whereRistheyieldofpigment(mg/mg),Tisthetemperature(ºC),andPispres-sure(bar).

7089_C006.indd 204 10/8/07 11:56:16 AM


Furthermore, the ratio of carotenoids to chlorophylls (carot/chlor) in theextracts isalwayshigher than thatobtainedwithmethanol(carot/chlor=0.043)anddecreaseswithpressure,withthehighestratioobtainedat200barand60°C(carot/chlor = 1.389). This leads the authors to conclude that supercritical CO2ismoreselectivefor theextractionofcarotenoidsinthepresenceofmorepolarpigmentslikechlorophylla.

The SC extraction of chlorophyll in this work contrasts previous work fromotherauthors,inwhichthenonextractionofthistypeofcompoundbysupercriticalCO2fromBotryoccocus braunii[33],Skeletonema costatum [46], andOchronomas danica[46]wasreported.However,inthestudyofsupercriticalCO2extractionofoilfromScenedesmus obliquus [20],theextractionofchlorophyllaismentioned.ThismatterisdiscussedbyBalabanetal.[23],whosuggestedthatentrainedparticlesofalgaemighthavecausedthediscrepancybetweenthisresultandpreviousreportsofinsolubilityofchlorophyllinsupercriticalCO2.

6.2.7 spirulina (arthrospira)

Spirulinaisoneofthemostpromisingmicroalgae.Itisrichintheessentialfattyacidall-cis-6,9,12-octadecatrienoicacid(GLA);pigments(phycocyanin,myxoxantho-phyl,zeaxanthin,andβ-carotene);proteins;andsulfolipids[77]. Likeotheralgae,suchasChlorella, Spirulina has alsobeenusedas functional food (foodderivedfromnaturalsourceswhoseconsumptionisbeneficialtohealthofthehumanbody).Inthiscase,algaeareprovidedeitherassupplementsorcompletefood[78].ResearchhasalsoshownthetherapeuticvalueofSpirulina anditsextractsinalargenumberofdiseases[79].

TheGLAfromSpirulina actuallycannotcompeteeconomicallywiththeGLAfromhigherplants(e.g.,eveningprimrose,blackcurrant,andborage),buttherearesomeadvantagestousingSpirulina microalgaeasaGLAproducer.Thiscompoundisfoundmainlyintheglycolipidfractionofthelipids,whicheasesitspurificationasapharmaceuticalcommodityand,ontheotherhand,unliketheGLAofthehigherplants,itisnotassociatedwithundesirablefattyacids[77].

GLApresentshigherEFAactivityandantithromboticandhypolipidemiceffectsthanlinoleicacid(18:2w6)[80].Italsohasbeenusedinseveralmedicalapplica-tions, such as the treatment of schizophrenia, multiple sclerosis, atopic eczema,premenstrualsyndrome,diabetes,andrheumatoidarthritis[80,81].Itcanalsoplayanimportantroleinthesynthesisofakindofprostaglandin,ahormoneinvolvedin essential tasksof thehumanbody,namely the controlof the arterial tension,cholesterol,andinflammation.

TheantioxidantactivityofextractsfromSpirulinaplatensis obtainedwithpres-surizedliquidextractionwasstudiedandattributedtothepresenceofcarotenoids,phenoliccompounds,anddegradationproductsofchlorophyll[82].

6.2.7.1 Spirulina maxima

SupercriticalCO2extractionoflipidsfromS. maxima wasstudiedbyMendesetal.[32],Canelaetal.[29],andValderramaetal.[30].ThefirstauthorsfocusedtheirworkontheextractionofGLA,thesecondonesontheextractionoffattyacidsand

7089_C006.indd 205 10/8/07 11:56:16 AM


carotenoids,andthethirdontheresidueoftheextractioninordertoobtainthepig-mentphycocyanin.

TheobjectiveoftheworkofMendesetal.[32]wastocarryoutthesupercriticalCO2extractionoflipids,focusingontheGLAcontentoftheselipidsandtoassessthe influenceofpressure, temperature,and theuseofethanolasentraineron theextractionyieldsandselectivityoftheseveralfattyacidsandclassesoflipids.TheextractionoftheSpirulina lipidswasalsoperformedwithorganicsolvents(ethanol,hexane,acetone,andamixtureofwater,chloroform,andmethanol),havinginviewthecomparisonofthetwotypesofextraction(SFEandorganic).Thesupercriticalexperimentswerecarriedoutinaflowapparatuson3.6gofash-free,freeze-driedArthrospira (Spirulina) maximaataflowrateof2g/minatpressuresof250barand300barandtemperaturesof50ºCand60ºC.At250barand50ºCwithpureCO2,ayieldof0.05%(GLA/drybiomasswt%)wasobtainedwhen1.4kgCO2wasused,but above a solvent-feed ratioof300g/g, the concentrationof lipids inwasverylow.At the same conditions, using supercritical CO2plus ethanol (10mol%), theyield improvedto0.17%,buthigheryieldsarepossiblebecause theconcentrationofthelipidsinthesolventgrewsteadilyevenforhighsolvent-feedratios.Ethanolcan have an entrainment effect on the extraction of the lipids, which are mainlypolar,increasingitssolubilityand,ontheotherhand,cancounterbalancethehydro-genbondsand ionic forcesbetween themembrane-associated lipids andproteins[83],allowingthelipidstobeavailableforextractionbythesupercriticalfluid.Theincreaseoftemperatureat250barledtoanincreaseinGLAyield,buttheincreaseinpressureto350barat60ºCledtothehighestGLAyieldobtained(0.44%).TheGLAyields reachedwithethanol, acetone,andhexanewere0.68%,0.63%,and0.01%,respectively.Inanotherstudy[31],inwhichethanolwasmixedwithSpirulina,GLAyield increasedwith the amountof ethanol and, for equalmassesof ethanol andmicroalgae,theyieldwasmorethan10timesthatobtainedwithsupercriticalCO2extractionfromnon-pretreatedSpirulina.

Valderramaetal.[30]extractedtheCO2-solublematerialfromSpirulina maxima,withtheaimofconcentratingthepigmentphycocyaninintheextractionresidue.ThefirstextractionwascarriedoutusingpureCO2at300barand60ºC,whichledtoalipidyieldof1.1%.Inasecondsetofexperiments,theco-solventethanol(10%)wasused,withanincreasedyieldof3%.ThecontentinphycocyanininthesamplesofSpirulina maximaincreasedfrom6.17wt%to8.3%whenpureCO2wasusedandfrom6.91%to8.4%whenthesolventwasCO2plus10%ethanol.

An empirical model for the concentration of phycocyanin correlates well thelipidextractionyield(Y)withthesolvent-feedratio(X):

Y e X= −( )−α β1 (6.9)

Inthemodel,αandβareempiricalconstantsobtainedfromtheexperimentalyield.Y = 0 for X = 0, and the yield has as a limit the empirical maximum, which isrepresentedbytheparameterα.

Canela et al. [29] studied the supercritical CO2 extraction of fatty acids andcarotenoids from S. maxima. The experiments were conducted at temperaturesbetween20ºCand70ºCandwithpressuresupto180bar.Theseauthorspreviously

7089_C006.indd 206 10/8/07 11:56:17 AM


determinedSpirulina composition(3.1%lipids,54.5%proteins,9.1%humidity,and12.2% ashes). The total amount of CO2-soluble material was determined, usinga3.5-cm3extractionat180bar (30ºCand40ºC)and150bar (30ºC,40ºC,60ºC,and70ºC).Thehighestamount,0.93%(dryweightbasisofSpirulina),wasobtainedat150barand60ºC.

KineticexperimentrunsofsupercriticalCO2extractionoflipidsfromS. maximawereperformedusingastandardextractionunitprovidedwithanextractioncellofabout 368 cm3. For each run, an amount of 175 g microalgae, mixed with equalamountofglassbeads,wasused.Thesolventflowratewasmaintainedat0.12kg/h.The effects of temperature andpressurewerequantifiedusing a factorial experi-mentaldesign.Theoptimalextractionconditionswere150barand60ºC.Buttakingintoaccount the targetcomponents(fattyacidsandcarotenoids),whicharepronetodegradationathightemperatures,atemperatureof50ºCorlowerissuggested.Canela et al. [29] reported the composition of the supercritical extracts obtainedin terms of total carotenoids. The yields are very low when compared with thecarotenoidcontentofthemicroalgaereportedbyCohen[77].Thisbehaviorcanbeattributedinparttothelowpressuresused,asthecarotenoidspresentaverylowsolubilityatthesepressures[84].

Supercritical extractions curves were modeled according to the Goto et al.

model [85], which was developed for the supercritical extraction of essential oilfrom peppermint. This model treats the solid substrate as a porous matrix. Thesoluteisextractedafteritsdesorptionfromthesolid.Diffusionoccursinsidetheparticlepores,andthereisamass-transferresistanceinthefilmsurroundingtheparticles.Themodelgaveagood representationof theexperimentaldata, andapartitioncoefficientandacombinedmass-transfercoefficientwereobtainedfromthefittingoftheexperimentalresultstothemainequationofthemodel.

6.2.7.2 Spirulina platensis

Santosetal.[26]performedexperimentsofSFEinsamplesofS. platensis usingCO2atatemperatureof40ºCandapressureof200bar.Bedsof10galgae(andsome-times20g)wereplacedinahalf-literextractionvessel.

Toevaluate theeffectof themoisturecontentof thealgae, threeexperimentswerecarriedout.Thefirstone,directlyonthemicroalgaesupplied(moisturecon-tentof3.24%),ledtoalipidyieldofabout1.1%(drybasiswt);thesecondtestwasperformed on a sample that had been dried in P2O5 desiccator for several hours;thethirdtestwasontheresiduefromthesecondone,whichhadbeendampedtogiveamoisturecontentof7%.Thedryingofthemicroalgaeproducedasubstantialfallinthelipidyield(0.3%inadryweightbasis).Furtherdampingoftheresidueenabledmorelipidicmaterialtobeextractedfromtheresidue,butstillalowyieldwasobtained(0.5%).

Inthiswork,thefattyacidsfromtheglyceridefractionobtainedintheexperi-mentswereidentified.Atypicalpercentageoftheobtainedfattyacidsissimilartothisone:palmitic(33%),palmitoleic(8%),stearic(2%),oleic(4%), linoleic(21%),andg-linolenic(32%).

7089_C006.indd 207 10/8/07 11:56:18 AM


InordertomodeltheSFE,theauthorsconductedexperimentsattwosuperficialvelocities. When the amount of extracted lipids is plotted against the CO2 massused,thecurvesforthehigherflowratefallbelowthatcorrespondingtothelowerflowrate,butwhenyieldsareplottedagainsttime,theexperimentalpointsfallina common curve. This is consistent with a mass transfer mechanism within thealgaeparticles(bydiffusion),whichissoslowthattheextractantphasecomposi-tionchangesvery littlealong thebedandneverapproachesclose to theequilib-riumvalue.Theexternalfilmresistanceisnegligible.Basedontheseassumptions,Santosetal.[26]appliedthewell-knownsinglesphereextractionmodelandfoundthatthebestfitforDe/r2,whereDeistheeffectivediffusivity(ofthelipids)andtheristhealgaeparticleradius,ledtothefollowingvalue:Deπ2/r2=0.00120,forthedataresultingfromtheSFEoflipidsfromS. platensisatapressureof200barandtemperatureof40ºC.

Qiuhui[27]usedsupercriticalCO2extractionoflipidsfromthesemicroalgae,withthegoalofremovingitsbadsmell,anobstacletothemarketingandacceptanceofSpirulina,whichissoldandexportedasapowder.Infact,thisauthorpretendedtoseparateandpurifytheactivecomponentsofthealgae.TheSFEexperimentswerecarriedoutinasemicontinuousapparatus,providedwithrecyclingofCO2,ataflowrateof24kg/h;pressuresof30,35,and40MPa;extractiontimeof2,3,and4h,andtemperatureof40ºC.

Thehighestlipidyield(7.2%)wasobtainedat35MPaand4hofoperation,avaluenearthatobtainedat40MPa(7%)forthesameextractiontime.However,foranextractiontimeof2h,theyieldobtainedat40MPa(6.5%)washigherthantheoneobtainedat35MPa(5.9%).TheyieldofGLAincreasedwithpressure,havingobtained0.12%at20MPa,whileat40MPa0.29%wasreached.Bothyieldvaluesarehigherthantheone(0.05%)obtainedbyMendesetal.[32]at25MPa/50ºCfromS. maxima.

Theremovingofthedeleterioussmellwasalsoreached.Moreover,aftertreat-ment with supercritical CO2,theprotein contentwaspracticallynot altered,withonlyaslightreductionofabout1%inessentialaminoacidshavingbeendetected,therebypreservingthenutritivevalueofSpirulina.

Carerietal.[28]carriedoutstudiesofSFEonthestrainpacifica(acarotenoid-richdietaryproduct)ofthemicroalgaeS. platensis.Thetargetcompoundsforthestudy were the carotenoidsβ-carotene,β-cryptoxanthin, and zeaxanthin, and theauthorsproposedtofindthebestexperimentalconditionsfortheirrecoverythroughan experimental design procedure. Four parameters were investigated: pressure(150,250,and350bar), temperature(40,60,and80ºC),dynamicextraction time(40,70,and100min),andpercentageofethanol(involume)addedtotheCO2(5,10,and15%).Otherexperimentalconditionswere0.5gofdrypowderedalgae(50mmparticlesize)ina7-cm3extractor.Theextractionbyorganicsolventwasperformedusingtetrahydrofuranandpetroleumether[86].

Forallthreecarotenoids,thehighestpressure(350bar)ledtothehighestrecov-eries.Thetemperaturesthatmaximizedtherecoverieswere80ºC,70ºC,and60ºCfor zeaxanthin,β-cryptoxanthin, andβ-carotene, respectively. The effects of thevariousfactorswereanalyzedand,forallthecompounds,thetemperaturewasfound

7089_C006.indd 208 10/8/07 11:56:18 AM


nottobesignificantasthemaineffect.Onthecontrary,thepressureofthesupercrit-icalfluidplaysanimportantrole,appearingtobesignificantforallthecarotenoids.

Atconstanttemperature,apressureincreasecausesanincreaseinfluiddensityandthuscouldhaveadoubleeffect: increasedsolventpowerofCO2andreducedinteraction between the fluid and the solid matrix, having as a consequence thedecreasingofthediffusioncoefficientathigherdensities.

Theamountofethanolwasalsosignificantforallthecarotenoids.Theincreaseinthepercentageoftheentrainerledtoanincreaseoftheextractionyields.Thiseffect is not only related to themodificationof thepolarityof the supercriticalfluidbutalso to the interactionof theethanolwith thesolidmatrixbecausethecarotenoidswithdifferentpolaritiesshowbetterrecoverieswhenethanolisaddedtothefluid.

Carerietal.[28]alsocomparedtheSFEobtainedatthebestconditionswiththatobtainedfromorganicsolventextraction.Intermsofyield,organicsolventextrac-tion showed a slight advantage, but because it is time-consuming (with multipleextractionandpurificationsteps)andveryexpensive,intheendSFE,provedtobeamoreeffectiveprocedure.

6.3 ConClusIon

The use of algae to obtain useful biochemicals for medical, pharmaceutical, anddietaryapplicationsshowsalargepotentialinthenearfuture.However,manyalgaealsoproduceharmfulcompounds.Althoughsomeofthesecanbedirectedtoobtainusefuldrugs,theymustbescreenedbeforehumanconsumptionoccurs.

Inmanycases,SFEshowsadvantagesovertheuseoforganicsolventstoobtaincompounds fromalgae,namelya shorter timeofextractionandbetteror similaryields,avoidingtheuseofexpensiveandpollutingsolvents.Moreover,SFEcanbeusedwithsupercriticalfluidfractionationandsupercriticalfluidchromatographytoseparateorpurifytheextractedcompounds.Becauseitisanexpensiveprocedure,SFEisonlyreservedforhigh-valuecompoundsincommercialapplications.

reFerenCes

1. Borowitzka,M.A.andBorowitzka,L.J.,Eds.,Micro-algal Biotechnology,CambridgeUniversityPress,Cambridge,1988.

2. Borowitzka,M.A.,inVitaminsandfinechemicalsfrommicro-algae,Micro-algal Bio-technology,Borowitzka,M.A.andBorowitzka,L.J.,Eds.,CambridgeUniversityPress,Cambridge,1998,153.

3. Burja,A.M.etal.,Marinecyanobacteria-aprolificsourceofnaturalproducts,Tetra-hedron,57,9347,2001.

4. Skulberg, O.M., Microalgae as a source of bioactive molecules: Experience fromcyanophyteresearch,J. of App. Phycol.,12,341,2000.

5. Kyle,D.J.,Specialtyoilsfrommicroalgae,New Perspectives in Biotechnology of Plant Fats and Oils,Ratray,J.,Ed.,AmericanOilChemists’SocietyPress,Champaign,Illi-nois,1991,130.

6. Cohen,Z.,Chemicals from Microalgae,Taylor&Francis,London,1999. 7. Borowitzka,M.A.,Fats,oilsandhydrocarbonsMicro-algal Biotechnology,Borowitzka,

M.A.andBorowitzka,L.J.,Eds.,CambridgeUniversityPress,Cambridge,1998,257.

7089_C006.indd 209 10/8/07 11:56:19 AM


8. Yamaguchi, K., Recent advances in microalgal bioscience in Japan, with specialreferencetoutilizationofbiomassandmetabolites:Areview,J. of Appl. Phycol., 8,487,1997.

9. Apt,K.E.andBehrens,P.W.,Commercialdevelopmentsinmicroalgalbiotechnology,J. Phycol.,35,215,1999.

10. Pulz, O. and Gross, W., Valuable products from biotechnology of microalgae, App. Microbiol. Biotechnol.,65,635,2004.

11. Dufossé,L.etal.,Microorganismsandmicroalgaeassourcesofpigmentsforfooduse:Ascientificoddityoranindustrialreality?,Trends Food Sci. Technol.,16,389,2005.

12. Smit,A.J.,Medicinalandpharmaceuticalusesofseaweednaturalproducts:Areview,J. of Appl. Phycol.,16,245,2004.

13. Margalith, P.Z., Production of ketocarotenoids by microalgae, App. Microbiol. Biotechnol.,51,431,1999.

14. Kyle,D.J.,Thefuturedevelopmentofsinglecelloils,inSingle Cell Oils, Cohen,Z.andRatledge,C.,Eds.,AmericanOilChemists’SocietyPress,Champaign,Illinois,2005,239.

15. Richmond,A.,Microalgalbiotechnologyat the turnof themillennium:Apersonalview,J. of Appl. Phycol.,12,441,2000.

16. Craig, R., Reichelt, B.Y. and Reichelt, J.L., Genetic engineering of micro-algae, inMicro-algal Biotechnology,Borowitzka,M.A.andBorowitzka,L.J.,Eds.,CambridgeUniversityPress,Cambridge,1998,415.

17. Borowitzka,M.A.,Carotenoidproductionusingmicroorganisms,inSingle Cell Oils,Cohen,Z.andRatledge,C.,Eds.,AmericanOilChemists’SocietyPress,Champaign,Illinois,2005,124.

18. Stahl,E.,Quirin,K.W.andBlagrove,R.J.,Extractionofseedoilswithsupercriticalcarbondioxide:Effectonresidualproteins,J. Agric. Food Chem.,32,938,1984.

19. Nakhost, Z., Karol, M. and Krukonis, V.J., Non-conventional approaches to foodprocessing in CELSS. I-Algal proteins, characterization, and process optimization,Adv. Space Res.,7,29,1987.

20. Choi,K.J.etal.,SupercriticalfluidextractionandcharacterizationoflipidsfromalgaeScenedesmus obliquus,Food Biotechnol.,1,263,1987.

21. Subra,P.andBoissinot,P.,Supercriticalfluidextractionfromabrownalgabystage-wisepressureincrease,J. Chromatogr.,543,413,1991.

22. Erazo,S. et al.,Estudiode labiomassayde lospigmentoscarotenoidescontenidosenunaespecienativadelamicroalgaDunaliella salina sp.,Rev. Agroquim. Tecnol. Aliment.,29,538,1989.

23. Balaban,M.O.,O’Keefe,S.andPolak,J.T.,Supercriticalextractionofalgae,inSuper-critical Fluid Technology in Oil and Lipid Chemistry,King,J.W.andList,G.R.,Eds.,AmericanOilChemists’SocietyPress,Champaign,Illinois,1995,247.

24. Wisniak,J.andKorin,E.,Supercriticalfluidextractionoflipidsandothermaterialsfrom algae, in Single Cell Oils, Cohen, Z. and Ratledge, C., Eds., American OilChemists’SocietyPress,Champaign,Illinois,2005,220.

25. Herrero,M.,Cifuentes,A.and Ibanez,E.,Sub-andsupercriticalfluidextractionoffunctionalingredientsfromdifferentnaturalsources:Plants,food-by-products,algae,andmicroalgae,areview,Food Chem.,98,136,2006.

26. Santos, R. et al., Extraction of valuable components from micro-algae Spirulina platensis withcompressedCO2,inProceedings of the Fourth Italian Conference on Supercritical Fluids and Their Applications,Reverchon,E.,Ed.,Capri(Napoli)-Italy,1997,217.

27. Qiuhui,H.,SupercriticalcarbondioxideextractionofSpirulina platensis componentandremovingthestench,J. Agric. Food Chem.,47,2705,1999.

7089_C006.indd 210 10/8/07 11:56:20 AM


28. Careri,M.etal.,Supercriticalfluidextractionforliquidchromatographicdeterminationofcarotenoids inSpirulinaPacificaalgae:achemometricapproach,J. Chromatogr.,912,61,2001.

29. Canela,A.P.R.F.etal.,SupercriticalfluidextractionoffattyacidsandcarotenoidsfromthemicroalgaeSpirulina maxima,Ind. Eng. Chem. Res.,41,3012,2002.

30. Valderrama,J.O.,Perrut,M.andMajewski,W.,Extractionofastaxanthinandphyco-cianinfrommicroalgaewithsupercriticalcarbondioxide,J. Chem. Eng. Data,48,827,2003.

31. Mendes,R.L.etal.,SupercriticalCO2extractionofgamma-linolenicacid(GLA)fromcyanobacteriumArthrospira(Spirulina)maxima:Experimentsandmodelling,Chem. Eng. J.,105,147,2005.

32. Mendes,R.L.,Reis,A.D.andPalavra,A.F.,SupercriticalCO2extractionofγ-linolenicacidandotherlipidsfromArthrospira(Spirulina)maxima:Comparisonwithorganicsolventextraction,Food Chem.,99,57,2006.

33. Mendes,R.L.etal.,SupercriticalcarbondioxideextractionofhydrocarbonsfromthemicroalgaBotryococcus braunii,J. Appl. Phyc.,6,289,1994.

34. Mendes,R.L.etal.,SupercriticalCO2extractionofcarotenoidsandotherlipidsfromChlorella vulgaris, Food Chem.,53,99,1995.

35. Mendes,R.L.etal.,Supercriticalcarbondioxideextractionofcompoundswithphar-maceuticalimportancefrommicroalgae,Inorganica Chimica Acta,356,328,2003.

36. Gamlieli-Bornshtein, I., Korin, E. and Cohen, S., Selective separation of cis-transi geometrical isomersofβ-caroteneviaCO2supercriticalfluidextraction,Biotechnol. Bioeng.,80,169,2002.

37. Mendes, R.L. et al., Solubility of synthetic and natural β-carotene in supercriticalcarbon dioxide, in Proceedings of the Fourth Italian Conference on Supercritical Fluids and Their Applications,Reverchon,E.,Ed.,Capri(Napoli)-Italy,1997,423.

38. Mendes, R.L. et al., Supercritical CO2 extraction ofβ-carotene from algae, in Pro-ceedings of the Sixth Conference on Supercritical Fluids and Their Applications,Reverchon,E.,Ed.,Maiori,Italy,2001,187.

39. Perretti,G.etal.,ExtractionofPUFArichoilsfromalgaewithsupercriticalcarbondioxide,inProceedings of the Sixth International Symposium on Supercritical Fluids,Brunner,G.,Kikic,I.andPerrut,M.,Eds.,Versailles,France,2003,29.

40. Nobre, B. et al., Supercritical carbon dioxide extraction of astaxanthin and othercarotenoidsfromthemicroalgaHaematococcus pluvialis,Eur. Food Res. Tecnol.,223,787,2006.

41. Machmudah,S.etal.,ExtractionofastaxanthinfromHaematococcus pluvialisusingsupercriticalCO2andethanolasentrainer,Ind. Eng. Chem. Res.,45,3652,2006.

42. Cheung, P.C.K., Temperature and pressure effects on supercritical carbon dioxideextractionofn-3fattyacidsfromredseaweed,Food Chem.,65,399,1999.

43. Sakaki,K.etal.,SupercriticalfluidextractionoffungaloilusingCO2,N2O,CHF3,andSF6,J. Am. Oil Chem. Soc.,67,553,1990.

44. Macías-Sánchez,M.D.etal.,Supercriticalfluidextractionofcarotenoidsandchloro-phyllafromNannochloropsis gaditana,J. Food Engin.,66,245,2005.

45. Andrici,G.etal.,SupercriticalfluidextractionofbioactivelipidsfromthemicroalgaNannochloropsis sp.,Eur. J. Lipid Sci. Technol.,107,381,2005.

46. Polak,J.T.,Balaban,M.B.andPhilips,A.J.,Supercriticalcarbondioxideextractionoflipidsfromalgae,ACS Symposium Series,406,449,1989.

47. Lim,G-B.etal.,SeparationofastaxanthinfromredyeastPhaffia rhdozymabysuper-criticalcarbondioxide,Biochem. Engin. J.,11,181,2002.

48. Radwan,A.etal.,SupercriticalfluidCO2extractionacceleratesisolationofhumicacidfromlivePilayella littoralis (Phaeophyta),J. of Appl. Phycol.,8,545,1997.

7089_C006.indd 211 10/8/07 11:56:20 AM


49. Cygnarowicz-Provost, M. et al., Supercritical fluid extraction of fungal lipids usingmixedsolvents:Experimentandmodeling,J. Supercritical Fluids,5,24,1992.

50. Bhattacharjee,P.andSinghal,R.S.,Extractionofsqualenefromyeastbysupercriticalcarbondioxide,World J. Microbiol. Biotechnol.,19,605,2003.

51. Calvin, M. and Taylor, S.E., Fuels from algae, in Algal and Cyanobacterial Biotechnology,Cresswell,R.C.,Rees,T.A.V.andShah,N.,Eds.,LongmanScientific&Technical,London,1989,137.

52. Templier, J., Largeau, C. and Casadevall, E., Mechanism of non-isoprenoid hydro-carbonbiosynthesisinBotryococcus braunii,Phytochemistry,23,1017,1984.

53. Yamaguchi,K.etal.,LipidcompositionofagreenalgaBotryococcus braunii,Agric. Biol. Chem.,51,493,1987.

54. Casadevall,E.,Productiond’hydrocarburesparl’algueBotryococcus braunii,Biomasse Actualités,3,64,1983.

55. Mendes,R.L.andPalavra,A.F.,Supercriticalextractionofcompoundsfrommicro-algae,inChemistry, Energy, and the Environment,Sequeira,C.A.CandMoffat,J.B.,Eds.,TheRoyalSocietyofChemistry,Cambridge,51,1998.

56. Gouveia,L.etal.,Evolutionof thepigments inChlorella vulgarisduringcaroteno-genesis,Bioresources Technol.,57,157,1996.

57. Davies,B.H.,Carotenoids,inChemistry and Biochemistry of Plant Pigments,Goodwin,T.W.,Ed.,AcademicPress,London,2,38,1976.

58. Bendich,A.,Non-provitaminAactivityofcarotenoids:Immunoenhancement,Trends in Food Sci. Technol.,2,127,1991.

59. Mendes,R.L.,PhDThesis,Extracçãosupercríticadelípidosdemicroalgas,UniversidadeTécnicadeLisboa,Portugal,1995.

60. Mendes, R.L. et al., Supercritical CO2 extraction of lipids from microalgae, inProceedings of the Third International Symposium on Supercritical Fluids,Brunner,G.andPerrut,M.,Eds.,Strasbourg,1994,2,477.

61. Mendes,R.L.etal.,Modelisationdel extractionsupercritiquedelipidesd algues,inProceedings 3eme Colloque sur les Fluides Supercritiques, Applications aux Produits Naturels,Pellerin,P.andPerrut,M.,Eds.,Grasse(France),1996,141.

62. King,M.B. et al.,Equilibriumand ratedata for the extractionof lipidsusing com-pressedcarbondioxide,Separation Sci. Technol.,22,1103,1987.

63. Hebuterne,X. et al., Intestinal absorption andmetabolismof 9-cis-beta-carotene invivo:Biosynthesisof9-cis-retinoicacid,J. Lipid Res.,36,1264,1995.

64. Levin,G. andMokady,S.,Antioxidant activityof9-cis compared toall-transbeta-caroteneinvitro,Free Radic. Biol. Med.,17,77,1994.

65. Ben-Amotz,A.,Lers,A.andAvron,M.,Stereoisomersofbeta-caroteneandphytoeneinthealgaDunaliella bardawil,Plant Physiol.,86,1286,1988.

66. Nobre,B.P.etal.,Separationoftheisomersofβ-carotenewithsupercriticalCO2andsilicagel,inProceedings of the Seventh Meeting on Supercritical Fluids,Perrut,M.andReverchon,E.,Eds.,Antibes,France,2000,2,781.

67. Olaizola, M., Commercial development of microalgal biotechnology: From the testtubetothemarketplace,Biomol. Eng.,20,459,2003.

68. Guerin, M., Huntley, M.E. and Olaizola, M., Haematococcus astaxanthin: Applica-tionsforhumanhealthandnutrition,Trends Biotechnol.,21,210,2003.

69. Østerlie,M.,Bjerkeng,B.andLiaaen-Jensen,S.,PlasmaappearanceanddistributionofastaxanthinE/Z andR/S isomers inplasma lipoproteinsofmenaftersingledoseadministrationofastaxanthin,J. Nutr. Biochem., 11,482,2000.

70. Denery, J.R.etal.,Pressurizedfluidextractionofcarotenoids fromHaematococcus pluvialisandDunaliella salinaandkavalactonesfromPiper methysticum,Analytica Chimica Acta,501,175,2004.

7089_C006.indd 212 10/8/07 11:56:21 AM


71. Ackman, R.G., Algae as a source of edible oils, in New Sources of Fats and Oils,Pryde,E.H.,Princen,L.H.andMukherjee,K.D.,Eds.,AmericanOilChemists’SocietyPress,Champaign,Illinois,1981,189.

72. Trautwein,E.A.,n-3 fattyacids:Physiologicaland technicalaspects for theiruse infood,Eur. J. Lipid Sci. Technol.,103,45,2001.

73. Becker, E.W., Microalgae: Biotechnology and Microbiology, Cambridge UniversityPress,1994,177.

74. Peet,M.etal.,Twodouble-blindplacebo-controlledpilotstudiesofeicosapentaenoicacidinthetreatmentofschizophrenia,Schizophr. Res.,49,243,2001.

75. Lubían,L.M.etal.,Nannochloropsis (Eutigmatophyceae)assourceofcommerciallyvaluablepigments,J. Appl. Phycol.,12,249,2000.

76. Chrastill,J.,Solubilityofsolidsandliquidsinsupercriticalgases,J. Phys. Chem.,86,3016,1982.

77. Cohen,Z.,ThechemicalsofSpirulina,inSpirulina platensis (Arthrospira):Physiol-ogy, Cell-Biology, and Biotechnology,Vonshak,A.,Ed.,Taylor&Francis,London,1997,175.

78. Otles,S.andPire,R.,FattyacidcompositionofChlorella andSpirulina microalgaespecies,J. AOAC Int.,84,1708,2001.

79. Khan, Z., Bhadouria, P. and Bisen, P.S., Nutritional and therapeutic potential ofSpirulina,Current Pharma. Biotechnol.,6,373,2005.

80. Nakahara,T.etal.,Gamma-linolenicacidfromgenusMortierella,inIndustrial Appli-cations of Single Cell Oils,Kyle,D.J.andRatledge,C.,Eds.,AmericanOilChemists’SocietyPress,Champaign,IL,1992,61.

81. Kennedy, M.J., Reader, S.L. and Davies, R.J., Fatty acid production characteristicsof fungi with particular emphasis on gamma linolenic acid production, Biotechno. Bioengin.,42,625,1993.

82. Jaime, L. et al., Separation and characterization of antioxidants from Spirulina platensis microalgacombiningpressurizedliquidextraction,TLC,andHPLC-DAD,J. Sep. Sci.,28,2111,2005.

83. Certik,M.,Andrasi,P.andSajbidor,J.,Effectofextractionmethodsonlipidyieldandfattyacidcompositionoflipidclassescontainingg-linolenicacidextractedfromfungi,J. Am. Oil Chem. Soc.,73,357,1996.

84. Mendes,R.L.etal.,Solubilityofβ-caroteneinsupercriticalcarbondioxideandethane,J. Supercritical Fluids,16,99,1999.

85. Goto,M.,Sato,M.andHirose,T.,Extractionofpeppermintoilbysupercriticalcarbondioxide,J. Chem. Eng. Jpn.,26,401,1993.

86. Hart,D.J. andScott,K.J.,DevelopmentandevaluationofanHPLCmethod for theanalysis of carotenoids in foods, and the measurement of the carotenoid content ofvegetablesandfruitscommonlyconsumedintheU.K.,Food Chem.,54,101,1995.

7089_C006.indd 213 10/8/07 11:56:22 AM

7089_C006.indd 214 10/8/07 11:56:22 AM

215

7 Application of Supercritical Fluids in Traditional Chinese Medicines and Natural Products

Shufen Li

Contents

7.1 Introduction................................................................................................. 2167.2 SpecialFeaturesofSFETechniqueinProcessingTCMand

NaturalProducts......................................................................................... 2177.3 StatusofSFEinProcessingTCMandNaturalProductsinChina............ 219

7.3.1 NationalSymposiumsonSCFTechnology..................................... 2197.3.2 SCFEquipmentMadeinChina....................................................... 2197.3.3 SummaryofApplyingSFEinProcessingTCMand

NaturalProducts..............................................................................2207.3.3.1 SFEwithPureSupercriticalCarbonDioxide....................2207.3.3.2 SFEwithCO2inPresenceofCosolvent............................2207.3.3.3 SFEwithCO2inPresenceofSurfactant............................ 2217.3.3.4 CombingSFEwithUltrasound-Enhanced

ExtractionMethod..............................................................2237.3.3.5 CombiningSFEwithEnhancedSeparationMethods.......2237.3.3.6 CombiningSFEwithOtherTechniquesto

MakeFullUseofHerbalMaterials....................................2247.4 SelectExamplesofSFEofTCMandNaturalProducts.............................225

7.4.1 ExtractionofEssentialOilfromCloveBudwithSC-CO2..............2257.4.2 ExtractionofMedicalIngredientsfromtheMixtureof

Angelica sinensisandLigusticum chuanxiongHortwithSC-CO2...2287.4.3 ExtractionofEdibleandMedicinalIngredientsfrom

GrapeSeedswithSC-CO2...............................................................2307.4.4 IsolationofOrganochlorinePesticidefromGinsengwithSC-CO2... 233

7.5 SummaryandProspect............................................................................... 236References.............................................................................................................. 237

7089_C007.indd 215 10/15/07 5:43:34 PM


7.1 IntroduCtIon

TraditionalChinesemedicine(TCM)isascientificsummaryofrichexperiencesoftheChinesenation’sstruggleagainstdiseaseforthousandsofyears.Itisoneoftheoldestandstrongesttraditionalmedicalsystemsinthehistoryoftheworld.ClassicalChinese herbal medicines encompass a large number of herbal formulations withknownmildpharmaceuticaleffectsandminimumsideeffectsthatareusedforthetreatmentofawidevarietyofdifficult-to-treatdiseases.Overthecourseofmanycenturies,TCMhasgreatlyformedauniquetheoreticalsystemanddiagnosingandtreatingtechniquesthathavemadeanindelibleandsubstantialcontributiontoboththehealthandprosperityoftheChinesepeople[1,2].TCMhasnotonlyenjoyedanexcellentreputationinChinabutalsointherestoftheworld.Itwillplayamoreandmoreimportantroleincontributingtothehealthandlongevityofmankind.

InChina,morethan11,000plantsareconsideredtobemedicineherbs.Almost2,000ChinesetraditionalpatentmedicinesarelistedintheofficialChinesePharma-copoeia.ThesemedicinesarewidelyusedasTCMinChina,eveninSoutheastAsia[1–4].Mostofthemareprocessedwithmanykindsofmedicineplantsaccordingtothetheoryofprescriptioncomposition.However,somearecomposedofonlyasingleplant.TheefficacyofChineseherbalmedicines is considereda synergismofmanyeffectivecomponents, includingnotonly thesmallmoleculecompoundssuchasvolatileoils,alkaloids,flavonoids,andsaponinsbutalsobiologicalmacro-moleculessuchaspolysaccharides,proteins,andpeptides.

Themosttraditionalmethodforprocessingherbsinvolvesboilingtheminwaterforhourssothatmostoftheingredientsaredissolved.Anothermethodinvolvestheuseofconventionalorganicsolventsforextractioninsteadofboilingwater;themostcommonlyusedorganicsolventsareethanol,ether,chloroform,andmethanol.Whenthetraditionalextractionmethodsforprocessingherbsareused,theextractsconsistofvariouscompounds,includingsomeundesiredsubstancesthatdissolvewiththedesiredproducts.Therefore,furtherpurificationstepsarenecessarytoremovethecoextractedimpurities.Intheseprocesses,longprocessingtimesof2to7daysaregenerallyneeded.Highboilingorextractiontemperaturesoftenleadtodegradationofheat-sensitivecompounds.Moreover,tracesoftoxicsolventsarehardlyremovedfromtheextracts,whichdirectlyinfluencesthequalityoftheproducts.Hydrodistil-lation(steamdistillation) isgenerallyusedforobtainingvolatileoilsfromplants.Itshighprocessingtemperaturecanalsoleadtodegradationofheat-sensitivecom-pounds. Therefore, alternative extraction techniques with better selectivity andefficiencyarehighlydesirable.

In recent years, the catchphrase “modernization and internationalization ofTraditionalChinesemedicine”hasoftenbeenpresentedinChinesepapers,maga-zines, and symposiums, and this concepthasbecomeaveryhot topic [5,6].Forthis reason, the pharmaceutical study of natural products has become one of themost interesting and active research areas in China. Some new chemical separa-tiontechnologies,suchassupercriticalfluidextraction(SFE),membraneseparation,ultrasonic-assist extraction,molecular distillation (MD), andpolymeric adsorbenttechnology,havebeenconsideredtohelpimprovetheproductiveprocessofTCM

7089_C007.indd 216 10/15/07 5:43:35 PM

Application of Supercritical Fluids in Traditional Chinese Medicines 217

[7,8].Theseeffortshavebeensuccessful.Amongthesenewtechnologies,SFEispresentlyconsideredasoneofthemostcleanandhighlyeffectivetechnologiesforprocessingTCM.

The high solvent power of supercritical fluid (SCF) was first reported over acentury ago. Demonstration of SFE technology for industrial applications wasreportedbyZoselin1970[9].Sincethen,thefundamentalandappliedaspectsofSCFandprocesseswithapplicationscoverawiderangeoftopicsinenergy,environment,medicine,chemicalindustries,andanalyticalfield.Ithasbeenalsorapidlyextendedtootherfields,suchaschemicalreaction,supercriticalfluidchromatography(SFC),andparticleformationinmaterialprocessing withSCF[10,11].

This chapter focuses on introducing the SFE techniques used in processingTCMandnaturalproducts.ThespecialfeaturesofSFEtechniquesandthestatusofSFEinprocessingTCMandnaturalproductsinChinaarebrieflyreviewed.FourtypicalexamplesofSCFapplicationinTCMandnaturalproductsfromourlabora-toryresearchhavebeenselectedtomakefurtherdescription.

7.2 speCIal Features oF sFe teChnIque In proCessIng tCM and natural produCts

Agas,whencompressedisothermallytopressuresgreaterthanitscriticalpressure,exhibits enhanced solvent power in the vicinity of its critical temperature. Suchfluid is called supercritical fluid. SCFs possess desirable specific characteristicsthatmakethemattractiveassolvents.Liquid-likedensitiesandgas-likeviscosities,coupledwithdiffusioncoefficientsthatareatleastanorderofmagnitudehigherthanthoseofliquids,contributetotheenhancementofmasstransfer.Inparticular,adjusting pressure and temperature can control the solvent density and hencesolventpower,becausethesolventpowerofaSCFrelatestothesolventdensityinthecriticalregion[12–14].

Among SCFs, supercritical CO2 (SC-CO2) remains the most commonly usedfluid for SFE application because of its mild critical properties (Tc = 31.1°C,Pc=7.38MPa),nontoxicity, chemical inertness, andavailability inhighpurity atlowcost.TheseexcellentpropertiesleadCO2tobeconsideredan“environmentallyfriendly”solvent forextractionofnaturalproducts,suchascoffee, tea,hops,andselectedspices[12–13].

Asisknown, thedipolemomentofCO2iszeroanditspolarizability isonly26.5× 10–25cm–3,which is less than thatofallofhydrocarbonsexceptmethane[14].Therefore, SC-CO2 is only a good solvent for extraction of nonpolar com-pounds,suchashydrocarbons,whileitslargequadripolemomentalsoenablesittodissolvesomemoderatelypolarcompounds,suchasalcohols,esters,aldehydes,andketones[9].WhenpureSC-CO2isemployedasasolventforprocessingnaturalproducts,mixturescontainingbothnonpolarandmoderatelypolarsubstancesaregenerallyextracted.

Both the properties of the solute and the solvent can affect the extractabilityof natural products. Vapor pressure, polarity, and molecular weight of solutesare themost important factors affecting the solubilityof solutes inSCF.Raising

7089_C007.indd 217 10/15/07 5:43:35 PM


the temperature raises the vapor pressure or sublimation pressure of solutes andhence increases the solubility of the solute. However, increasing the temperaturealsocausesasimultaneousdecreaseinthedensityofCO2,whichtendstodecreasesolubility.Twocompetingfactorsneedtobeconsideredtofindthesuitabletempera-ture.RaisingthepressureincreasesthedensityofthesolventofSC-CO2andhencethesolventpowerofthesolutes.However,thebenefitofthisincreaseisoftenlimitedbymanufacturingabilityofhigh-pressureequipmentandcapitalcosts.Selectingasuitablecosolventorentrainerthatcanmaintainorimproveselectivityandincreasesolubilitymaybeoneof thekeystoexpandingtheapplicationofSFE.Theaddi-tionofacosolventcannotonlyshift thecriticalpropertiesfromthepuresolventcriticalpropertiesandhenceaffect thepropertiesof theSCFbutcanalso inducecosolvent-soluteinteractionsorassociations,suchasacid-baseinteractions,depend-ingonthepropertiesofthesolute,solvent,andcosolvent,anyofwhichmayenhancethesolubility[15–19].

Inmostcasesofsolventextractionfrombotanicalsubstances,fourstepsofmasstransportoccur:

1.Diffusionofsolventintothebotanicalsubstance 2.Solvationofsolute 3.Diffusionofsoluteintobulkfluidphase 4.Transportofsoluteandthebulkfluidphasefromtheextractionzone

Usually,thediffusionofthesolutesoutofthematrixisthelimitstep.Inordertoreducethediffusiondistanceofsolutesthroughthebotanicalsubstrateandfurtherrupturingofthecellwall,henceeliminatingsomediffusionbarriers,itisnecessarytogroundthenaturalrawmaterialintotheoptimumsizebecauseparticlesizehassomeeffectonyieldandrateofrecovery[20–25].

SFEprocessesneedtoconsiderbothextractionandseparation.Threebasicoper-ationmodelscanbeusedtoseparatesolutesfromSCFsolvents:pressurereduction,temperaturevariation,andadsorption.Eachoperationalmodelhas itsadvantagesandlimitations.SFEwithextractseparationbyvaryingthetemperatureisoperatedin isobaric state.Extract separationby adsorption allows theSFEprocess to runisobaricallyandisothermally,andsoSCFcanbecirculatedwithoutrecompression.Thesetwomodesrequirelessenergyconsumption.However,pressurereductionisthemostusedmodeinprocessingofTCMbecausetheoperationcanbemademorestablebyeffectivelycontrollingtheliquidCO2levelwhenreducingtheseparationpressureandtemperaturetobelowthecriticalvalues.

ForsuccessfulSFE,somefactorsmustbetakenintoconsiderationpriortotheexperiments.Thesefactorsincludethetypeofrawmaterials,methodoffeedprepara-tion,typeoffluid,choiceofcosolvents,methodoffeedingcosolvents,andextractionandseparationconditions,includingpressure,temperature,flowrate,andextractiontime.TooptimizeSFEconditions,astatisticalexperimentaldesignbasedonortho-gonalexperimentsiscommonlyusedandreported,wheretheyieldandthecontentoftheactivecompoundintheextractsareoftenconsideredasthetargetindex.

7089_C007.indd 218 10/15/07 5:43:36 PM


7.3 status oF sFe In proCessIng tCM and natural produCts In ChIna

7.3.1 NatioNal SympoSiumS oN SCF teChNology

Asoneofthegreenchemistryandengineeringtechnologies,SCFscienceandtech-nologycallsgreatattentionfromthegovernment.Manynationalprojectsrelatingto SCF science and technology were supported by the government. Many enter-prisesalsocarryoutsomeresearchanddevelopmenttogetherwiththeresearchersofuniversitiesandinstitutes.Inaddition,nationalsymposiumsonSCFscienceandtechnologyhavebeenheldevery2yearssince1996.Uptofivesymposiumproceed-ingshavenowbeenpublishedinChina[26–30].

Table7.1summarizesthepresentationsfromthefiveChinesesymposiumsandarticlesappearingintheChinesecorejournalsinVIPChineseDatabank,whichwastheauthoritativeprofessionaldatabaseinChinathrough1998.ItcanbeseenfromTable7.1 that, although theapplicationofSCFhasalsobeen rapidlyextended tosupercriticalchemicalreaction,particleformation,SFC,andotherfields,theearliestandmostactiveresearchfieldcentersonSFE,especiallyitsapplicationsinextract-ingactivecomponentsfromChineseherbs.

7.3.2 SCF equipmeNt made iN ChiNa

Assupercritical statesoffluidsareathighpressures, the industrializationdesignandscale-upplantarethecoreissuesformostenterprisesandresearchinstitutions.Currently, at least seven setsof large-scale industrialSFEequipmentwere intro-duced fromEurope,ofwhich the largestonewasmade inGermany(UhdeHighPressure Technologies GmbH) and the extraction vessels’ configuration for eachof the threevesselsare3500L, respectively.Aside fromfor importing large-sizeinstruments, the domestic instruments for SCF technology especially in SFE arebeingdeveloped.Therearemorethan30setsofhomemadeSFEinstrumentswithextractorsizesover100L,and the largestsize is2000L.Additionally, thereare

table 7.1presentations in the Five Chinese symposiums and VIp on sCF technology

1st, 1996

2nd, 1998

3rd, 2000

4th, 2002

5th, 2004 VIp total

SFE 23 31(4) 42(1) 58 68 129 351

SCR 13(6) 16(4) 18(4) 17(6) 21 76 155

Particleformation 3 5 11 10 24 38 91

SFC 2 1 2 1 2 8 16

Others 1 2 7 8 13 21 52

Theoreticalstudy 12 13 19 18 16 37 125

Papersineachsymposium 48 68 99 112 144 309 790

7089_C007.indd 219 10/15/07 5:43:37 PM


morethan150SFElaboratoryunitswithexactorvolumeslessthan25LlocatedinthemanyprovincesofChina[6].

There exist some obstacles to developing homemade SFE instruments. Forexample,theauto-controlsystemisstillrelativelybackwardandsomemanufacturelevelsforhigh-pressurepartscannotreachinternationalstandards.

7.3.3 Summary oF applyiNg SFe iN proCeSSiNg tCm aNd Natural produCtS

Atleast150kindsofChinesetraditionalherbalplantswereselectedasrawmaterialsto investigate withSFE technology inChina. The research methods used canbeclassifiedintosixdifferentcases:

1.SFEwithpureSC-CO2

2.SFEwithCO2inpresenceofcosolvent 3.SFEwithCO2inpresenceofsurfactant 4.CombingSFEwithultrasound-enhancedextractionmethod 5.CombingSFEwithotherseparationmethods 6.CombiningSFEwithothertechniquestomakefulluseofherbalmaterials.

7.3.3.1 sFe with pure supercritical Carbon dioxide

Anoverviewof recentpublicationsonapplicationsofpureSC-CO2 inTCMandnaturalproductsisgiveninTable7.2.Thetargetextractsaremostcommonlyvola-tileessentialoils,whichareamixtureofnonpolarcomponentsandmoderatelypolarsubstances.Theextractiontemperaturesaregenerallyfrom30°Cto60°C,andtheinvestigatedpressureswerefrom8to40MPa,dependingonthepropertiesofboththerawmaterialusedandthedesiredextracts.Thesekindsofapplicationscanfullyshow the advantages of SFE over traditional solvent extraction, hydrodistillation,and steam distillation, such as higher yield with better quality, less hydrocarbonpollution,greatersafety,lowerproductioncost,andnodegradationofheat-sensitivecompounds.Asweknow,traditionalprocessingmethodsforthesekindsofherbalmaterialsaremostlyhydrodistillationandsteamdistillationorsolventextraction,inwhichhighboilingtemperaturesoftenleadtodegradationofheat-sensitivecom-pounds.Furthermore,tracesoftoxicsolventsarehardlyremovedfromtheextractswhensolventsareused,whichdirectlyinfluencesthequalityoftheproducts.

7.3.3.2 sFe with Co2 in presence of Cosolvent

SomeexamplesofusingSC-CO2inthepresenceofacosolventarelistedinTable7.3.Mostoftheextractsobtainedaremiddle-polarsubstances,suchasalkaloids,saponins,andflavonoids.Themostcommonlyusedcosolventsareethanolanddifferentcon-centrationsofaqueousethanolsolutions.Asisknown,comparedwithmethanolandchloroform, ethanol is less toxic andmoreacceptable forprocessingTCM.Wateris themost acceptableandcheapest solvent.By regulating the ratioofwater andethanol,onecanreadilymanipulatethepropertiesofthefluids.Usually,additionof

7089_C007.indd 220 10/15/07 5:43:37 PM


asmallamountofaliquidcosolventcansignificantlyenhancetheextractioneffi-ciencyandconsequentlyreducetheextractiontimeorpressure.

7.3.3.3 sFe with Co2 in presence of surfactant

TheresearchandprogressofSC-CO2microemulsionverifythepossibilityofextract-ing polar compounds with SC-CO2. When an appropriate surfactant is added intotheSC-CO2,a reversemicroemulsioncanform,whichfacilitates thedissolutionofhydrophilic molecules in SC-CO2.The formation of SC-CO2 microemulsion needs

table 7.2overview on the extraction of active Compounds from Chinese herbals with sC-Co2

raw Materials Conditions extracts Yield (%) references

Arnebiaeuchroma(Royle)Johnst 35°C,27MPa Naphthaquinoniccompounds

4.1–4.6 [31]

AtractylodesmacrocephalaKoidz 50°C,28MPa Volatilecomponents 4.27 [32]

Beepollen 55°C,30MPa Lipophiliccomponents

5.0 [33]

Cortexalbiziae 35°C,30MPa Lipophiliccomponents

5.4 [34]

Curcumakwangsiensis 60°C,26MPa β-elemene 0.0271 [35]

EarofSchizonepetatenifoliaBriq. 50°C,20MPa Essentialoil 6.31 [36]

Figresidues 45°C,30MPa Anticancercomponents

2.53 [37]

GlycyrrhizauralensisFisch. 50°C,25MPa Essentialoil 1.69 [38]

LeavesofArtemisiaeargyi 32°C,15MPa Essentialoil 2.71 [39]

Liliumbrownii 50°C,18MPa Essentialoil 2.92 [40]

OcimumbasilicumL. 45°C,16MPa Essentialoil 4.96 [41]

Orris 55°C,26MPa Orrisoil 12.71 [42]

Perillafrutescens(L.)Britton 50°C,15MPa Essentialoil 2.5 [43]

RadixAngelicaedahuricae 35°C,25MPa Essentialoil 3.6 [44]

RadixLitseaeCubebae 55°C,30MPa Lipophiliccomponents

2.6 [45]

SalviacastaneaDielsf.tomentosaStib.

65°C,35MPa Tanshinones 2.9 [46]

Saposhnikoviadivaricata(Turcz)Schischk

35°C,22MPa Lipophiliccomponents

4–4.5 [47]

SchisandraChinensis(Turcz)Baill

50°C,25MPa Schizandrin Notavailable

[48]

StromaofCordycepskyushuensis 50°C,20MPa Essentialoil 9.72 [49]

Wheatplumule 35°C,20MPa Wheatplumuleoil Notavailable

[50]

Zanthoxylumseed 35°C,40MPa Zanthoxylumseedoil

10.32 [51]

7089_C007.indd 221 10/15/07 5:43:38 PM


surfactantwithCO2-philicgroups,suchassiloxane,fluoroalkane,fluoroether,tertiaryamine,andalkyonol[69].

SC-CO2extractioninthepresenceofsurfactantandcosolventhasnotbeenwidelyusedyetinthefieldofTCM.However,someresearchersinChinahaveexploredthiskindofextraction.Forexamples,Chenetal.[70]observedtheeffectofsurfactantonenhancingtheefficiencyofSC-CO2extractionofephedrinefromephedra.Dioctylsodium sulfosuccinate (DSS), sodium dodecyl sulfate (SDS), 1-heptanesulfonate(SHS),andcarboxymethylcellulosesodium(CMC-Na)wereusedasthesurfactants,

table 7.3extraction of active Compounds from Chinese herbals by sC-Co2 in the presence of Cosolvent

raw Materials Condition Cosolvent extracts Yield (%) references

ApliniaOxyphyllaMiquelseeds

35°C,25MPa Ethanol Volatileoil 3.21 [52]

Astragalusroot 45°C,40MPa 95%ethanol AstragalosideIV 0.27 [53]

BranchesandneedlesofTaxusyunnanensis

40°C,34MPa Methanol Taxol 0.0057 [54]

CornusofficinalisSieb.etZucc.

45°C,35MPa Ethanol Urosolicacid 0.239 [55]

CorydalisyanhusuoW.T.Wang

40°C,15MPa 95%ethanol Tetrahydro-palmatine

0.039 [56]

Curcumalonga 55°C,25MPa Ethanol Curcumin 0.0024 [57]

Iristectorum 50°C,25MPa Chloroform Irone Notavailable

[58]

LigusticumchuanxiongHort.

45–65°C,30–50MPa

Ethanol Femlicacid 0.735 [59]

Polygonumcuspidatum

50°C,25MPa 95%ethanol Resveratrol Notavailable

[60]

PolygonummultiflorumThunb

50°C,30MPa Chloroform+methanol

Phospholip 3.11 [61]

PricklyashPeel 35°C,20MPa Ethanol Essentialoil 13 [62]

Propolis 40°C,35MPa 95%ethanol Flavonoids 34.9 [63]

PterissemipinnataL. 60°C,25MPa Ethanol Diterpenoids 0.117 [64]

RhizomeofCoptischinensisFranch

60°C,50MPa 1,2-propanediol Berberine 7.53 [65]

Salviamiltiorrhizabunge

60°C,25MPa Methanol TanshinoneIIA 0.038 [66]

Sinomeniumacutum(Thumb)RehdetWils

60°C,30MPa Methanol Sinomenine 0.747 [67]

Taxusmaireibark 45–50°C,30–35MPa

Ethanol Taxoids Notavailable

[68]

7089_C007.indd 222 10/15/07 5:43:39 PM


andtheirinfluencesonextractionofephedrinefromephedrabySFEwerestudied.TheresultsindicatethatDSS,SDS,SHS,andCMS-NaenhancedtheefficiencyofSFEby246.8%,123.4%,83.0%,and53.2%, respectively,whichwasdue to theirmolecularconstitutions.Themoreliposolublepartsthesurfactantshave,thehighertheefficiency.TheapplicationofsurfactantsofferedavaluablewayforSFEofalka-loid.Geet al. [71] studied theuseofTween-80andSpan-80 in theextractionofmatrinesfromKuh-seng.Andtheyfoundthattheyieldis1.8–2.2timesmorethanthatof themethodwithout the surfactant.Satisfactory resultswerealsoachievedwhen Wang et al. [72] use nonionic surfactant, Span-80, and Tween-80, togetherwithwaterandethanol,inacertainproportionasmodifierstoextractlactonesfromatractylodesmacrocephalaKoidz.Thecontentofthelactonesintheextractivecanreachto87.78%atextractiontemperatureof15°Candpressureof30MPa.

7.3.3.4 Combing sFe with ultrasound-enhanced extraction Method

Most TCMs are solid materials and the mass transfer rate of the solid materialsis limitedby thediffusion inside theparticles.Thatmakes themass transfer rateslowintheSCFandleadstoalongerextractiontimeasaresult.ThisbottleneckofSFE,however,canbesolvedbyintroducingultrasoundintotheSCF.Anumberofphysicaleffects(turbulence,particleagglomeration,andbiologicalcellrupture)aswellaschemicaleffects(freeradicalformation)possessedbyultrasoundfacilitatethemasstransferinSCF.

Dingetal.[73]useddouble-frequencyultrasoundsalternatelytoenhanceSFEofflavonoidsfromToonasinensis.Ethanolwasalsousedasacosolvent.Dingetal.demonstrated in their research that thesuccessiveorderofdifferenteffect factorsontheyieldiscosolventamount>ultrasonicfrequency>extractiontemperature>extractionpressure>ultrasonicpower,andtheoptimumconditionsoftheextrac-tionincludetemperature50°C,pressure20MPa,cosolventamount2mL/g,ultra-sonicfrequency20kHz,andpower150W.Whenextractingoilandcoixenolidefromadlayseeds,Huetal.[74]foundenergysavingsaftertheintroductionofultrasoundbecausetheextractiontemperature,pressure,andrateofCO2couldbedecreasedandextractiontimecouldalsobeshortened.

7.3.3.5 Combining sFe with enhanced separation Methods

7.3.3.5.1 SFE Combined with Pressured Fractional DistillationSFEcoupledwithpressuredfractionaldistillationhasreportedlybeenusedtocon-centratew-3fattyacidsfromfishoils[75].Itisnowusedinseparationandpurifica-tionofTCMandnaturalproducts.Forexample,Liuetal.[76]usedSFEcoupledwithdistillationtoextractandconcentratevitaminEfromsoybeanoildeodorizer.Li[77]has successfully used this combined technology for extracting the lipid fractionsfromadlayseeds.Theindustrialscale-upofthisprocesshasledtoreplacementoftheoriginalsolventextractionmethodandhasobtainedapprovalfromChina’sFoodandDrugAdministrationforapplicationinthepharmaceuticalindustryformanu-facturingTCM.

The lipidfractionsofadlayseedsare theactivepharmaceutical ingredientsofKanglaiteInjection,aparenteraldrugapprovedinChinaandRussiafortreatmentof

7089_C007.indd 223 10/15/07 5:43:40 PM


advancednon-small-celllungcancer.ExtractionofadlayseedoilintheextractiontankwasfirstlycarriedoutwithSC-CO2attemperaturesof30°Cto45°Candpressuresof22MPa.TheCO2withdissolvedcrudeadlayseedoiltheninturnenteredtothesepa-rationcolumnandtwoseparationtankstoremoveimpurities,includingfattyacids,moisture,andpigments.WhenSFEiscombinedwithpressuredfractionaldistilla-tion,thechangeoftemperatureandpressurecanresultinachangeofrelativesepara-tionfactor.Asaresult,thequalityofadlayseedoilextractedwithSC-CO2extractionhasattainedthestandardofrefinedoilintermsofitsqualityspecifications.

7.3.3.5.2 SFE Combined with MD or HSCCC TechniquesSFE extracts are generally not a single compound but rather a complex mixtureof effective components, including some impurities. Sometimes, in order to geta component of high purity, it is necessary to use other separation techniques tofurthertreatmentextractsofSFE.MD,high-speedcountercurrentchromatography(HSCCC),silicagelcolumnseparation,solidphaseextraction,andotherprocessesarepresentlybeinginvestigatedforthispurpose,withMDandHSCCCbeingthemostwidelyinvestigatedprocessesinChina[78–88].

MDisaliquid-liquidextractiontechniqueinahigh-vacuumcondition,whichhasthefeatureoflowdistillationtemperature,shortheatingtime,andhighselectivity.ThecombinedtechniqueofSFEandMDismostlyusedtoextracttheessentialcom-ponentsofTCM.AfterMDprocessing, thecomponentsof lowmolecularweightintheSFEextractscanbeconcentrated.Zhangetal.[78–83]usedthiscombinedtechniquetoextracttheeffectivecomponentsofrhizomaatractylodismacrocephala,garlic, forsythia suspense, radix angelicae pubescentis, Spirulina, and ligusticumwallichiiFranch,andalltheresultsweresatisfactory.Forexample,whengarlicwasextracted with SC-CO2, 16 compounds in the extractive were identified, whereasonly 4 active compounds (diallyl disulfide, 3-ethenyl-1,2-dithia-cyclohex-5-ene,2-ethenyl-1,3-dithia-cyloohex-5-ene,anddiallyltrisulfide)wereobtainedbymolecu-lardistillationoftheextractive[80].

HSCCCisauniqueliquid-liquidpartitionchromatographytechniquethatusesnosolidsupportmatrix.Iteliminatestheirreversibleadsorptivelossofsamplesontothesolidsupportmatrixthatoccurswithuseoftheconventionalchromatographiccolumn[84]. Cao et al. [85–86] used HSCCC to purify the catechins and free fatty acidsextractedbySFEfromcratoxylumprunifoliumDyerandgrapeseedsandobtainedpuritiesof98%and99%,respectively.Wangetal.[87]gotpsoralenandisopsoralenpurities of above 99% when they combined SFE with HSCCC. Similarly, Pengetal.[88]gotflavonoidsof97.6~99.2%purityfromPatriniavillosaJuss.Theliteraturementions[84]thatcountercurrentchromatographycouldbeusedinalittlelargerscale,whichsuggestsabrightfutureforcombinedSFEandHSCCCtechniques.

7.3.3.6 Combining sFe with other techniques to Make Full use of herbal Materials

Sometimes,medicalplantshavemorethanonekindofeffectivecomponent.Inordertomakefulluseoftheplant,researcherstrytoisolatedifferenteffectivecomponentswithdifferentmethods.SFEhastheadvantageofextractingnonpolarandmoder-atelypolarsubstances,soitisusuallyusedtoextractthelipophiliccompounds.

7089_C007.indd 224 10/15/07 5:43:40 PM


Zhangetal.[89]obtainedlipophiliccomponentsoftanshinoneandhydrophiliccomponentsofdanshensuandprotocatechualdehydeatonetimebycombiningSFEwithwaterboiling.Ourlabalsodidsomeworktomakefulluseoftherawmateri-als.Yeetal.[90]usedSC-CO2toextractoilfromgrapeseeds,theresiduesofwhichwereextractedwithhotwater and thendepositedwithappropriatealcohol togetproanthocyanidin.Xiaoetal.[91]combinedSC-CO2extractionwithsolventextrac-tiontoobtainbothessentialoilandalkaloidsfromNelumboNuciferaGaertn.

Another case is that the active compounds with stronger polar in herbs aredesired,buttheherbsalsocontainacertainamountoflipophilicsubstances,whichwereonceremovedasimpuritieswithtraditionalsolventextractionmethods.Withthefeatureofconvenientoperation,highsafety,highremovalratio,andeasyiso-lationofsolvent,SFEisnowadopted toremovethe lipophiliccompoundsbeforeextractionofeffectivecomponentswithothertechniques.

Inorder toextractpolysaccharidefrommongoliamushroom,Wangetal.[92]firstinvestigatedtheeffectofpretreatmenttodegreaseanddecolarmongoliamush-roombySC-CO2orbysolventextraction.Theexperimentalresultsindicatedthat,whensuitableextractionconditionswereusedwithCO2,theeffectofdegreasinganddecolarwasexcellent.Moreover,thepretreatmentprocessfavoredtheextractionofpolysaccharide.TheextractionyieldofthepolysaccharidewithpretreatmentbySFEis1.8foldthatwithsolventpretreatmentand4.2timesthatwithoutpretreatment.

7.4 seleCt exaMples oF sFe oF tCM and natural produCts

Inourlaboratory,morethan20kindsofherbalplantswereselectedasrawmaterialstoinvestigateSFEprocessing.Fourtypicalexampleswerebrieflyreported,whichindicatethatdifferentSFEprocessesandparameterscanbedevelopeddependingonprocessingpurposeandthepropertiesoftherawmaterials.

7.4.1 extraCtioN oF eSSeNtial oil From Clove Bud with SC-Co2

Clove(Eugenia caryophyllataThunb.) iswidelycultivated in thesouthofChina.Clove bud oils contain high contents of eugenol, which give it strong biologicalactivity and antimicrobial activity. Beside eugenol, clove bud oils also containsomeamountofotheractivecompoundsofeugenolacetateandβ-caryophyllene.Clovebudoilhasseveraltherapeuticeffects,includingantiphlogistic,antivomiting,analgesic,antispasmodic,carminative,kidneyreinforcement,andantisepticeffects.Italsoisusedasaflavoringagentandantimicrobialmaterialinfood[93–95].

Extraction of clove oils from clove bud with SC-CO2 was investigated [96].TheherbalmaterialsofclovebudweregroundbyaFW80SampleMillmachinein different periods to get different particle distribution, which was measured bymechanical sieving after extraction and calculated by weight of different size ofclovebud particle.Gradesof particle size were classified on the following scale:1=<10mesh;2=10~20mesh;3=20~40mesh;4=40~60mesh;5=60~80mesh;6=80~100mesh;7=100~120mesh;8=>120mesh.Particlesizeindexwascalcu-latedbythefollowingformula:

7089_C007.indd 225 10/15/07 5:43:41 PM


Particle size indexweight of each grade= × gradetotal weight highest grade×∑ (7.1)

Theparticlesize indexesofmaterial in thisexperimentwere0.7944,0.6430,and0.5223,namedas1#,2#,and3#respectively.

Thefollowingparameterswereused:temperature,30°C,40°C,and50°C;pres-sure,10MPa,20MPa,and30MPa;andparticlesize,1#,2#,and3#.Alltheselectedfactorswereexaminedusingathree-levelorthogonalarraydesignwithanOA9(33)matrix,asshowninTable7.4.Itcanbeseenfromtheorderofthemaximumdiffer-encesthatparticlesizehadthemostinfluenceontheoilyield,thentemperatureand

table 7.4three-level orthogonal design and experimental results for extraction of Clove oil with sC-Co2 [96]

run no.Factor a (t/°C)

Factor b (p/Mpa)

Factor C (particle size/#)

Yield (kg extract/

kg feed)

eugenol Content

(%)

1 1(30) 1(10) 1(1#) 0.2056 53.69

2 1(30) 2(20) 2(2#) 0.1943 54.22

3 1(30) 3(30) 3(3#) 0.1830 55.64

4 2(40) 1(10) 3(3#) 0.1910 56.20

5 2(40) 2(20) 1(1#) 0.2224 54.52

6 2(40) 3(30) 2(2#) 0.2043 55.30

7 3(50) 1(10) 2(2#) 0.1956 58.77

8 3(50) 2(20) 3(3#) 0.1827 57.83

9 3(50) 3(30) 1(1#) 0.2395 56.97

Yield(%)

K1 0.5830 0.5923 0.6676

∑=1.8186

K2 0.6178 0.5995 0.5942

K3 0.6179 0.6269 0.5568

K1/3 0.1943 0.1974 0.2225

K2/3 0.2059 0.1998 0.1981

K3/3 0.2060 0.2090 0.1856

R 0.0117 0.0116 0.0369

Eugenolcontent(%)

K1 163.55 168.66 165.18

∑=503.14

K2 166.02 166.57 168.29

K3 173.57 167.91 169.67

K1/3 54.52 56.22 55.06

K2/3 55.34 55.52 56.10

K3/3 57.86 55.97 56.56

R 3.34 0.25 1.04

ReprintedfromFood Chemistry,101,1558–1564,©2007.WithpermissionfromElsevier.

7089_C007.indd 226 10/15/07 5:43:42 PM


pressure.However,thesequenceoftheinfluencesoftheparametersontheeugenolcontentintheoilswastemperature,particlesize,andthenpressure.Thefactoroftemperatureshowsthemaximuminfluenceontheeugenolcontentintheoils.

Basedontheabovedatafromthethree-levelorthogonalarraydesign,Figure7.1furthershowstheeffectoftemperature,pressure,andparticlesizeontheyieldandeugenolcontentofcloveoilextractedbySC-CO2.Itcanbeobservedthatincreaseoftemperaturefrom30°Cto40°Cresultsinanincreaseoftheextractionyieldandhigheugenolcontentintheoils,whiletheincreaseoftemperaturefrom40°Cto50°Cdoesnotresultinanincreaseoftheoilyield,andthereisanincreaseofeugenolcontentincloveoil.Theextractionyieldenhancedsignificantlywith increaseofpressureduetotheincreaseofthesolubilityoftheoilcomponents.Thisincreaseisattrib-utedtotheincreaseoftheCO2density,whichresultsinanincreaseofitsdissolvingability.However,becausethehigh-molecular-weightcompoundsinclovebuds(fattyacids,fattyacidsmethylesters,sterols,etc.)werealsocoextractedwithincreaseofpressure,theeugenolcontentofthecloveoildoesnotobviouslychange.Theextrac-tionyieldincreasesbydecreasingtheparticlesizeofthecomminutedclovebudsduetothehigheramountofoilreleasedwhenthebudcellsaredestroyedbymilling,andthisamountofoiliseasilyextractedfordirectexposuretotheSC-CO2.However,theeugenolcontentinthecloveoilincreasesasparticlesizeincreases.Therefore,theparticlesizeshouldnotbetoosmallinordertoavoidcoextractionofmorecom-poundswithhigh-weightmolecules.

Gas chromatography with Mass spectrometry (GC/MS) analysis was used toidentifythecompoundsinthecloveoilsextractedwithSC-CO2.Twenty-threecom-poundswereidentified.Comprehensivecomparisonofthecloveoilsobtainedbydif-ferentmethodsislistedinTable7.5.ThecontentofthemainbiologicalingredientsofeugenolpluseugenolacetateinthecloveoilbySoxhletextractionislowest,althoughitsyieldofcloveoilishighestamongthefourextractionmethods.Furthermore,the

Pressure (MPa) Particle Size (index)Temperature (°C)50

60

58

50

52

54

56

2# 3#1#4030

0.23

0.18

0.19

0.20

0.21

0.22

Euge

nol i

n O

il (%

)

Yield

(kg

extra

ct/k

g fe

ed)

10 20 30

Eugenol Content Yield

FIgure 7.1 Effectoftemperature,pressure,andparticlesizeonyieldandeugenolcontentofcloveoilextractedbySC-CO2.(ReprintedfromFood Chemistry, 101,1558–1564,©2007.WithpermissionfromElsevier.)

7089_C007.indd 227 10/15/07 5:43:43 PM


extractbySoxhletmethodisbrownointment,whichmeansmoreundesiredimpuri-tiesandorganicsolvent residuemayhaveexisted.SFEoffers themost importantadvantagesoverothermethods.ExtractionyieldofSFEwasabouttwotimesashighasthatobtainedbysteamandhydrodistillation.Thehighestcontentofeugenolpluseugenolacetateintheextractedoilwasalsoobtained.PaleyellowoilisdesiredandshortestextractiontimeisneededforSFEcomparedwiththeotherthreeextractionmethods.

7.4.2 extraCtioN oF mediCal iNgredieNtS From the mixture oF AngelicA sinensis aNd ligusticum chuAnxiong hort with SC-Co2

Angelica sinensis (Oliv.)DielsandLigusticum chuanxiong horthavebeenwidelyusedasTCMtotreatpathologicalconditionssuchasatherosclerosisandhypertension.TheirphytochemicalprofilesanalysessuggestthatAngelica sinensisandLigusticum chuanxionghortcontainsimilarsubstances,suchasferulicacidandessentialoil.FerulicacidisoneofthemostimportantmedicalcomponentsinAngelica sinensisandLigusticumchuanxiongbecauseitpossessesantioxidativepropertiesbyvirtueofthephenolichydroxylgroupinitsstructure.Studieshaveshownthatferulicacidcouldinhibitmalondialdehyde(MDA)productionfromplatelets,inhibiterythrocytelysesinducedbyMDAandhydroxylradical,andinhibitlipidperoxidationinducedbyH2O2andO2[97–99].

ExtractionofferulicacidfromamixtureofsimilarportionsofAngelica sinensisand Ligusticum chuanxiong hort was firstly carried out with SC-CO2. As ferulicacid was considered to be the active component for preventing heart disease, itscontentinextractswasanalyzedbyhigh-performanceliquidchromatographyandtheanalyzingresultswereusedasthequalitycontrolindexformedicalefficiency.

Theeffectsofextractiontemperature,extractionpressure,particlesize,andmate-rialsourcesontheextractyield(E)andthecontentofferulicacidinextracts(C)usingpureCO2wasfirstexperimentallyinvestigated,aslistedinTable7.6.AsshowninTable7.6,theextractyieldsincreasedfrom2.95%to3.95%andthecontentofferulicacidinextractsincreasedfrom0.25%to0.28%whenpressureincreasedfrom20to50MPaatconstanttemperatureof65°C.Thesechangesoccurredbecauseincreas-ing the pressure at constant temperature increases the density of SC-CO2, whichfurtherincreasesthesolvationpoweroftheSCF.Whentemperaturesincreasedfrom

table 7.5Characteristic of the Clove oils obtained by different Methods [96]

Clove oil Yield (%)

eugenol plus eugenol acetate

(%)extraction period (h)

Color and texture

organic solvent used

SFE(50°C,10MPa) 19.6 58.8+19.6 2 Paleyellowoil No

Steamdistillation 10.1 61.2+10.2 8–10 Paleyellowoil Yes

Hydrodistillation 11.5 50.3+3.2 4–6 Brownyellowoil Yes

Soxhletextraction 41.8 30.8+9.3 6 Brownointment Yes

ReprintedfromFood Chemistry,101,1558–1564,©2007.WithpermissionfromElsevier.

7089_C007.indd 228 10/15/07 5:43:43 PM


35°Cto65°Catapressureof30MPa,theextractyieldsincreasedfrom3.18%to3.63%andthecontentofferulicacidinextractsincreasedfrom0.18%to0.26%.

TheeffectofparticlesizeontheextractyieldisalsoshowninTable7.6.Extractyields increased from 3.63% to 5.52% with particle size decreasing from 20–40meshesto60–80meshes.Therefore,itisnecessarytogroundthenaturalrawmate-rialsintotheoptimumsizeinordertoreducethediffusiondistanceandtoimproveextractionefficiency.However,thecontentofferulicacidintheextractsdecreasedfrom0.26%to0.21%,indicatingothercomponentsmayalsobeextracted.

AlthoughSC-CO2hasbeenwidelyinvestigated,itisapoorextractantforpolarsubstances.Inordertoincreasethepowerofsolventforextractingpolarferulicacid,threecosolventswereemployed:ethanol,ethylacetate,andn-butylalcohol.Differ-ent ratiosofcosolvents to rawmaterial (w/w)werealsostudied.Cosolventsweredirectlyadded into the rawmaterialsandsoaked for4hoursbeforecarryingoutSC-CO2extractionundertheconditionsof65°Cand30MPa.AftertheSFEprocess,theextractswerevaporizedwithanevaporatorinavacuumtoremovethesolvent.TheexperimentalresultsarelistedinTable7.7.

Table7.7 shows that all of three cosolvents not only greatly enhanced thecontentsofferulicacid in theextractsbutalso increased theextractyieldgreatly

table 7.6experimental data of extraction of Ferulic acid from the Mixture with pure Co2

temperature (°C)

pressure (Mpa)

particle size (mesh) e(%) C(%)

65 20 20–40 2.95 0.25

65 30 20–40 3.63 0.26

65 40 20–40 3.79 0.28

65 50 20–40 3.95 0.28

35 30 20–40 3.18 0.18

45 30 20–40 3.32 0.23

55 30 20–40 3.50 0.24

65 30 20–40 3.63 0.26

65 30 40–60 4.40 0.22

65 30 60–80 5.52 0.21

table 7.7experimental results of different Cosolvents

Cosolvent t (°C) p(Mpa) e (%) C (%)

PureCO2 65 30 3.63 0.26

Ethanol 65 30 5.16 0.58

Andn-butylalcohol 65 30 5.13 0.52

Ethylacetate 65 30 4.69 0.31

7089_C007.indd 229 10/15/07 5:43:44 PM


compared with pure CO2extraction. Because ethanol is one of the few acceptedorganicsolventsinthefoodandmedicineindustries,itwasemployedtostudytheratioofcosolvent.Theexperimentalresultsshowthattheextractyieldof7.12%andthecontentofferulicacidof0.83%inextractivewereobtainedwhentheratiooftheethanoltorawmaterialswas1.6.

ThepercolationmethoddescribedinthePharmacopoeiaofPeople’sRepublicofChina(2000year)wasemployed.Ethanolwithconcentrationof95%wasusedasasolvent.Beforepercolation,thepoweredmixturewassoakedwithsolventfor24hours.Thepercolationflowratewas15drops/minandthepercolationtimewasabout8hours.Afterpercolation,theextractswerevaporizedwithavacuumrotatoryevaporatortoremovethesolvent.Thematerialwasgroundusingamixer-grinder.ComparisonsofSFEwithpercolationmethodarelistedinTable7.8.

It can be seen that both the extract yields and the content of ferulic acid inextractsbypureCO2arethelowestamongthethreeprocessingmethods.Addingasuitablecosolvent,suchasethanolinthisstudy,couldgreatlyincreasethecontentofferulicacidinextracts,whichissuperiortothetraditionalpercolationmethodofextractingpolarferulicacidfromthemixtureofAngelica sinensisandLigusticum chuanxiong hort. This method may be one key way to make use of a suitablecosolventforincreasingthesolventpowerofCO2andtoexpandtheapplicationofSFE.However,SFEextractsfromtheherbsofthemixtureofAngelica sinensisand Ligusticum chuanxiong hortaregenerallyacomplexmixtureof thecomponents,whichmayhavesomedifferencesinbothcompositionandcontentscomparedwiththeextractiveobtainedbytheoriginalpatentedtraditionalmethods.Therefore,fur-therresearchonpharmacologyandmedicineefficiencyisneededforthesafeandeffectiveuseofthismethodforTCM.

7.4.3 extraCtioN oF ediBle aNd mediCiNal iNgredieNtS From grape SeedS with SC-Co2

Inourlab,wealsoinvestigatedtheuseofSC-CO2intheextractionofactivecom-poundsfromgrapeseeds.Grapeseedscontainseedoilandprocyanidins,whicharegenerallynamedplantpolyphenol.Theweightproportionofgrapeoilinthetotalgrapeseedisabout10–15%andthatoil is rich in linoleicacid,whichbelongs tounsaturatedfattyacid.Inthefieldsoffood,cosmetics,andmedicine,itisconsid-eredtobebeneficialtouseoilshighinlinoleicacid.Grapeprocyanidinshavebeenincreasinglypaidmuchattentionasoneofthetenmostpopularherbalmedicinesintheworldandcanbeusedformedicine,hygienicfood,andcosmeticsduetotheir

table 7.8Comparison of sFe with percolation Method

extraction Methodtemperature

(°C)pressure (Mpa) e (%) C (%)

SFEwithpureCO2 65 30 3.63 0.26

Percolation 4.54 0.61

SFEwithethanol 65 30 7.12 0.83

7089_C007.indd 230 10/15/07 5:43:45 PM


biological and pharmacological actives, such as anti-oxidation and anti-mutation[101–103].

The influencesof extraction temperatureandpressureon theextractionyieldoftheseedoilwereinvestigated[104].Figure7.2illustratestheeffectofextractionpressureontheextractionyieldofseedoilat45°C.WithpureCO2,weobtainedthequalifiedgrapeoils.Theresultsshowthattheyieldofoilseedwasupto9.5%at45°Cand30MPa.However,whenusing seeds supplied fromanother source area, theresultshowsthattheyieldofoilseedwasupto13.51%at55°Cand30MPa.GC-MSanalysisshowsthattheunsaturatedfattyacidintheextractedoilconstituentwasupto90.1%.Therefore,itisimportanttoinvestigatesourceareaoftherawmaterialsandmakesomenecessaryanalysisfortheactivecomponents.

Afterextractingoil from thegrape seedswithSC-CO2, theextractionofpro-cyanidinswasfurtherstudiedwithSC-CO2inthepresenceofthecosolventofethanol.ThreekindsofmethodsforaddingcosolventwereinvestigatedinordertoenhancethesolventpowerofCO2forincreasedyieldandpurityofprocyanidins.Thethreemethodsincludedaddingcosolventtorawmaterialinstaticmode,addingcosolventtoSC-CO2inflowingmode,andacombinationofthetwomodes.Theeffectsofextrac-tiontemperature,pressure,theconcentrationanddosageofcosolvents,andsoakingtimeontheextractionyieldandpurityofprocyanidinswerestudied.Theexperimen-talresultsshowthat,whenthemassratioofcosolventaddedtotherawmaterialwas1.2:1(w:w)andsoakedfor60minutesbeforetheSC-CO2extractionwascarriedout.Attemperatureof55°Cand30MPaandwhenaconcentrationof60%cosolventinCO2wasappliedinflowingmode,ayieldof10.9%withapurityof95.9%ofpro-cyanidinscouldbeobtained (Figure7.3).Whenaconcentrationof60%cosolventinCO2wasusedasextractionsolventsinflowingmodetomakeSFEatextraction

0

2

4

6

8

10

0Amount of CO2 Used (L)

Yiel

d of

Gra

pe S

eed

Oil

(%)

20 MPa30 MPa40 MPa

200 400 600 800

FIgure 7.2 Plotofextractionyieldofgrapeseedoilvs.SC-CO2amount(45°C).

7089_C007.indd 231 10/15/07 5:43:45 PM


temperatureof55°Candextractionpressureof35MPa,ayieldof10.9%withapurityof95.9%ofprocyanidinscouldbeobtained.Whenthemassratioof1.2:1(w:w)forcosolventaddedtotherawmaterialwasusedandsoakedfor60minutes,andthen25%concentrationofcosolventinSC-CO2inflowingmodewasusedasextractionsolvents for SFE at the extraction temperature of 55°C and extraction pressure of35MPa,thehighestyieldof11.73%,withapurityof96.6%ofprocyanidins,couldbeobtained(Figure7.4).Therefore,addingcosolventtorawmaterialinstaticmodecombinedwithaddinga suitableconcentrationof cosolvent toSC-CO2 inflowingmodemaybethebestmethodamongthethreeforaddingcosolvents.

0

1

2

3

0.5 1.0Mass Ratio of Entrainer to Material

Yiel

d, %

0

20

40

60

80

100

Purit

y, %

YieldPurity

1.5

FIgure 7.3 Influenceofmass ratioofcosolvent tomaterialonyieldandpurityofpro-cyanidins(T=55°C,P=30MPa,soakingtime=60min).

8

10

12

10Concentration of Cosolvent in Solvent

Mixture (wt%)

Yiel

d (%

)

70

80

90

100

Purit

y (%)

YieldPurity

20 30 40 50

FIgure 7.4 Effectofdosageofcosolventflowingonyieldandpurityofprocyanidins(T=55°C,P=35MPa,massratioofcosolventrestingtomaterial=1.2:1,soakingtime=90min).

7089_C007.indd 232 10/15/07 5:43:46 PM


Forcomparison,anotherintegratedtechnologywasalsoinvestigatedforobtain-ing procyanidins from grape seeds by using the combined method of SFE andmacroporous resinadsorption technology [105].SFEwithSC-CO2wasfirstusedtoremovethegrapeseedoil.Thenmacroporousresinadsorptiontechnologywasusedtopurifythecrudeprocyanidinsextractedbyhotwaterextractionwithalcoholdeposition.Theexperimentalresultsshowthatayieldof4.88%havingapurityof95%ofprocyanidinscouldbeobtained,whichisfarlessthantheyieldobtainedbyaddingcosolvent to rawmaterial in staticmodecombinedwithaddinga suitableconcentration of cosolvent to SC-CO2 in flowing mode. It reveals that extractingnaturalproductswithSFEhasobviouspredominance.

7.4.4 iSolatioN oF orgaNoChloriNe peStiCide From giNSeNg with SC-Co2

ManyChinesetraditionalandherbaldrugsarebeingexportedabroadasfoodaddi-tivesorplantdrugs.RadixginsengisarareChinesetraditionalmedicinematerialwhichhastherapeuticeffectsthatcanbeusedtotreatmanydiseasesandcanalsobeusedasahealthfood.However,ahighcontentofresiduesofprohibitoryorgano-chlorinepesticides,suchashexachlorocyclohexanes(BHC),existsinradixginseng,whichexceedsthelimitedlevelgreatlyaccordingtotheinternationalstandardregu-lation[106–108].

ThesafetyissueofChineseherbalmedicinesisasubjectofscientificinterest.SCFs as “environmentally friendly” alternatives to liquid solvents for samplepreparation inanalyticalchemistryhave receivedmuchattention in thepast fewyears.SFEhasbeenshown tobeanefficientand rapidmethod for the isolationoforganochlorinepesticidesfromvegetables[109].However,nostudiesreportontheprocessof removalofBHCfrom radixginsengwithSFE.The feasibilityofremovingBHCpesticideresiduesfromradixginsengwithSC-CO2wasexploredinourlab[110–113].

The rootswithhairsof radixginsengwerepowderedandsiftedoutprior toextraction, in which radix ginseng with sizes of 550 to 1120 µm was selected.ExtractionswereperformedwithaSpe-edSFE instrument (AppliedSeparationsInc.,Allentown,PA).

ForthedeterminationofBHC,GasChromatographwithElectricalConductivityDetector(GC-ECD)analysiswascarriedoutusinganAgilent6890plus(U.S.)gaschromatographequippedwith63Nielectron-capturedetector,usingaBPX608cap-illarycolumn(25m×0.32mm).Thechromatographicconditionswereasfollows:injectortemperature,280°C;detectortemperature,320°C;andnitrogenflow-rates,10.0mL/min(carriergas).Thecolumntemperaturewasprogrammedasfollows:increasedatarateof25°C/minfrominitialtemperatureof50°Cto150°C,retainedfor1min,andthenincreasedatarate6°C·min–1to240°C.

DeterminationofBHCinradixginsengsampleswascarriedout.TheBHCcon-tentwas2.380mg·kg–1feeds.Accordingtotheinternationalstandardregulationforfoodanddrugs,thecontentofthepesticideresiduesofBHCinradixginsengshouldbelowerthan0.1mg·kg–1.Therefore,atleast95.2%ofBHCshouldberemovedfromtheradixginsengsamplesinthiswork.

7089_C007.indd 233 10/15/07 5:43:47 PM


ExtractionofBHCfromradixginsengwasfirstinvestigatedwithpureSC-CO2intemperaturesrangingfrom60°Cto80°Candpressurerangingfrom25MPato50MPa.However,theresultsofGC-ECDanalysesindicatedthatpureCO2couldnotreduceBHC contenttothelevelof0.1mg·kg–1tomeettheBHCpermissionlimit,sothatacertainmodifierwasnecessary.

Removal of BHC residues from radix ginseng with CO2 in the presence ofcosolventwasinvestigatedusingthreekindsofsolvents:water,ethanol,andhexane.Whenasuitableamountofwaterisaddedintothefeedstockbeforeextraction,BHCcontentinradixginsengcouldbereducedto0.0394mg·kg–1at60°Cand30MPa,whileadditionofthesameamountofethanolorhexaneascosolventsdidnotservesuchapurpose.

The effect of extraction pressure on removal of BHC from radix ginseng bySC-CO2inthepresenceofcosolventwaterat60°CisshowninFigure7.5.TheBHCcontentinradixginsengwasreducedtolevelslowerthan0.1mg·kg–1,whichwere0.08mg·kg–1and0.04mg·kg–1separatelyinthepressuresof20MPaand30MPa.

00.020.040.060.08

0.10.120.140.160.18

20Extraction Pressure (MPa)

BHC

Isom

ers C

onte

nt(m

g/kg

)

α-BHC

β-BHC

γ-BHC

δ-BHC

BHC(∑)

30 40 50

FIgure 7.5 EffectofpressureonremovalofBHCwithSC-CO2inthepresenceofwaterat60°C.(FromLi,S.F.andQuan,C.,Chinese Journal of Chemical Engineering, 13,433,2005.Withpermission.)

00.020.040.060.08

0.10.120.140.16

40Extraction Temperature (°C)

BHC

Isom

ers C

onte

nt (m

g/kg

)

β-BHC

γ-BHC

δ-BHC

BHC(∑)

α-BHC

60 80

FIgure 7.6 EffectoftemperatureonremovalofBHCwithSC-CO2inpresenceofwaterat30MPa.(FromLi,S.F.andQuan,C.,Chinese Journal of Chemical Engineering, 13,433,2005.Withpermission.)

7089_C007.indd 234 10/15/07 5:43:48 PM


However,furtherincreaseinpressureincreasedtheBHCresidues,sotheoptimalpressureobtainedinthisstudywas30MPa.

Theeffectofextractiontemperaturewasstudiedataconstantpressureof30MPawithwater as cosolvent at temperatures ranging from40°C to80°C (Figure7.6).IncreasingtemperatureresultedindecreasingcontentofBHCinradixginseng.Atatemperatureof40°C,BHCresiduesinradixginsengwerehigherthan0.1mg·kg–1.However,theBHCcontentwasreducedto0.04mg·kg–1whentemperatureincreasedto60°C.However,inlightofthethermo-sensitivepropertiesofthenaturalplant,theoptimaltemperatureis60°C.

Toseektheminimumcosolventdosage,influencesofdosageofwateronSFEofBHCinradixginsengwerestudiedatatemperatureof60°Candpressureof30MPa.ThecontentofBHCinextractedradixginsengdecreasedrapidlyfrom1.43mg·kg–1to0.04mg·kg–1(Figure7.7)whenthedosageofwaterincreasedfrom0to0.5[water(g)/ginseng(g)],sodosageofcosolventhadsignificanteffectonremovalofBHCwithSFE.Whenthedosageofwaterwas0.4,contentofBHCwas0.11mg·kg–1,andwhenitwas0.5,contentofBHCwas0.04mg·kg–1.Therefore,thedosageofwatermustbemorethan0.4.However,anexcessiveamountofwaterdoesnothaveaposi-tivefunctionbecauseitcannotbeabsorbedbythepowderedginseng,sothesuitabledosageofwaterwasabout0.5gpergramofginseng.

At60°C,30MPa,andadosageofwaterof0.5g,influencesoftheamountofCO2onremovalofBHCfromradixginsengwereinvestigated.AsshowninFigure7.8,α-BHC, β-BHC, and γ-BHC change less while δ-BHC decreased obviously withincreasingamountofCO2forcertainginsengmaterial.Asthetotalresults,about30 to50standard literCO2pergramginsengcouldmatch the totalBHCresiduepermissionlimit—thatis,150LormoreofCO2couldreducethecontentofBHCtolessthan0.1mg·kg–1for5gginseng.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0Modifier Dosage (water(g)/ginseng(g))

BHC

Isom

ers C

onte

nt (m

g/kg

)α-BHC

β-BHC

γ-BHC

δ-BHC

BHC(∑)

0.1 0.2 0.3 0.4 0.5

FIgure 7.7 Influences of dosage of water on removal of BHC from radix ginseng at atemperatureof60°Candpressureof30MPa.(FromLi,S.F.andQuan,C.,Chinese Journal of Chemical Engineering, 13,433,2005.Withpermission.)

7089_C007.indd 235 10/15/07 5:43:48 PM


7.5 suMMarY and prospeCt

Fromtheabove-statedexamples,wefindthatSFEhassufficientlyadvancedtech-nologyandsuperiorefficiencywhenextractingnonpolarorlow-polarcompounds.Withthemodificationofcosolventandsurfactant,thelimitationofSFEinextract-ingmoderatelypolar and intensivelypolar compounds is somewhat improved. Inaddition, thecombinationofSFEwithother techniqueswidenstheapplicationofSFE in thefieldsofTCMandnaturalproducts.Asa cleanandgreen separationtechnique,SFEhasapromisingfutureinitsapplicationinthefieldsofTCMandnaturalproducts.However,wealsolearnedthatSFEextractsfromherbsaregener-allynotasinglecompoundbutacomplexmixtureofcomponents,whichcommonlyhavesomedifferencesincompositionorincontentscomparedwiththeextractiveobtainedbyothertraditionalmethods.Therefore,mostoftheresearchresultsstatedin this chapter aregenerally in the laboratory level, fewof themhave scaledup.FurtherresearchonpharmacologyandmedicineefficiencyisnecessarytoensurethesafetyandefficacyofcombinedextractionprocessesforTCM.

Additionally,somefactorsthatlimitedtherapiddevelopmentofSFEshouldnotbeoverlooked.AslackofadeeperunderstandingtoSCFstateitself,muchexperi-mentalworkisneededtodeterminetheprocessconditionsthatcannotbepredicted.TheoreticalresearchofthethermodynamicanddynamiccontrolmechanismsoftheSFEprocessalsoneedtobeinvestigatedbecausethisresearchcanprovideimpor-tant guidance for optimizing the industrial production of SC-CO2 extraction. AsoperatingSFEprocessisathighpressurewhichdemandsequipmenttoguaranteesafety,howto increase thequalityof theequipmentsand toreduce theoperatingcostsalsoneedthemanufacturerstotaketheirefforts.

SCFtechnologyisapromisingtechnologywithexcitingcommercialpotential.It is replacing older solvent technologies and creating new technologies for pro-cessingTCMandnaturalproductsbecauseitissafe,environmentallybenign,and

0

0.02

0.04

0.06

0.08

0.1

0.12

20Amount of CO2(L/g)

BHC

Isom

ers C

onte

nt (m

g/kg

)α-BHC

β-BHC

γ-BHC

δ-BHC

BHC(∑)

30 40 50

FIgure 7.8 ImpactontheamountofCO2onBHCcontent.(FromLi,S.F.andQuan,C.,Chinese Journal of Chemical Engineering,13,433,2005.Withpermission.)

7089_C007.indd 236 10/15/07 5:43:49 PM


cost-effective.Itfascinatesmanyresearchersanddeservesfurtherinvestigationtoexpanditsapplication.

reFerenCes

1. Chinese Pharmacopoeia Commission, Chinese Pharmacopoeia (Chinese), Vol. 1,ChemicalIndustryPress,Beijing,2000.

2. Zheng, H.Z., Dong, Z.H. and She, J., Modern Study and Application of Chinese Medicine(Chinese), Vol.2,XueyuanPress,Beijing,1997.

3. Huang,T.K., Traditional Chinese Patent Pharmacology (Chinese),3rded.,ChinaPressofTraditionalChineseMedicine,Beijing,1996,610.

4. Xiao,C.H.,Chinese Medicine Chemistry (Chinese),ShanghaiScientificandTechnicalPublisher,Shanghai,1994.

5. Gan,S.J.etal.,Development Strategy in Modernization of Traditional Chinese Medicines (Chinese),ScientificandTechnicalDocumentsPublishingHouse,Beijing,1998,110.

6. Luo,G.A.etal.,ModernizationoftraditionalChinesemedicineandmateriamedica.World Sci. Technol.,5,11,1999.

7. Li,S.F.,Supercriticalfluidextractiontechnology,inCritical Technologies in Modern-ization of Traditional Chinese Medicines (Chinese),Yuan,Y.J.,Liu,M.Y.andDong,A.J.,Eds.,ChemicalIndustryPress,Beijing,2002,11.

8. Zhu, Z.Q., Introduction, Supercritical Fluids Technologies-Principles and Applica-tions (Chinese),ChemicalIndustryPress,Beijing,2000,chapt.1,p.2.

9. Zosel,K.(StudiengesellschaftKohle),DBP2005293The process for the decaffeination of green coffee beans,1970.

10. Han,B.X.,ExtractionandseparationtechnologyofSCFScience and Technology of Supercritical Fluid (Chinese), China Petrochemical Press, Beijing, 2005, chapt. 8,p.219.

11. McHugh,M.A.andKrukonis,V.J.,Introduction,Supercritical Fluid Extraction: Prin-ciples and Practice,2nded.,Butterworth,Boston,1993,chapt.1,p.5.

12. Bruno,T.J.andEly,J.F.,Supercriticalfluidtechnology,inReviews in Modern Theory and Applications. Boston,MA:CRCPress,1991.

13. King,J.W.,Fundamentalsandapplicationofapplicationsofsupercriticalfluidextrac-tioninchromatographyscience,J. Chromatogr., 27,355,1989.

14. Zhang, J.C., The application of SFE in the research and development of traditionalChinese medicines, Supercritical Fluid Extraction Technology (Chinese), ChemicalIndustryPress,Beijing,2002,chapt.6,p.105.

15. Walsh,J.M.,Ikonomou,G.D.andDonohue,M.D.,Supercriticalphasebehavior:Theentrainereffect,Fluid Phase Equil.,33,295,1987.

16. Ariel,A.C.,Solute-soluteandsolute-solventcorrelationsindilutenear-criticalternary-mixtures:Mixed-soluteandentrainerseffects,J. Phys. Chem. B,97,2740,1993.

17. Mijeong,L.J. andDavid, J.C., Investigationofmodifier effects in supercriticalCO2extractionfromvarioussolidmatrixes,J. Supercrit. Fluids,16,33,1999.

18. Kerry,M.D.,Chien-Ping,K.andRobert,P.G.,Theuseofentrainersinthesupercriticalextractionofsoilscontaminatedwithhazardousorganics,Ind. Eng. Chem. Res.,26,2058,1987.

19. John,J.L.etal.,Roleofmodifiersforanalytical-scalesupercriticalfluidextractionofenvironmentalsamples,Anal. Chem.,66,909,1994.

20. Marentis,R.T.,In Supercritical Fluid Extraction and Chromatography,Charpentier,B.A.andSevenants,M.R.,Eds.,ACS,Washington,1988,127.

21. Motonobu, G., Bhupesh, C.R. and Tsutomu, H., Shrinking-core leaching model forsupercriticalfluidextraction.J. Supercrit. Fluids,9,128,1996.

7089_C007.indd 237 10/15/07 5:43:50 PM


22. Tan,C.andLiou,D.,Modelingofdesorptionatsupercriticalconditions,AICHE J.,35,1029,1989.

23. Sovová,H.,RateofthevegetableoilextractionwithsupercriticalCO2,IModellingofextractioncurves,Chem. Eng. Sci.,49,409,1994.

24. Özkal,S.G.,Yener,M.E.andBayïndïrlï,L.,Masstransfermodelingofapricotkerneloilextractionwithsupercriticalcarbondioxide, J. Supercrit. Fluids,35,119,2005.

25. Reverchon, E. and Marrone, C., Modeling and simulation of the supercritical CO2extractionofvegetableoils,J. of Supercritical Fluids, 19,161,2001.

26. Shen, Z.Y. et al., The First National Symposium on Supercritical Fluids (Chinese), Shi-Ja-Zhuang,China,1996.

27. Zhang,J.C.etal.,The Second National Symposium on Supercritical Fluids (Chinese), GuangZhou,China,1998.

28. Chen,K.X.etal.,The Third National Symposium on Supercritical Fluids (Chinese), Xi-An,China,2000.

29. Yu,D.S. et al.,The Fourth National Symposium on Supercritical Fluids (Chinese), GuiYang,China,2002.

30. Han, Y.Q. et al., The Fifth National Symposium on Supercritical Fluids (Chinese), QingDao,China,2004.

31. Liang,R.H.,Xie,M.Y.andLiu,W.,Responsesurfaceanalysisstudyonnaphthaquinoniccompounds yield by supercritical CO2 extraction of Amebia euchroma (Royle) John,Food Sci.,25,76,2004.

32. Wu,S.X.,Lv,G.Y.andLi,W.L.,SupercriticalCO2extractionprocessofatractylodesmacrocephala Koidz and the determination of the extracts, Chin. Trad. Pat. Med. (Chinese),27,885,2005.

33. Lei,H.P.etal.,SupercriticalCO2extractionoffattyoilsfrombeepollenanditsGC-MSanalysis,J. Chin. Med. Mater. (Chinese),27,177,2004.

34. Wu,G.etal.,GC-MSanalysisoflipophiliccomponentsofcortexalbiziaeextractedbysupercriticalCO2,Chin. Trad. Herbal Drugs (Chinese),36,832,2005.

35. Wu,L.H.,Du,X.andLiu,H.M.,β-Elemenefromvolatileoilofcurcumakwangsiensisbysupercriticalfluidextraction,Chin. Trad. Herbal Drugs (Chinese),37,368,2006.

36. Chen,Y.,Jiang,Z.H.andTian,J.K.,GC-MSanalysisofvolatileoilfromtheearofSchizonepeta tenifolia Briq.,J. Chin. Medic. Mater. (Chinese),29,140,2006.

37. Wang,Z.B.andMa,H.L.,Studyonanti-cancercomponentsoffigresidueswithsuper-criticalfluidCO2extractingtechnique,Chin. J. Chin. Mater. Med. (Chinese),36,1443,2005.

38. Fu,Y.J.,Wang,W.andZu,Y.J.,StudyonvolatilecomponentsofseedoilfromGlycyrrhiza uralensisfisch.by supercritical carbondioxideextraction-gaschromatography-massspectrometry,Chin. J. Anal. Chem. (Chinese),33,498,2005.

39. Zeng,H.Y.andLi,J.H.,TechnologystudyonthevolatileoilsfromtheleavesofArtemisiae argyiextractedbysupercriticalCO2ormicrowave,Food Sci.,25,124,2004.

40. Liu,C.M.,Huang,G.S.andLiu,X.H.,ExtractionofvolatileoilfromLilium brownii bysupercriticalCO2,Nat. Prod. Res. Develop. (Chinese),17,485,2005.

41. Yan,G.H.andLin,Z.Y.,ThevolatileoilsfromOcimum basilicum L.bysupercriticalCO2extraction,Res. Pract. Chin. Med. (Chinese),19,56,2005.

42. Li, C.H. et al., Study of supercritical CO2 extraction of orris oil, Nat. Prod. Res. Develop. (Chinese),17,773,2005.

43. Qiu,Q.etal.,GC-MSanalysisofchemicalconstituentsoftheessentialoilfromPerilla frutescens (L.)britton.bydifferentextractionmethods,Chin. J. Pharm. Anal.,26,114,2006.

44. Mi, H. et al., Analysis of volatile components in essential oil of radix angelicaedahuricaeofsupercriticalfluidextractionbygaschromatographymassspectrometry,Chin. J. Anal. Chem. (Chinese),33,366,2005.

7089_C007.indd 238 10/15/07 5:43:51 PM


45. Liu,J.Z.andZhong,Z.J.,GC-MSanalysisoflipophiliccomponentsofradixlitseaecubebae extracted by supercritical CO2, J. Chin. Med. Mater. (Chinese), 29, 142,2006.

46. Mo, S.Z. et al., Extraction of lipophilic components from salvia castanea diels f.tomentosastib.bysupercriticalCO2,J. Chin. Med. Mater. (Chinese),27,735,2004.

47. Gao,Y.etal.,Hemostaticeffectsoftheextractsofsaposhnikoviadivaricata(Turcz.)schischk by supercritical extraction, Chin. Trad. Herbal Drugs (Chinese), 36, 254,2005.

48. Zhang,Y.,Extractionofschisandrachinensis(Turcz)bysupercriticalcarbondioxide,Chin. Tradit. Patent Med. (Chinese),27,880,2005.

49. Zhang,G.Y.,Lin,J.Y.andQiu,Q.,SupercriticalcarbondioxideextractionandGC-MSofessentialoilfromthestromaofcordycepskyushuensis,Chin. J. Pharm. Anal.,26,191,2006.

50. Zhang,X.W.etal.,Supercriticalcarbondioxideextractionofwheatplumuleoil,J. Food Engin.,37,103,1998.

51. Wang, J.P. et al.,SupercriticalCO2extractionof zanthoxylumseedoil,Chin. Trad. Herbal Drugs (Chinese),35,997,2004.

52. Liu, H. et al., Extraction of aplinia oxyphylla miquel seed essence via supercriticalcarbondioxideandantioxidantactivityoftheextracts,J. South Chin. Univ. Technol. (Natural Science Ed.) (Chinese),34,54,2006.

53. Sun,H.Y.,Guan,S.andHuang,M.,Studyonnewextractiontechnologyofastragalo-sideIV,J. Chin. Med. Mater. (Chinese),28,705,2005.

54. Liu,L.etal.,Supercritical-CO2fluidextractionoftaxolfrombranchesandneedlesoftaxusyunnanensis,Chin. Tradit. Patent Med. (Chinese),28,480,2006.

55. Han, Z.H. et al., Supercritical fluid CO2 extraction of urosolic acid from cornusofficinalis,Chin. Trad. Herbal Drugs (Chinese),36,1159,2005.

56. Chen, F., Guo, L.W. and Jin, S.L., Supercritical CO2 fluid extraction of tetrahydro-palmatinefromcorydalisyanhusuoW.T.Wangbyorthogonaldesign,Tradit. Chin. Drug Res. Clin. Pharm. (Chinese),16,137,2005.

57. Xiu, S.L., Wu, Q.N. and Zhen, O.Y., Study on the SFE condition for curcumin incurcumalonga,Chin. J. Chin. Mater. Med. (Chinese),29,857,2004.

58. Guo,T.andChen,J.,Theprocessstudyontheextractionofironebysupercriticalfluidsextraction,J. Chin. Med. Mater. (Chinese),27,768,2004.

59. Sun,Y.Y.etal.,ExtractionofmedicalcomponentsfromLigusticum chuanxiong hort.withsupercriticalCO2,Chem. Engin. (Chinese),34,60,2006.

60. Zhou,J.K.,Li,J.H.andGe,F.H.,ExtractionofresveratrolfromPolygonum cuspidatum bysupercriticalCO2,J. Chin. Med. Mater. (Chinese),27,675,2004.

61. Geng,Y.L.etal.,StudyonsupercriticalCO2extractionofphospholipidfromPolygonum multiflorum thumb,Food Sci.,27,140,2006.

62. Huo, W.L., Study on technology of supercritical CO2 extraction of pricklyash peelvolatileoils,Food Sci.,26,153,2005.

63. Gu, Y.H. et al., Extraction of flavonoids from propolis by supercritical CO2, Chin. Traditi. Herbal Drugs (Chinese),37,380,2006.

64. Deng, Y.F. and Liang, N.C., Extraction of diterpenoids from Pteris semipinnata bysupercriticalCO2fluidandtheiranalysiswithHPLC-MS,Chin. Trad. Herbal Drugs (Chinese),35,145,2004.

65. Liu,B.etal.,Extractionofberberinefromrhizomeofcoptischinensisfranchusingsupercriticalfluidextraction,J. Pharm. Biomed. Anal.,41,1056,2006.

66. Dean,J.R.,Liu,B.andPrice,R.,ExtractionoftanshinoneIIAfromsalviamiltiorrhizabunge using supercritical fluid extraction and a new extraction technique, phytosolsolventextraction,J. Chromatogr. A,799,343,1998.

7089_C007.indd 239 10/15/07 5:43:52 PM


67. Liu,B.etal.,SupercriticalfluidextractionofsinomeninefromSinomenium acutum (thumb)RehdetWils, J. Chromatogr. A,1075,213,2005.

68. Cui,H.W.andGe,F.H.,StudiesonconstituentsfromTaxus mairei bark,J. Chin. Med. Mater. (Chinese),27,566,2004.

69. Johnston, K.P. et al., Water-in-carbon dioxide microemulsions: An environment forhydrophilesincludingproteins,Science,271,624,1996.

70. Chen,B.etal.,Roleofsurfactantinsupercriticalfluidextraction,Acad. J. Second Mil. Med. Univ. (Chinese),21,463,2000.

71. Ge,F.H.etal.,Effectofnon-ionogenicsurfactantsontheextractionofmatrinesfromkuh-seng, J. Chin. Med. Mater.,26,426,2003.

72. Wang,F.,Li,X.Y.andZhen,X.H.,Theaffectionofmodifierinextractionandisola-tionofsesquiterpenesolidefromtheessentialoilofatractylodesmacrocephalaKoidz,J. Xian Instit. Technol. (Chinese),25,465,2005.

73. Ding,C.M.,Qiu,T.Q.andLu,H.Q.,Double-frequencyultrasoundsalternatelyenhancedsupercritical fluid extraction of effective components of plants, Chem. Eng., 33, 67,2005.

74. Hu,A.J.etal.,Ultrasoundassistedsupercriticalfluidextractionofoilandcoixenolidefromadlayseed,Ultrasonics Sonochemistry,14,219,2007.

75. Eisenbach,W.,Ber. Bunsenges Phys. Chem.,88,882,1984. 76. Liu,Y.,Ding,X.L., andZhu,D.H.,ExtractionandconcentrationnaturalvitaminE

usingsupercriticalCO2,Chem. Eng. (China),34,59,2006. 77. Li,D.P.,ChinesePatentZL02137312.4,2004. 78. Zhang,Z.Y.etal.,Analysisofessentialoilfromrhizomaatractylodismacrocephalae

by GC-MS with supercritical CO2 extraction and molecular distillation, J. Instrum. Anal. (Chinese),22,61,2003.

79. Zhang, Z.Y., Lei, Z.J. and Wang, P., Studies on chemical composition of forsythiasuspensabysupercriticalfluidextractionandmoleculardistillation,J. Instrum. Anal.(Chinese),22,60,2002.

80. Wang,P.,Zhang,Z.Y.andWu,H.Q.,Studiesonvolatileoilofgarlicbysupercriticalfluidextractionandmoleculardistillation,Chin. Hosp. Phatm. J.,22,253,2002.

81. Gu,W.X.etal.,ExtractionofactiveprinciplesofradixangelicaepubescentisbyCO2SFE-MD,Acad. J. Guangdong Coll. Pharm. (Chinese),18,85,2002.

82. Shi,Y.,Gu,W.X.andZhang,Z.Y.,ExtractionofactiveprinciplesofspirulinabyCO2SFE-MD,Guangdong Pharm. J. (Chinese),13,10,2003.

83. Zhou,B.J.,Zhang,Z.Y.andShi,Y.,Analysiswithgaschromatographyandmassspec-trography of volatile components of Ligusticum wallichii franch extracted by CO2supercriticalfluidextractionincombinationwithmoleculardistillation,J. First Milit. Med. Univ. (Chinese),22,652,2002.

84. Li,Z.C.,Developmentandfutureofcountercurrentchromatograph,Chem. Ind. Eng. Prog.,24,816,2005.

85. Cao,X.L.etal.,Supercriticalfluidextractionofcatechinsfromcratoxylumprunifoliumdyer and subsequent purification by high-speed counter-current chromatography,J. Chromatogr. A,898,75,2000.

86. Cao,X.L. andYoichiro, I., Supercriticalfluid extractionof grape seedoil and sub-sequentseparationoffreefattyacidsbyhigh-speedcounter-currentchromatography,J. Chromatogr. A,1021,117,2003.

87. Wang,X.etal.,Anefficientnewmethodforextraction,separation,andpurificationofpsoralenandisopsoralenfromFructus psoraleae bysupercriticalfluidextractionandhigh-speedcounter-currentchromatography,J. Chromatogr. A,1055,135,2004.

88. Peng,J.Y.etal.,Efficientnewmethodforextractionandisolationofthreeflavonoidsfrom patrinia villosa juss. by supercritical fluid extraction and high-speed counter-currentchromatography,J. Chromatogr. A,1102,44,2006.

7089_C007.indd 240 10/15/07 5:43:52 PM


89. Zhang, H. et al., Extraction of effective components in salvia miltiorrhiza bge. bysupercritical extraction combined with water decoction, Chin. Trad. Herbal Drugs (Chinese),35,1360,2004.

90. Ye, C.H., Li, S.F. and Tang, S.K., Extraction and purification of procyanidins fromgrapeseedwithsupercriticalCO2andmacroporousadsorptionresin,Chem. Ind. Eng. (Chinese),23,220,2006.

91. Xiao, L. et al., Combining supercritical CO2 extraction with solvent extraction forobtaining active ingredients from nelumbo nucifera gaertn, presented at the ThirdInternationalSymposiumonSupercriticalFluidTechnologyforEnergyandEnviron-mentApplications,China,Oct.24–26,2004,74.

92. Wang,D.W.,Shan,Y.L.andTolgor,B.,EffectofextractionrateofsupercriticalCO2extractiononmongoliamushroompolysaccharide,Food Sci.,27,107,2006.

93. Deans,S.G.andRitchie,G.,Antibacterialpropertiesofplantessentialoils, Int. J. Food Microbiol.,5,165,1987.

94. Kim, H. M., Lee, E.H. and Hong, S.H., Effect of syzygium aromaticum extract onimmediatehypersensitivityinrats,J. Ethnopharmacol.,60,125,1998.

95. DellaPorta,G.etal.,IsolationofclovebudandstaraniseessentialoilbysupercriticalCO2extraction,Lebensm.-Wiss. u.-Technol.,31,454,1998.

96. Guan,W.Q. et al.,Comparison of essential oils of clove buds extracted with super-critical carbondioxideandother three traditional extractionmethods,Food Chem.,101,1558–1564,2007.

97. He, D.P. and Song, G.S., The development report on the extraction of Ligusticum chuangxiong Hort.assolvent,Chin. Oil (Chinese),21,10,1996.

98. Shi, L. F., Deng, Y. Z. and Wu, B. S., Studies on chemical constituents and theirstabilityoftheessentialoilfromLigusticum chuangxiong Hort.,Chin. J. Pharm. Anal. (Chinese),15,26,1995.

99. Sun,Y.Y.andLi,S.F.,Solubilityofferulicacidandtetramethylpyrazineinsupercriticalcarbondioxide,J. Chem. Eng. Data (Chinese),50,1125,2005.

100. Daniel,S.,Bailey’s Industrial Oil and Fat Products,AWiley-IntersciencePublication,WileyJ.,NewYork,1979,449.

101. Bagchi,D.etal.,Freeradicalsandgrapeseedproanthocyanidinextract:Importanceinhumanhealthanddiseaseprevention,Toxicology,148,187,2000.

102. Castillo,J.etal.,AntioxidantactivityandradioprotectiveeffectagainstchromosomaldamageinducedinvivobyX-raysofflavan-3-ols(procyanidins)fromgrapeseeds(vitisvinifera):Comparativestudyversusotherphenolicandorganiccompounds,J. Agric. Food Chem.,48,1738,2000.

103. Gabor,M.,Engi,E.andSonkodi,S.,Effectofflavonederivativesonthearterialwalland its resistance in rats with spontaneous hypertension, Kiserl Orvostud, 39, 425,1987.

104. Tang,S.K.etal.,ExtractionofgrapeseedoilfromgrapeseedwithsupercriticalCO2,J. Chem. Eng. Chin. Univ. (Chinese),18,23,2004.

105. Ye, C.H., Li, S.F. and Tang, S.K., Extraction and purification of procyanidins fromgrapeseedwithsupercriticalCO2andmacroporousadsorptionresin,Chem. Indust. Eng.,23,220,2006.

106. Liu,F. andHe,L.C.,Studieson thequalitativeandquantitativemethodsofqualitycontrolofPanaxginseng,Medicament Analysis (Chinese),22,173,2002.

107. Wang, Y.H. and Rong, H., Determination of organic chloride pesticide residue inginsengwithcapillarygaschromatography,Chin. J. Anal. Chem.,22,931,1994.

108. Yang, S.L. et al., Determination of the benzene hexachloride residue in Ligusticum chuanxiong hort. and other 6 Chinese herbal drugs, Chin. Trad. Herbal Drugs (Chinese),33,423,2002.

7089_C007.indd 241 10/15/07 5:43:53 PM


109. Jian,H.W.,Qiang,X.andKui,J.,Supercriticalfluidextractionandoff-lineclean-upfortheanalysisoforganochlorinepesticideresiduesingarlic,J. Chromtogr. A,818,138,1998.

110. Quan,C.etal.,Determinationoforganochlorinepesticidesresidueinginsengrootbyorthogonalarraydesignsoxhletextractionandgaschromatography,Chromatographia,59,89,2004.

111. Quan,C.etal.,Supercriticalfluidextractionandclean-upoforganochlorinepesticidesinginseng,J. Supercrit. Fluids,31,149,2004.

112. Li,S.F.andQuan,C.,IsolationoforganochlorinepesticidefromGinsengwithsuper-criticalCO2,Chin. J. Chem. Eng.,13,433,2005.

113. Li, S.F. et al., Determination and clean-up of the pesticides residue in the Chinesemedicinalmaterials,Chin. Trad. Herbal Drugs (Chinese),35,232,2004.

7089_C007.indd 242 10/15/07 5:43:54 PM

243

8 Extraction of Bioactive Compounds from Latin American Plants

M. Angela A. Meireles

Contents

8.1 Introduction:ExamplesofLatinAmericanBioactiveCompounds............ 2438.1.1 ExamplesofSFEfromNativeLatinAmericanPlants....................244

8.2 ExtractingBioactiveCompoundsbySFE................................................... 2528.2.1 RelevantProcessInformationforCOMEstimation........................2548.2.2 SelectingParametersIntendedforCOMEstimation......................254

8.2.2.1 PressureandTemperatureProcesses................................. 2558.2.2.2 TheKineticParameters...................................................... 257

8.2.3 TheCOMforLatinAmericanPlants..............................................2608.3 Conclusions................................................................................................. 262References.............................................................................................................. 262

8.1 IntroduCtIon: examples of latIn amerICan BIoaCtIve Compounds

In this chapter, a brief review of supercritical fluid extraction (SFE) of bioactivecompoundsfromsolidsubstratumispresented.ThestateoftheartofSFEinLatinAmerica is described. Examples of research in development, embodying experi-mentalandmodelingofmasstransferandthermodynamics,forseveralsystemsarediscussed.Forpreliminarystudiesof technicalandeconomical feasibility,averysimpleempiricalmodelcanbeusedtodescribethemasstransferintheextractorcell.Tocalculatethecostofmanufacturing(COM),nosolubilitydataarerequired,andconsideringtheflashseparatorideal,therequiredinformationistheglobalyieldinextractatagivenconditionoftemperatureandpressurealongwithanestimatetimeintervalforanextractioncycle.COMestimatedthiswayisprovidedforsomeLatinAmericanplants.

LatinAmerica(LA)isformedby33countries:AntiguaandBarbuda,Argentina(AR),Bahamas,Barbados,Belize,Bolivia(BO),Brazil(BR),Chile(CH),Colombia(CO), Commonwealth of Dominica, Costa Rica, Cuba, Dominican Republic,Ecuador, El Salvador, Granada, Guatemala, Guyana, Haiti, Honduras, Jamaica,Mexico,Nicaragua,Panama,Paraguay(PA),Peru(PE),SaintKitts,SaintVincentand the Grenadines, Santa Lucia, Suriname, Trinidad and Tobago, Uruguay, and

7089_C008.indd 243 10/15/07 5:45:46 PM


Venezuela.FewofthesecountriesareintheAmazonianregion;thosethatareincludeBrazil,Bolivia,Colombia,Ecuador,Guyana,Peru,Suriname,andVenezuela.Therichnessof theAmazonianbiodiversitymayhelp the regiondevelopmentaswellas its devastation. Presently, the governments of the Amazonian countries havedemonstrated their concernwith thedevastationof theirnatural resources.Manyinitiatives of sustained development are available from governmental agencies,nongovernmental organizations, and private companies. These initiatives includesustainedharvestingofnativeplantsbylocalcommunities.Addingvaluetotherawmaterialbyprocessingithasalsobeenstimulated.

Besidesthat,LAcountriesareproducersofcondiments,aromaticherbs,roots,andtropicalfruitsusedbythefood,pharmaceutical,andcosmeticindustries.Someof theseproducts areused locally, andothers are exported.Among the exportedproductsareblackpepper,clovebuds,andginger.Essentialoilsandoleoresinsofvetivergrass,eucalyptus,cinnamon,mint,andotherplantsarealsoexported.BrazilandParaguayarelargeproducersofstevia,aplantwhoseaqueousextracthasbeenusedforyearsasasucrosesubstituteinspecialdiets[1].Inaddition,severalotherplantspossess lipids, starches,andcellulose thatcanpotentiallybeeconomicallyexplored.Examplesof these are turmeric, saffron, andbacuri. In addition,Chilecultivates certain microalgae, such as Spirulina maxima, which maybe used assourceoffattyacids[2–3].

Questionsrelatedtotheuseoftechniquesthatavoidorminimizedamagestotheenvironmentarecurrentlybeingdebated.Consumers’demandsindicatethat, inthenearfuture,productsofbetterqualitywillberequestedmoreandmore.ThistendencycanbeexploredthoroughlybytheLAcountries.Totakeadvantageoftheirpotential,thesecountriesneedtodeveloporadapttechnologiesthatareeconomicallyviableandecologicallyresponsible.ProductsobtainedbySFEarefreefromtoxicresiduesandgenerallypossesshigherqualitythanproductsobtainedbyconventionaltechniques.

Therefore, rawmaterials fromLAcountries represent abusinessopportunityforproducersofvegetableextracts,moreoveriftheseextractsarepreparedbySFE.Combining this rich biodiversity with an ecologically correct technology wouldrepresenttheidealmarriage!

8.1.1 ExamplEs of sfE from NativE latiN amEricaN plaNts

Compilations of literature on SFE were done recently by Meireles [5], Rosa andMeireles[6],Diáz-Reinosoetal.[7],anddelValleetal.[8].Therefore,theinforma-tionpresentedinthischapterrepresentsanupdateofthepreviousworks.Inspiteof that, thecompilationof literaturedatawasnotmeant tobeexhaustive; insteadit focused on including information that was not easily accessible. Table8.1 toTable8.3 listLatin-Americanplants (spontaneousor cultivated) studied; theSFEstudiesonthemicroalgaeSpirulina maximawerealsoincluded[2,3].Thecommonnameswereconfirmed in theU.S.DepartmentofAgriculturePlantDatabase[9],theRainforestDatabase[10],w3TROPICOSofTheMissouriBotanicalGarden[11],SearchableWorldWideWebMultilingualMultiscriptPlantNameDatabase [12],andCropINDEX[13].ThescientificnamespellingswereconfirmedusingthesamedatabasesandtheFlorabrasiliensis[14].Theregionsofoccurrence(spontaneousor

7089_C008.indd 244 10/15/07 5:45:47 PM

Extraction of Bioactive Compounds from Latin American Plants 245

taB

le 8

.1B

ioac

tive

Com

poun

ds fr

om l

atin

am

eric

a pl

ants

(sp

onta

neou

s an

d In

trod

uced

): v

olat

ile o

il, o

leor

esin

, and

o

ther

aro

ma

Com

poun

ds

Com

mon

nam

esc

ient

ific

nam

epa

rt u

sed

Bio

acti

ve C

ompo

und(

s)

reg

ion

of

occ

urre

nce

(spo

ntan

eous

or

Cul

tiva

tion

)sf

e C

ondi

tion

s/

mpa

/K/C

osol

vent

Yiel

d (%

)r

efer

ence

Agu

arib

aySc

hinu

s m

olli

sFr

actio

natio

nof

ste

am

dist

illat

ion

vola

tile

oil

β-Pi

nene

,α-p

inen

e,a

ndli

mon

ene

LA

9/32

3—

[15]

Ann

atto

Bix

a or

ella

naSe

eds

Vol

atile

oil

and

oleo

resi

n:b

ixin

and

no

rbi

xin

BR

-N20

–30/

313–

333/

EtO

H1–

45[1

6,1

7]

Bam

boo

pipe

ror

pi

men

ta-l

onga

Pip

er a

dunc

umL

eave

sα-

hum

ulen

e,a

sari

cin,

β-

cary

ophy

llene

SA10

–30/

303–

313

1.4–

1.8

[18]

Bas

il(s

wee

t)O

cim

um g

rati

ssim

umL

eave

sE

ugen

olB

R-S

E10

–30/

313

1–1.

8[1

9]

Bla

ckp

eppe

rP

iper

nig

rum

L.

Seed

sβ-

cary

ophy

llene

,lim

onen

e,

3-δ-

care

ne,s

abin

ene

BR

,PA

15–3

0/30

3–32

30.

5–2.

1[2

0–22

]

Bus

hyli

ppia

Lip

pia

alba

Lea

ves

Car

vone

and

lim

onen

eB

R-S

E/N

E8–

12/3

13–3

231.

5–5

[23]

Cha

mom

ileC

ham

omil

la r

ecut

ita

[L]

R.

Flow

ers

Azu

lene

and

cha

maz

ulen

eB

R-S

10–2

0/30

3–31

30.

82–4

.3[2

4]

Citr

onel

laC

ymbo

pogo

n w

inte

rian

us,J

owitt

Lea

ves

Citr

onel

lal,

citr

onel

lolg

eran

iol

BR

-SE

7–16

/289

–298

0.45

–1[2

5]

Clo

veb

uds

Eug

enia

car

yoph

yllu

sFr

uit

Eug

enol

,β-c

aryo

phyl

lene

,and

α-

hum

ulen

e

BR

-NE

6.7–

10/2

83–3

08~

14[2

6,2

7]

cont

inue

d

7089_C008.indd 245 10/15/07 5:45:48 PM


taB

le 8

.1 (c

onti

nued

)B

ioac

tive

Com

poun

ds fr

om l

atin

am

eric

a pl

ants

(sp

onta

neou

s an

d In

trod

uced

): v

olat

ile o

il, o

leor

esin

, and

o

ther

aro

ma

Com

poun

ds

Com

mon

nam

esc

ient

ific

nam

epa

rt u

sed

Bio

acti

ve C

ompo

und(

s)

reg

ion

of

occ

urre

nce

(spo

ntan

eous

or

Cul

tiva

tion

)sf

e C

ondi

tion

s/

mpa

/K/C

osol

vent

Yiel

d (%

)r

efer

ence

Cof

fee

Cof

fea

arab

ica

Frui

tA

rom

aco

mpo

unds

(py

razi

nes,

py

ridi

nes,

and

fur

and

eriv

ativ

es)

LA

24–3

1/31

5–37

1—

[28]

Cor

iand

erC

oria

ndru

m s

ativ

umL

.Se

eds

Vol

atile

oil,

phe

nolic

com

poun

dsL

A20

–30/

298–

331

0.8–

2.3

[29,

30]

Cro

ton

Cro

ton

zehn

tner

iPa

xet

Hof

fL

eave

s(E

)-A

neth

ole

BR

-NE

/S6.

7–7.

9/28

3–30

12.

1–3.

8[3

1]

Erv

aba

leei

rao

rw

ilds

age

Cor

dia

verb

enac

eaL

eave

sβ-

cary

ophy

llene

and

α-h

umul

ene

BR

-SE

7.8–

30/2

99–3

230.

11–5

.5[3

2]

Euc

alyp

tus

Euc

alyp

tus

citr

iodo

ra,

Hoo

kL

eave

sC

itron

ella

land

citr

onel

lol

BR

-SE

7–16

/289

–298

0.31

–0.6

8[2

5]

Euc

alyp

tus

Euc

alyp

tus

tere

tico

rnis

Lea

ves

Aro

mad

endr

ene,

1,8

-cin

eola

nd

glob

ulol

BR

-NE

6.7–

7.9/

283–

298

0.45

–1.1

3[3

3]

Fenn

elFo

enic

ulum

vul

gare

Seed

sA

neth

ole,

fen

chon

e,a

ndf

atty

aci

dsB

R-S

E10

–30/

313

3–12

.5[3

4]

Gin

ger

Zin

gibe

r of

ficin

alis

R.

Rhy

zom

eβ-

pine

ne,m

-die

thyl

-ben

zene

,o-

diet

hyl-

benz

ene,

ar-

curc

umen

e,

α-zi

ngib

eren

e,β

-ses

quip

hella

ndre

ne

BR

-SE

15–3

0/29

3–31

32–

3[3

5–37

]

Gre

enp

eppe

rba

sil

Oci

mum

sel

loi

Lea

ves

Vol

atile

oil

BR

-SE

10–3

0/30

3–32

30.

71–2

.2[3

8]

Hor

seta

il(g

iant

)E

quis

etum

gig

ante

umL

.A

eria

lpar

tsO

leor

esin

BR

-S12

–30/

303–

313

1.44

[39]

Kho

aSa

ture

ja b

oliv

iana

Lea

ves

Pule

gone

,iso

men

thon

e,ty

mol

BO

6.5–

7/28

9–29

4/E

tOH

2–

4.6

[40]

7089_C008.indd 246 10/15/07 5:45:49 PM


Lem

onv

erbe

naA

loys

ia tr

iphy

lla

Lea

ves

Ner

al(

orZ

-citr

al)

and

gera

nial

(o

rE

-citr

al),

and

spa

thul

enol

BR

-SE

10–3

5/30

8–31

80.

6–1.

5[

41]

Lem

ongr

ass

Cym

bopo

gon

citr

atus

Aer

ialp

arts

Ner

ala

ndg

eran

ial

BR

(N

E,S

E,

and

S)6.

9–7.

4/28

8–29

70.

21–4

2[4

2]

Lip

pia

sido

ides

Lip

pia

sido

ides

C.

Lea

ves

Tim

olB

R-N

E6.

7–7.

9/28

3–29

82.

2–3.

3[4

3]

Mac

ela

Ach

yroc

line

sa

ture

ioid

esa

nd

A. a

lata

Lea

ves

α-hu

mul

ene,

β-c

aryo

phyl

lene

,qu

erce

tin

BR

-SE

10–3

0/30

3–31

31.

2–4.

2[4

4]

Mar

igol

dC

alen

dula

offi

cina

lis

Flow

ers

Ole

ores

inB

R-S

12–2

0/29

3–31

3K

2–2.

8[4

5]

Mas

tran

toH

ypti

s su

aveo

lens

Lea

ves

Spat

ulen

e,G

erm

acre

neB

,C

aryo

phyl

lene

V

E8–

9/30

8–31

80.

1–0.

3[4

6]

Ora

nge

(sw

eet)

Cit

rus

sine

nsis

(L

.)Sh

ells

Vol

atile

oil

SA20

/313

0.6–

0.15

[47,

48]

Ore

gano

Ori

ganu

m v

ulga

reL

.L

eave

sC

is-s

abin

ene

hydr

ate,

thym

ol,

carv

acro

lL

A10

–20/

293–

313

0.4–

1.3

[49]

Palm

aros

aC

ymbo

pogo

n m

arti

ni

Rox

b.L

eave

sG

eran

iol,

linal

ool

BR

-AM

7–16

/289

–298

0.07

–0.2

[25]

Pipr

ioca

Cyp

erus

ses

quifl

orus

Rhy

zom

esSp

athu

leno

l,tr

ans-β-

guai

ene,

ge

rmac

rene

D

BR

-AM

10–1

2/33

3–35

30.

3–0.

4[5

0]

Ros

emar

yR

osm

arin

us o

ffici

nali

sL

eave

sC

amph

or,c

arno

sic

and

rosm

arin

ic

acid

s,p

heno

licd

iterp

enes

BR

-SE

10–3

0/30

3–31

31–

5[5

1,5

2]

Stev

iaSt

evia

reb

audi

ana

B.

Lea

ves

Aus

troi

nulin

,n-t

etra

cosa

ne,

n-pe

ntac

osan

eB

R/P

A25

/303

1.4–

1.6

[53,

54]

Vet

iver

gras

sVe

tive

ria

ziza

nioi

des

(L.)

Nas

hR

oots

Khu

zym

olB

R-N

E/S

E20

/313

3.2

[50,

55,

56]

Xyl

opia

ar

omat

ica

Xyl

opia

aro

mat

ica

Frui

tβ-

Phel

land

rene

,β-m

yrce

ne,α

-pin

ene

LA

7.5/

318

1.5

[57]

LA

:L

atin

Am

eric

a; S

A:

Sout

hA

mer

ica;

BR

-AM

:A

maz

onia

nre

gion

of

Bra

zil;

BR

-NE

:N

orth

east

of

Bra

zil;

BR

-S:

Sout

hof

Bra

zil;

BR

-SE

:So

uthe

ast

of B

razi

l;A

R: A

rgen

tina;

BO

:Bol

ivia

;PA

:Par

agua

y; V

E: V

enez

uela

7089_C008.indd 247 10/15/07 5:45:49 PM


taB

le 8

.2B

ioac

tive

Com

poun

ds fr

om l

atin

am

eric

a pl

ants

(sp

onta

neou

s an

d In

trod

uced

): l

ipid

s an

d li

pid-

solu

ble

Com

poun

ds

Com

mon

nam

esc

ient

ific

nam

epa

rt u

sed

Bio

acti

ve C

ompo

unds

reg

ion

sfe

Con

diti

ons/

m

pa/K

/Cos

olve

ntYi

eld

(%)

ref

eren

ce

Bac

uri

Pla

toni

a in

sign

isSh

ell

Free

fat

tya

cids

BR

-AM

6.3–

7.0/

289–

294

0.15

–0.4

7[5

8]

Bur

itiM

auri

tia

flexu

osa

L.

Frui

tC

arot

enoi

ds,t

ocop

hero

ls,a

nd

lipid

s:f

atty

aci

ds,e

tc.

BR

-AM

20–3

0/3

13–3

28

4.7–

7.8

[59]

Cup

uass

uT

heob

rom

a gr

andi

floru

m

Seed

sL

ipid

s:f

atty

aci

ds,e

tc.

BR

-AM

24.8

–35.

2/32

3–35

3So

lven

ts:C

O2/

Eth

ane

2–6/

5–6

[60]

Jojo

baSi

mm

onds

ia c

hine

nsis

Seed

sFa

ttya

cids

AR

40/3

13–3

530.

34–0

.4[6

1]

Oliv

ehu

sk—

Hus

kV

eget

able

oil

CH

30/3

13

7.5–

12.5

[62]

Palm

E

laei

s gu

inee

nsis

L.

Pres

sed

palm

fib

ers

ork

erne

lC

arot

enoi

ds,t

ocop

hero

ls,

fatty

aci

ds,e

tc.

BR

-AM

15–3

0/31

8–32

81.

8–4.

9[6

3–66

]

Papr

ika

pow

der

Cap

sicu

m a

nnuu

m—

Car

oten

oids

AR

30/3

330.

9[6

7]

Pass

ion

frui

tPa

ssifl

ora

edul

isSe

eds

Lip

ids,

fat

tya

cids

,etc

.B

R-A

M/S

E20

–30/

317–

343

13.7

–27.

7[6

8]

Pejib

aye

orp

upun

haG

uili

elm

a sp

ecio

sa o

r B

actr

is g

asip

aes

Frui

tFa

ttya

cids

SA-A

M8–

30/2

93–3

23

9–13

[69,

70]

Rap

esee

dB

rass

ica

napu

sSe

eds

Veg

etab

leo

ilC

H30

/313

6–

12[6

2]

Ric

ebr

anO

ryza

sat

iva

Parb

oile

dri

ceb

ran

Toco

trie

nola

ndto

coph

erol

sB

R-S

/SE

15–3

0/29

8–33

38

[71,

72]

Ros

ehip

Ros

a ca

nina

L.

Seed

sC

arot

enoi

dsa

ndf

atty

aci

dsC

H10

/301

4–6.

5[6

2]

Tuc

uman

Ast

roca

ryum

vul

gare

Seed

sFa

ttya

cids

BR

-AM

20–3

0/31

3–34

331

[73]

Ucu

uba

Viro

la s

urin

amen

sis

Seed

sT

rim

irys

tinSA

20–2

5/32

344

[74]

SA:S

outh

Am

eric

a;S

A-A

M:A

maz

onia

nre

gion

of

Sout

hA

mer

ica;

BR

-AM

:Am

azon

ian

regi

ono

fB

razi

l;B

R-S

E:S

outh

east

of

Bra

zil;

AR

:Arg

entin

a;C

H:C

hile

7089_C008.indd 248 10/15/07 5:45:50 PM


taB

le 8

.3B

ioac

tive

Com

poun

ds fr

om l

atin

am

eric

a pl

ants

(sp

onta

neou

s an

d In

trod

uced

): m

isce

llane

ous

Com

mon

nam

esc

ient

ific

nam

epa

rt u

sed

Bio

acti

ve c

ompo

und(

s)r

egio

nsf

e co

ndit

ions

/ m

p a/K

/Cos

olve

ntYi

elds

/%r

efer

ence

And

read

oxa

And

read

oxa

flava

Lea

ves

8-m

etho

xy-N

-met

hyl-

flind

ersi

neB

R-N

E20

.4–3

0.6/

313

—[7

5]

Arn

ica

Soli

dago

chi

lens

isL

eave

sIs

oque

rcet

in,r

utin

,vite

xin

BR

10/3

33/E

tOH

12.5

[76]

Aro

eira

Schi

nus

ther

ebin

thif

oliu

sL

eave

s,

flow

ers

and

stem

s

Ana

card

ica

cids

BR

-NE

13.6

–27.

2/31

3–32

3—

[77]

Arr

uda

das

erra

or

arru

dab

rava

Poir

etia

bah

iana

C.M

ulle

rL

eave

sW

axes

,iso

flavo

nes,

rot

enon

es

and

mon

oter

peno

idB

R-N

E20

.4–2

3.8/

313

—[7

8]

Art

emis

iao

rsw

eets

agew

ort

Art

emis

ia a

nnua

Lea

ves

Art

emis

inin

BR

-SE

15–3

5/30

3–32

35–

6.5

[79]

Avo

cado

Pers

ea a

mer

ican

aL

eave

sQ

uerc

etin

,iso

quer

cetin

,rut

in,

vite

xin

LA

10/3

33/E

tOH

14[7

6]

Bac

char

iao

rva

ssou

raB

acch

aris

dra

cunc

ulif

olia

D

CL

eave

s(E

)-ne

rolid

ola

nds

path

ulen

olSA

9–12

/313

–333

0.

4[8

0]

Bas

ilO

cim

um b

asil

icum

Lea

ves

Ant

ioxi

dant

com

poun

dsB

R-S

E10

–35/

303–

323

K/H

2O:1

/10/

20%

1–2/

6–11

/12–

24[8

2]

Bla

ckw

attle

or

blac

kac

acia

Aca

cia

mea

rnsi

iB

ark

Tann

inB

R15

–20/

313–

353

4.8–

23.7

of

tann

in[8

1]

Bra

zilia

ngi

nsen

gP

faffi

a gl

omer

ata

Rhi

zom

eE

cdys

tero

neB

R20

/303

0.6

[83]

Bol

doPe

umus

bol

dus

M.

Lea

ves

Bol

dine

CH

6–15

/303

–333

/H2O

0.5–

3.4

[84]

Cac

aoT

heob

rom

a ca

cao

Frui

tC

affe

ine,

theo

brom

in,f

atty

ac

ids,

and

met

yilx

antin

esL

A24

.8/3

23

3–13

[85] co

ntin

ued

7089_C008.indd 249 10/15/07 5:45:51 PM


taB

le 8

.3 (c

onti

nued

)B

ioac

tive

Com

poun

ds fr

om l

atin

am

eric

a pl

ants

(sp

onta

neou

s an

d In

trod

uced

): m

isce

llane

ous

Com

mon

nam

esc

ient

ific

nam

epa

rt u

sed

Bio

acti

ve c

ompo

und(

s)r

egio

nsf

e co

ndit

ions

/ m

p a/K

/Cos

olve

ntYi

elds

/%r

efer

ence

Cas

hew

Ana

card

ium

occ

iden

tale

L.

Nut

sC

arda

nol,

anac

ardi

cac

id,

cate

quin

BR

-NE

9.8–

30/3

13–3

33

2–22

[8

6,8

7]

Chi

lean

hop

H

umul

us lu

pulu

sL

eave

sA

lpha

-aci

dsC

H20

/313

6–

14[8

8]

Coc

aE

ryth

roxy

lum

coc

a L

am.

Lea

ves

Coc

aine

CO

17–2

2/31

3/M

eOH

+

H2O

0.17

–0.6

0[8

9]

Cop

aiba

Cop

aife

ra s

p.L

eave

sPh

enol

icc

ompo

unds

BR

-AM

10–2

5/32

3–33

30.

5–4

[90]

Gra

peVi

tis

vini

fera

Frui

tski

nR

esve

ratr

olA

R,C

H15

/313

/EtO

HR

ecov

ery

of

20–1

00%

of

resv

erat

rol

[91]

Gra

pefr

uit

Cit

rus

para

disi

L.

Peel

Nar

ingi

nA

R9.

5/33

1.8/

15%

EtO

HR

ecov

ery

of

14.4

%o

fna

ring

in

[92]

Gua

coM

ikan

ia g

lom

erat

aL

eave

sR

utin

BR

10/3

33/E

tOH

8.8

[76]

Gua

rana

Paul

lini

a cu

pana

Seed

sC

affe

ine

BR

-N10

–40/

313–

343

Rec

over

yof

98%

of

caf

fein

e[9

3,9

4]

Jack

frui

tA

rtoc

arpu

s he

tero

phyl

lus

Lea

ves

Isoq

uerc

etin

,vite

xin

LA

10/3

33/E

tOH

6[7

6]

Jala

peno

pep

per

orr

edp

eppe

rC

apsi

cum

ann

uum

L.

Frui

tC

apsa

icin

CH

32/3

13

14[9

5–98

]

Man

goM

angi

fera

indi

caL

eave

sPh

enol

icc

ompo

unds

SA25

/318

1[1

00]

Mar

igol

d(p

ot)

Cal

endu

la o

ffici

nali

sFl

ower

sFa

radi

ol-3

-O-l

aura

te,p

alm

itate

an

dm

yris

tate

LA

50/3

231.

2–2.

5[4

5,1

01]

7089_C008.indd 250 10/15/07 5:45:52 PM


Mat

eIl

ex p

arag

uari

ensi

s A

.St.-

Hil.

Lea

ves

Caf

fein

ean

dm

ethy

l-xa

nthi

nes

SA-S

40/3

432–

4[1

02]

Pass

ion

flow

ero

rm

aypo

psPa

ssifl

ora

inca

rnat

eL

eave

sQ

uerc

etin

,vite

xin

LA

10/3

33/E

tOH

9.2

[76]

Pink

trum

pett

ree

Tabe

buia

ave

llan

edae

Woo

dL

apac

hol

AR

,BR

9–20

R

ecov

ery

of

lapa

chol

0.

2–1.

9%

[103

]

Pita

nga

or

Suri

nam

che

rry

Eug

enia

uni

flora

Lea

ves

Isoq

uerc

etin

,rut

inL

A10

/333

/EtO

H10

.6[7

6]

Stev

iaSt

evia

reb

audi

ana

B.

Lea

ves

Dite

rpen

esg

lyco

side

s:s

tevi

osid

ean

dre

baud

iosi

deA

B

R,P

A

12–2

5/28

3–31

8/H

2O,

EtO

H0.

3–1.

2[5

3,5

4]

Tabe

rnae

mon

tana

Ta

bern

aem

onta

na

cath

arin

ensi

s

Bar

kan

dle

aves

Alk

aloi

ds:C

oron

arid

ine,

vo

acan

gine

,etc

.B

R-S

E20

–300

/308

–328

/E

tOH

,Iso

C3/

H2O

0.4–

15[1

05,1

06]

Tur

mer

icC

urcu

ma

long

a L

.R

oots

Cur

cum

inoi

dsa

ndte

rpen

oids

BR

-C20

–30/

303–

318/

EtO

H+

Iso

C3

5–22

[51,

10

7–11

1]

Vin

cao

rM

adag

asca

rpe

riw

inkl

e

Cat

hara

nthu

s ro

seus

Lea

ves

Isoq

uerc

etin

,rut

in,v

itexi

nL

A10

/333

/EtO

H15

.3[7

6]

LA

:L

atin

Am

eric

a;S

A:

Sout

hA

mer

ica;

SA

-S:

Sout

hof

Sou

th A

mer

ica;

BR

:B

razi

l;B

R-C

:C

entr

alr

egio

nof

Bra

zil;

BR

-NE

:N

orth

east

of

Bra

zil;

BR

-SE

:So

uthe

ast

of

Bra

zil;

AR

: Arg

entin

a;C

H:C

hile

;CO

:Col

ombi

a

7089_C008.indd 251 10/15/07 5:45:52 PM


cultivationplaces)wereindicated;thisinformationwasgatheredfromthepublica-tionsorthedatabasespreviouslymentioned.Plantswereclassifiedasproducersofvolatileoilsand/oroleoresins(Table8.1)[15–57],lipids,andlipid-solublesubstances(carotenoids, tocopherols, etc.) (Table8.2) [58–74]. Plants that produce miscella-neouscompoundssuchasphenoliccompounds,isoflavones,andsoonaregroupedinTable8.3[75–111].TheexperimentaldatainTable8.1toTable8.3wereobtainedfocusedintheSFEprocess,thus,yields,kineticbehavior,andchemicalcompositionoftheextracts(orthecontentofthetargetcomponent)and,infewcases,thebio-logicalactivities(antioxidant,anticancer,antimycobacterium)ofthevarioussystemsweredetermined.Someplants,inspiteoftheireconomicalimportancefortheLAcountries(suchastheorangeforBrazil)havereceivedlittleattention;nonetheless,aninterestingstudyonthefractionationoftheoily-fractionofconcentrated-frozenorangejuiceoilwasdonebyMarques[112].

Table8.4summarizesthephaseequilibriumdata[113–133]measuredforsomeofthesystemsinTable8.1toTable8.3;someentirelypredictivestudieshavealsobeenincluded[122,125–127].Phaseequilibriummeasurementandmodelingweremostlydoneforlipidsystems[5,118,132].Alkaloids+CO2phaseequilibriumdatawere measured for caffeine in CO2 and CO2 + cosolvent [115] and predicted forpurinealkaloids[130].PhaseequilibriumdataforartemisinininCO2weremeasuredandfittedtodensitybasedmodelandcubicequationofstate[113].Thephaseequi-libriumofquercetin+CO2+ethanolwasmeasuredandthedatawerefittedtogroupcontributionandequationofstate(EOS)models[131].ThesolubilitiesofoleoresincompoundssuchasboldineandcapsaicininCO2weremeasuredandthedatawerefitted todensity-basedmodels [114,117].ThephaseequilibriaofSFEextractsofclove and fennel in CO2 [119, 120, 133] showed liquid-vapor and liquid-liquid-vaporphasesplit;thephaseequilibriumofvetivergrassSFEextract+CO2showedliquid-vaporsplit.Thephaseequilibriaofcamphor+CO2,camphor+propane,andcamphor + CO2 + propane were measured and fitted to the Peng-Robinson EOS(PR-EOS)[116].Thesehighlyasymmetricalsystemsshowliquid-vaporphasesplitaswellasliquid-liquid-vaporthatwerequantitativelydescribedbythePR-EOS.Theexperimentalphaseequilibriumoflimoneneoxidationproducts+CO2wasalsowelldescribedbythePR-EOS[123,124].

BecauseoftheimportanceofSFEasananalyticaltool,Table8.5isgivenheretopresentsomeanalyticalapplicationsindevelopmentinLA[134–146];thesestudiesareconcentratedinBrazilandChile.

Otherapplicationsofsupercriticalfluids(SCFs)arerelatedtotheuseofcurcumi-noidstoimpregnatePolyethyleneTerephthlate(PET)films[156],tohydrolyzestarchymatrices such as ginger and turmeric [111, 157], to hydrolyze cellulosic matrices[99,104],andtofractionvolatileoilsthatcoupleSFEandmembranes[158].

8.2 extraCtIng BIoaCtIve Compounds BY sfe

SFEfromvegetablematricesiscomplexbynature.Therefore,hardlyanycharacter-isticofthesystemcanbedescribedbyasimplemodel.Nonetheless,averysimpli-fiedmodelcanbeextremelyusefulforCOMestimation.Inthissimplifiedmodel,thevegetablematerialdescribedhasbeenformedbyacellulosicstructure(CS)and

7089_C008.indd 252 10/15/07 5:45:53 PM


asolutemixture.TheCScontainsallinsolublematerials,includingproteins,carbo-hydrates, and salts, and is insoluble in the solvent but strongly interactswith thesolutemixture.Thesoluteisformedbyamulticomponentmixturecontainingcom-poundsfromavarietyofchemicalfunctionsfromlow-molecular-masssubstancessuch as, for instance, ethanol (this substance occurs naturally in orange oil fromcertain varieties cultivated in Brazil [112]), terpenoids, and high-molecular-masssubstances such as stevia glycosides [53, 54] and curcuminoids [51, 107–109]. Inthis definition, the presence of cosolvent modifies the composition of the solutemixtureaswellasthatoftheCS.Yet,thesolidmatrixcanbeviewedaspreviouslydescribedbyincorporatingintothesolutethesubstancesthatarenowsolubleduetothepresenceofthecosolvent.Analogously,intheCSphaseremainsonlythesolvent(SCF+cosolvent)insolublematerial.Therefore,intheextractorvessel,themixtureCS + solute + solvent can be treated as a pseudoternary system. The solute is a

taBle 8.4phase equilibrium (or solubility) for Bioactive Compounds at High pressures

pure Component or mixture solvent type of equilibrium/mpa/K reference

Artemisinin CO2 Solubility/10–25/308.2–328.2 [113]

Boldine(Hydro-alcoholicboldleafextracts)

CO2 Solubility/8–40/298–333 [114]

Caffeine CO2+EtOHandIsoC3 Solubility/15–30/323–343 [115]

Camphor CO2/propane LV/3.2–13.6/304–354 [116]

Capsaicin CO2 Solubility/6–40/298–318 [117]

Castoroilandtheirfattyacidethylesters

CO2 LV/1.7–25.4/313–343 [118]

CloveSFEextract CO2 LV,L1L2V/303–328/5.8–13.2 [119]

FennelSFEextract CO2 LV,L1L2V/4.7–22/303–333 [120]

Fishoil CO2 Solubility/14.7–29.4/301–323 [121]

L-dopa CO2 * [122]

Limoneoxidation CO2 LV/4.9–14/313–343 [123,124]

Orangepeeloil CO2 LV/4–11/313–333 [125–127]

Palmfattyaciddistillate CO2 Solubility/20–35/313–363 [128,129]

Purinealkaloids CO2 * [130]

Quercetin CO2+EtOH Solubility/8–12/313 [131]

Soybeanoilanditsfattyacidethylesters

CO2 LV/1.3–26.4/313–343 [118]

Triglycerides CO2 LV/3.3–14/278.3–368.5 [132]

VetivergrassSFEextract CO2 LV/7.7–30.8/303–333 [133]

EtOH:ethanol;IsoC3:isopropylalcohol;LV:Liquid-vaporequilibrium;L1L2V:Liquid-Liquid-Vaporequilibrium;Sol:Solubilitydata*Noexperimentaldataareavailable.

7089_C008.indd 253 10/15/07 5:45:54 PM


multicomponentmixturewhosenaturedependsonthevegetablematerialused.TheCSisalsoamulticomponentmixtureand,thus,apseudocomponentthatisentirelyinert to theactionof thesolvent(orsolventmixture);nonetheless, itdoesinteractwiththesolutemixture.Thesysteminaverysimplifiedconceptioncanbeconsid-eredasatwo-phasesystem.Thelight,orsolvent,phaseconstitutesofsolutemixture+solvent,andtheheavyphasecontainsthecellulosicstructure+solutemixture.

8.2.1 rElEvaNt procEss iNformatioN for com EstimatioN

ThehugeamountofdataonSFE fromseveraldifferent solidmatricespublished[5–8]indicatesthatSFEhasbeenproventobetechnicallyfeasibleforvirtuallyanysolidsubstratum.Nonetheless,inspiteoftherecentdevelopmentofnewindustrialplantsallovertheworld,LAhasnone.OneofthereasonsistherestraintimposedbythefixedcostofinvestmentofanSFEunit.Therefore,tofulfillthecurrentpursuitofcleantechnology,onemustshowinvestorsthatinadditiontobeingtechnicallyviable,SFEisindeedanattractivechoiceforanextractionprocess.Inordertodoso,wemustbuildabenchmarkfortheCOMofSFEtobecomparedwiththeCOMofconventionalprocesses.So,asimpleandyetreliablemethodtoestimateCOMforpreliminaryanalysisorbusinessplananalysisisneeded.Forthis,asimpleproce-durethatemploysminimumexperimentalinformationwouldbeadequate.RosaandMeireles[147]havedemonstratedsuchmethodologybasedonthemethoddescribedbyTurtonetal. [148]; theseauthorsestimatedCOMforclovebudoilandgingeroleoresin. In the next section, we describe in detail the required information toemploytheprocedureadoptedbyRosaandMeireles[147].

8.2.2 sElEctiNg paramEtErs iNtENdEd for com EstimatioN

For a business plan, that is, at the very early stages of process development, thefollowingquestionsmustbeansweredforeachvegetablematrix:

taBle 8.5analytical applications

substratum Bioactive Compound reference

Honey Pesticides [134]

Fish Mercury [135]

Lactealmatrices Fatsolublevitamins [136]

Organotin Organotin [137]

Sausages N-nitrosamines [138]

Soilsamples Butyltin [139]

Soilsamples Pesticides [140–142]

Vegetableoils Polycyclicaromatichydrocarbons [143]

Humanhair Cadmium [144]

Urine Chromium [145]

Urine Nitrofurantoin [145]

7089_C008.indd 254 10/15/07 5:45:55 PM


1.Whatisthebestprocessforobtainingthedesiredextract?(Atthispoint,thealternativeextractionprocessesshouldbeconsidered,includingSFE,low-pressure solvent extraction [LPSE]withavarietyof solvents, and steamdistillation[forvolatileoilonly].)

2.Foragivenextractwithspecifiedfunctionalproperties,wouldSFEbeagoodchoice?

3. IfSFEisanalternative,whatarethepressureandtemperatureofextraction? 4.Atthispressureandtemperature,whatistheprocessyield? 5.Howlongdoesittaketoobtainsuchyield?

Inordertoanswerthesequestions,twotypesofexperimentaldatamustbeavailable:

1.Globalyield,ortotalamountofsolublesubstancespresentinthevegetablematrixforagivenconditionoftemperatureandpressure.

2.SFEkineticsforthesystemunderconsideration.

To develop mass transfer and phase equilibrium models, several types ofinformationarerequired[36,61,97,149–151].Forinstance,thecharacteristicsofthe solidmatrix, suchashumidity,contentof solublematerial, structure,particlesize,anddistribution,are required forevaluationof themass transferparametersfromdifferentmodels.Tochoosea thermodynamicmodelsuitable fordescribingphaseequilibrium,experimentaldatamustbeavailable.Theseparametersmustbemeasured,estimated,orboth,preferablyusingstandardprocedures.Nonetheless,forthebusinessplan,theneededinformationisless.Forinstance,withtheknowledgeofthebedapparentdensity,therequiredbedvolumeforagivenproductioncanbeestimated.Or,ifextractorsofagivenvolumeareavailable,itispossibleusingthebedapparentdensitytocalculatetherawmaterialdemandtobeprocessed.Addingtothissimpleinformation,theglobalyieldandthetimeforanextractioncycleisenoughforCOMestimation[147].

Additionally, the compositionof the extract in termsof itsmajor compoundsandonefunctionalpropertywouldhelpthedecisionmakers.Toobtaintherequiredinformation for process design, identification of the solute mixture is necessary;therefore, the chemical composition of SFE extracts must be determined byappropriate methods, such as gas chromatography with flame ionization detector(GC-FID), Gas Chromatography-Mass Spectrometry (GC-MS), high-performanceliquidchromatography,orultraviolet spectrophotometry.Forextracts thatwillbeusedasnutraceuticals,biologicalactivitymustalsobemonitored.ThiscanbedoneusingasimpletechniquetoaccesstheantioxidantactivityoftheSFEextract.OnesuchmethodisthatofHammerschmidtandPrat[152],whichhasbeenadaptedtobeusedforSFEextracts[51].AnotherkeyissueistheoptimizationoftheseparationstepthatcanbesimulatedusingasimplecubicEOS,suchasthePR-EOS.

8.2.2.1 pressure and temperature processes

Theselectionoftheprocessconditions,suchastemperature,pressure,solventflowrate, cosolvent (if required), solid matrix preparation, and so on, is required forprocessoptimization.Nonetheless,beforeselectingtheparametersrelatedtomass

7089_C008.indd 255 10/15/07 5:45:56 PM


transfer(i.e.,tothekineticsthatwillultimatelybeusedforprocessoptimization),itisinterestingtochoosethepressureandtemperatureofextraction.Thiscanbedoneconsidering the thermodynamicsof the system aswell as the composition of theextractatagivenconditionoftemperatureandpressure.Inordertoselectthepres-sureandtemperatureofprocess,thephaseequilibriumorsolubilityofthesystemsolute+SCF,thesolubilityofthepseudoternarysystemCS+solute+SF,ortheglobalyieldcanbeused.Atthispoint,itisimportanttorememberthatinspiteoftheCSbeinginerttothesolvent,itstronglyinteractswiththesolute;therefore,theinteractionofthesolutewiththesolvent,aswellaswiththeCS,mustbeconsidered.Thus, thesolubilityof thesolute insolventmeasured in thepseudobinarysystemformedbysoluteandsolvent,andthesolubilityofthesolutemeasuredforthesolidmatrix-solvent system will be quite different. In the second case, as reported byBrunner[4],thesolubilitycanbeanorderofmagnitudesmallerthanthesolubilitymeasured for thefirst case.As theCSstrongly interactswith the solutemixture,itcan be expected that the various compounds that form the solute mixture willhavedifferentaffinitiesfortheCS;therefore,thechoiceofprocesstemperatureandpressurewillbebetterdoneconsideringparametersmeasuredforthepseudoternarysystem.Thephaseequilibriumofseveralpseudobinarysystems(solute/solvent)hasbeensystematicallyreportedinliterature[5];thesedataareveryhelpfulinoptimiz-ingtheSFEseparationstep.Otherauthorshavemeasuredthesolubility(Y*)ofthepseudoternarysystemCS+solute+SFusing thedynamicmethod.AsdiscussedbyRodriguesetal.[27],toobtaincorrectvaluesofY*wouldrequireatediousandcostlyworktodeterminethesolventflowrate thatcanbesafelyusedtomeasurethisparameter.Thesolubilityhasalsobeenreportedbyseveralauthorsastheinitialslopeofanoverallextractioncurve(OEC)intermsoftotalyieldasafunctionoftheratioofsolventmass(S)tothefeedmass(F);thisparameterisreferredasY*S/F.ThedifferencebetweenY*andY*S/Fcanbeunderstoodbyrecallingthatinordertomea-sureY*trueequilibriumforthepseudoternarysystemisexpectedtobeobtainedwhileY*S/Fismeasuredatagivenratioofsolventmasstofeedmass.TheresultsofMouraetal.[34]forfennel+CO2haveshownthatY*S/Fcanbeafunctionofthefixedbedgeometry,i.e.,theratioofthebedheight(HB)tothebeddiameter(DB).TheseauthorsobtainedincreasingvaluesofY*S/FastheratioHB/DBincreasedfrom2.21to8.84.Ontheotherhand,similarexperimentsdonebyCarvalhoetal.[52]forrosemary+CO2showedthatY*S/FvariedinanarrowrangeasHB/DBincreasedfrom0.67to8.4.Evenso,asreportedbyMouraetal.[34]andCarvalho[52],theseresultsweredependentonthesolventflowrate.Moreprecisely,theinterstitialvelocityinthefixedbedplaysanimportantroleintheprocess,thusaffectingthemeasurementofY*S/F.Additionally,toobtainreliableresultsofeitherY*orY*S/F,theexperimentalassaysmustbeperformedinSFEunitscontainingextractorvesselswithvolumesofatleast50mL,sinceanOECmustbebuilt.Alternatively,thechoiceoftheoperatingtemperatureandpressurecanbedoneconsideringtheglobalyieldsisotherms(GYI)asreportedbyRodriguesetal.[154]andMouraetal.[34],amongothers.Thetotalorglobalyield(Xo)canbeobtainedthroughoutexhaustiveextractioninaSFEunit.ThereisnoneedtobuildanOEC;therefore,extractorvesselsofsmallvolumes(VE<50mL)andsmallamountsoffeedarerequired.Thisisaveryconvenientchoicewhenonlysmallamountsofthevegetablematerialareavailable,whichisoftenthe

7089_C008.indd 256 10/15/07 5:45:57 PM


caseforspontaneouscrops.Atrickquestioniswhatshouldbetheratioofsolventmass tofeedmass?Excesssolventmustbeusedinorder toobtainthetruevalueofXo.Consideringthatthetotalyieldshouldbeanintensivepropertyasdiscussedelsewhere[43,154],itshoulddependonlyontemperatureandpressure,therefore,itisenoughtoestablishasuitablevaluefortheratioS/Ftomeasuretheyieldinsuchawayastoguarantyitsusabilityforselectingtheoperatingpressureandtempera-ture.SincetheglobalyieldwasdeterminedataselectedratioofS/FitshouldbedenotedasXo,S/F.Basedonourresults[100],valuesofS/Fgreaterthan15aregoodenoughfortheselectionoftheoperatingtemperatureandpressure.Consideringthatan extraction experiment to build an OEC for some vegetable matrices can takeseveralhourswhileglobalyieldsassaysareshorter,theusageoftheGYItoselecttheoperatingtemperatureandpressureinsteadcansavehoursofexperimentalwork.IfOECsareavailableandglobalyieldinformationismissing,thentheglobalyieldcanbeestimatedusingthesplinefittingoftheOEC,aswillbediscussednext.

8.2.2.2 the Kinetic parameters

Fortheproductionofessentialoils,oleoresins,vegetableoilsfromexoticvegetableseeds,sweeteners,andsoon,ingeneral,amultipurposeplantcontainingatleasttwofixed-bedextractorswillbeemployed.Atthelaboratorylevel,theanalysisofsuchaprocesscanbedoneconsidering theOEC.Theeffectsof theprocessvariablespressure, temperature, and solvent flow rate on the total yield as well as on thechemicalprofileoftheextractarenoteasilyseenfromtheOEC.Therefore,forfirstapproximations,itwouldbeinterestingtoestablishasimpleproceduretoanalyzetheeffectsoftheprocessvariables.Afterward,theconditionscanbeoptimizedcon-sideringtheglobalprocess.

AnOECisobtainedconsideringtheamountextracted(massofextractoryield)asafunctionoftime.TheinformationprovidedbyanOECisthetimerequiredforanextractionbatch.AtypicalOECcanbedescribedbythreesteps:

1.Aconstantextractionrateperiod(CER) 2.Afallingextractionrateperiod(FER),whichrepresentsthestepforwhich

bothconvectionanddiffusioninthesolidsubstratumcontrolstheprocess 3.Adiffusion-controlledrateperiod(DC)

PriortoSFE,thesolidsubstratumrequirespreprocessingthatatleastincludescomminution.Inordertoavoidchanneling,theparticlesizeusedinSFEwilldependon the ratio of bed diameter to particle diameter, which has been reported to bebetween50and250[5].BecauseofthestronginteractionbetweenwaterandCS,solute dehydration is required if the water content is more than 20%, wet basis.Therefore, thepreprocessingof the solid substratumpromotes the ruptureofcellwalls;thus,thesolidmatrixsubjectedtoSFEwillcontainrupturedaswellasunrup-turedcells.Evenso,forcertainsolidmatrices,theseveretreatmentsufferedduringthe pretreatment results in about 100% of ruptured cells, as for instance, for therecoveryofcarotenoidsfrompressedpalmoilfibers[63,64].However,forspecialsolidmatricesforwhichthesoluteislocatedverysuperficially,nocomminutionis

7089_C008.indd 257 10/15/07 5:45:58 PM


needed.Indeed,Silva[17]hasshownthatextractionofbixinandnorbixinfromuru-cum(Bixa orellana)seedsismoreeffectiveusingwholeseedsthanmilledseeds.

TheCERperiod is characterizedby theextractionof the solutecontained inthesurfaceofthesolidsubstratumparticlesorincellsthatwerebrokenduringpre-processing.Sovová [149]called thesolute removedduring theCERperiodeasily accessible solute. Themasstransferintheexternalfilmneartheparticle’ssurfaceiscontrolledbyconvection.TheCERperiodischaracterizedbythefollowingkineticparameters: (1) themass transfer rate (MCER), (2) thedurationof theCERperiod(tCER),(3)theyieldduringtheCERperiod(RCER),and(4)massratioofsoluteinthefluidphaseattheextractorvesseloutlet(YCER).About70%toasmuchas90%ofthesolublematerialcanbeextractedfromthesubstratumduringtheCERperiodifcarefulpretreatmentisused[20,26].IntheFERperiod,aconsiderableportionofthesolidparticlesisnolongercoatedwithsoluteorthenumberofbrokencellsisnolongeruniform.Thus,themasstransferratediminishesasaresultofthedecreaseintheeffectivemasstransferareaaswellastheincreaseinimportanceofthedif-fusionalmechanism.IntheDCperiod,thesolutecoatingofthesolidparticleshasbeencompletelyremovedand,thus,theextractionprocessiscontrolledbythediffu-sionofthesolventtotheinnerpartsoftheparticlesfollowedbythediffusionofthesolute-solventmixturetothesurfaceoftheparticles.

8.2.2.2.1 Describing the OEC by a SplineAnOECcanbedescribedbyafamilyofstraightlines.Themassofextract(ortheyield)canbeobtainedfromthefollowingequations.

ForNlines:

m b C b b tExt i i

i

i N

i

i

i N

= −

++=

=

=

=

∑ ∑0 1

1 1

(8.1)

Fortwostraightlines:

m b C b b b tExt = −( ) + +( )0 1 2 1 2 (8.2)

Forthreestraightlines:

m b C b c b b b b tExt = − −( ) + + +( )0 1 2 3 3 1 2 3 (8.3)

wherebifori=0,1,2arethelinearcoefficientsoflines1,2…andCifori=1,2aretheinterceptsoftheselines(forinstance,C1istheinterceptofthefirstandsecondlines,andC2istheinterceptofthesecondandthirdlines),mExtisthemassofextract(ortheyield),andtistime.

Figure8.1 shows that two straight lines can quantitatively describe the OECforginger+CO2 [36],whereasFigure8.2shows that three linesare required forchamomile+CO2[24].ThefirstlinerepresentstheentireCERplusthebeginningoftheFERperiod.Theslopeofthelinerepresentsthemass-transferrateoftheCERperiod,MCER.Thetimecorrespondingtotheinterceptionofthetwolinesisdenoted

7089_C008.indd 258 10/15/07 5:46:01 PM


0.00

0.50

1.00

1.50

2.00

2.50

0 60 120 180 240

tCER

300 360 420

Extraction Time/min.

Yiel

d/%

fIgure 8.1 OECforginger+CO2:20MPa,308K,5.91×10–5kg/s[36].

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

0 100 200 300 400

tFERtCER

500 600Extraction Time/min.

Yiel

d/%

fIgure 8.2 OECforchamomile+CO2:20MPa,313K,6.67×10–5kg/s[24].(FromPovh,N.P.,Marques,M.O.M.andMeireles,M.A.A.,J. Supercrit. Fluids, 21,245,2001.Withpermission.)

7089_C008.indd 259 10/15/07 5:46:04 PM


bytCERandroughlyrepresentstheminimumtimeaSFEcycleshouldlast.Themassratioofsoluteinthesupercriticalphaseatthebedoutlet(YCER)isobtainedbydivid-ingMCERby themeansolventflowrate for theCERperiod.Theyieldrelative totheCERperiodisRCER,orminimumyieldexpectedfromSFEprocessatagiventemperature,pressure,solventflowrate,andsolidsubstratumpreprocessing.IftheGYIismissingbutanOECisavailable,thentheglobalyieldcanbeestimatedusingEquation8.1orEquation8.2bycalculatingthemExtatt=3tCER;thisapproximationwasusedbyPovhetal.[24]forchamomile+CO2.

ThedatafittingcanbeperformedusingthesplinemethodofFreudandLittle[153]andSAS6.12software.Oncethisisestablished,thekineticparametersshouldbeassociatedwithaphenomenologicalmodeltodescribetheOEC.

Forcertainsolidsubstrataassociatedwithunusuallylowsolventflowrates,theOECwillshowalag-phasebeforethesystemreachesthepseudosteadystate;thistimeintervalwillbeidentifiedbytLAGandiscalculatedfromthesplinemodelbysettingthemassofextractequaltozero(mExt=0)(Figure8.3).

8.2.3 thE com for latiN amEricaN plaNts

Table8.6 shows the COM for selectedLA plants. COM was estimated using theprocedureofRosaandMeireles[147]thatappliesthemodelofTurtonetal.[148],inwhichCOMiscalculatedasasumofdirectcosts(DMC),fixedcosts(FMC),andgeneralexpenses(GE):

COM DMC FMC GE= + + (8.4)

DMC C C C C FCI COMRW WT UT OL= + + + + +1 33 0 069 0 03. . . (8.5)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

0 60 120 180 240 300Extraction Time/min

Yiel

d/%

tLAG tCER

fIgure 8.3 OECforginger+CO2:20MPa,313K,1.60×10–5kg/s[36].

7089_C008.indd 260 10/15/07 5:46:08 PM


taB

le 8

.6C

om

for

sfe

extr

acts

from

sel

ecte

d pl

ants

raw

mat

eria

lB

otan

ic n

ame

targ

et C

ompo

nent

sfe

Con

diti

ons

mpa

/K/C

osol

vent

Yiel

d(%

)*t

ext(m

in.)

Co

m

(us

$/kg

)r

efer

ence

Ani

seP

impi

nell

a an

isum

Ane

thol

e10

/303

7.9

100

21.2

1[1

60]

Bra

zilia

ngi

nsen

gP

faffi

a gl

omer

ata

Ecd

yste

rone

20/3

030.

614

01,

648.

00[8

3]

Clo

veE

ugen

ia c

aryo

phyl

lus

Vol

atile

oil

10/2

8812

.970

9.18

[147

]

13.5

909.

88

14.1

120

10.9

7

Fenn

elFo

enic

ulum

vul

gare

Ane

thol

e25

/303

12.5

808.

81[1

60]

Gin

ger

Zin

gibe

r of

ficin

alis

Ole

ores

in20

/313

2.7

150

99.8

0[1

47]

Ros

emar

yR

osm

arin

us o

ffici

nali

sV

olat

ileo

il30

/313

510

042

.69

[160

]

Tabe

rnae

mon

tana

Tabe

rnae

mon

tana

cat

hari

nens

isC

oron

arid

ine

and

voac

angi

ne35

/308

–318

/EtO

H1.

490

440.

31[1

61]

*tE

xt:e

xtra

ctio

ntim

e

7089_C008.indd 261 10/15/07 5:46:08 PM


FMC C FCIOL= +0 708 0 168. . (8.6)

GE C FCI COMOL= + +0 177 0 009 0 16. . . (8.7)

whereCRWisthecostrawmaterial,CWTisthecostofwastetreatment,COListhecostofoperationallabor,andFCIisafractionoftheinvestment.

COMestimationwasdoneusingthesoftwareTecanalysisv.2.0developedinLASEFI–DEA/FEA-UNICAMP.Incalculatingthecost,theyieldsandthemini-mumtimeoftheSFEcycleswereestimatedasdiscussedinSection8.2.2.2.1.TheSFEunitchosenasthebenchmarkcontainstwoextractorvesselsof400L(approx-imate cost U.S. $2 million). The total annual operating time was assumed to be7920h,whichcorrespondsto330daysperyearbasedona24-hshift.ThecostofoperationallaborwasestimatedtobeU.S.$3.00/h.TheSFEunitismultipurpose;therefore,itshouldbeoperatingtheassumed7920hperyearregardlessoftherawmaterialused.Thecostofwastetreatmentwasassumedtoequalzero.Table8.7andTable8.8showtheTecanalysisreportfortheinputdataandresults,respectively,forCOMestimationofclovevolatileoil.Theextractiontimes(tExt)wereassumedequalor1.10to1.90tCER.

8.3 ConClusIons

SFEcanbeatruealternativeforobtaininghighqualityextractsfromseveralLAplants.ComparingtheCOMestimatedwiththesellingprices,itisclearthatthereisabusinessopportunity.

referenCes

1. Meireles,M.A.A.,Wang,G.-M.,Hao,Z.-B.,Shima,K.andTeixeiradaSilva,J.,Stevia(SteviarebaudianaBertoni):Futuristicviewofthesweetersideoflife,inFloriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues, TeixeiradaSilva,J.,Ed.,GSB,2006,chap.46,pp.415–425.

2. Canela,A.P.R.F.,Rosa,P.T.V.,Marques,M.O.M.andMeireles,M.A.A.,SupercriticalfluidextractionoffattyacidsandcarotenoidsfromthemicroalgaeSpirulina maxima, Ind. Eng. Chem. Res., 41,3012–3018,2002.

3. Valderrama,J.O.,Perrut,M.andMajewski,W.,Extractionofastaxantineandphyco-cyaninefrommicroalgaewithsupercriticalcarbondioxide,J. Chem. Eng. Data, 48,827–830,2003.

4. Brunner,G.,Gas Extraction: An Introduction to Fundamentals of Supercritical Fluids and the Application to Separation Processes, Springer,NewYork,1994.

5. Meireles,M.A.A.,Supercriticalextractionfromsolid:Processdesigndata(2001–2003),Curr. Opin. Solid St. M. Sci., 7,321–330,2003.

6. Rosa,P.T.V.andMeireles,M.A.A.,SupercriticaltechnologyinBrazil:Systeminvesti-gated(1994–2003),J. Supercrit. Fluids, 34,109–117,2005.

7. Diáz-Reinoso, B., Moure, A., Dominguéz, H. and Parajoá, J.C., Supercritical CO2extraction and purification of compounds with antioxidant activity, J. Agric. Food Chem.,54,2441–2469,2006.

7089_C008.indd 262 10/15/07 5:46:11 PM


taBle 8.7technical-economical analysis report: Input data

raw material: Clove Buds

Initial Investment

Price(US$):SFEunitwithtwoextractors 2,000,000.00

Extractorvolume(m³) 0.40

Totalannualoperationtime(h) 7920

Operationlaborcost(US$/h) 3

Rawmaterialcost(US$/MT) 505

Initialhumidity(%) 10

Finalhumidity(%) 10

Grindinganddryingcost(US$/MT) 30

CO2cost(US$/kg) 0.1

LossofCO2(%oftotalusedinacycle) 2

Electricalpowercost(US$/Mcal) 0.0703

Coolingwatercost(US$/Mcal): 0.0837

Saturatedsteam(5barg)Cost(US$/Mcal) 0.0133

Depreciation(%/Year) 10

Seafreightcost(US$/MT⋅km) 0.01

Seafreightdistance(km) 0

Totalroadfreightcost(US$/MT⋅km) 0

operational data

Extractiontime(min) 120

Extractiontemperature(°C) 15

Extractionpressure(MPa) 10

Flashtankpressure(MPa) 4

CO2flowrate(kg/h) 90

Beddensity(kg/m³) 520

scale-up model

Yield(kgextract/kgfeed) 0.1414

Waste treatment Cost

Solidwaste(US$) 0

Liquidwaste(US$) 0

Gaswaste(US$) 0

7089_C008.indd 263 10/15/07 5:46:12 PM


taBle 8.8technical-economical analysis report: results

raw material: Clove Buds

fraction of Investment

Totalinvestment(US$)-IT 2,000,000.00

Columnvolume(m³)-Cv 0.40

operational labor Cost

equipment Hmo/Hop total Hmo (h) Cost (us$)

Extractor 1 7920 23,760.00

Flashdistillation 0.1 792 2,376.00

Condenser 0.1 792 2,376.00

CO2tank 0.5 3960 11,880.00

Pump 0.05 396 1,188.00

Heatexchanger 0.1 792 2,376.00

Total 43,956.00

Hmo/Hop:Man-laborhoursperequipmentperhourofoperationofthesystem;Hop:Annualoperatinghoursoftheequipment

raw material Cost

Solidmattercost(US$) 415,958.40

CO2usedinprocess(kg) 712,800.00

LossofCO2(%) 2.00

CO2-specificcost(US$/kg) 0.10

CO2cost(US$) 1,425.60

Preprocessingcost(US$) 24,710.40

Seacargocost(US$) 0.00

Roadcargocost(US$) 0.00

Rawmaterialcost(US$) 442,094.40

utility Cost

equipment energy (mcal) specific

Cost (us$/mCal) Cost (us$)

Flashdistillation 33,854.79 0.0133 450.27

Condenser –36,385.97 0.0837 3,045.51

Pump 1,013.96 0.0703 71.28

Heatexchanger 1,922.80 0.0133 25.57

Total 3,592.63

Waste treatment Cost

Solidwaste(US$) 0.00

Liquidwaste(US$) 0.00

7089_C008.indd 264 10/15/07 5:46:12 PM


8. delValle,J.M.,delaFuente,J.C.andCardarelli,D.A.,ContributionstosupercriticalextractionofvegetablesubstratesinLatinAmerica,J. Food Eng., 67,35–57,2005.

9. U.S. Department of Agriculture Natural Resources Conservation Service, PlantDatabase,http://plants.usda.gov/, accessedJune2006.

10. Raintree Nutrition, Rainforest Plant Database, http://www.rain-tree.com/, accessedJune2006.

11. MissouriBotanicalGarden,w3TROPICOS,http://mobot.mobot.org/W3T/Search/vast.html,accessedJune2006.

12. SearchableWorldWideWebMultilingualMultiscriptPlantNameDatabase(M.M.P.N.D.)http://www.plantnames.unimelb.edu.au/Sorting/Frontpage.html,accessedJuly2006.

13. CenterforNewCropsandPlantProducts,CropINDEX,PurdueUniversity,http://www.hort.purdue.edu/newcrop/morton/pejibaye.html,accessedJuly2006.

14. Florabrasiliensis,http://florabrasiliensis.cria.org.br/,accessedJune2006. 15. Daghero,J.etal.,FractionationofSchinus molle L.(aguaribay)essentialoilwithsuper-

critical CO2, in V Brazillian Meeting on Supercritical Fluids, Ferreira, S.R.S., Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

16. Nobre, B.P. et al., Supercritical carbon dioxide extraction of pigments from Bixa orellanaseeds(experimentsandmodeling),Braz. J. Chem. Eng., 23,251–258,2006.

17. Silva,G.F.,ExtraçãodecorantesdourucumcomdióxidodedarbonoSupercrítico,PhDthesis,StateUniversityofCampinas,Campinas,Brazil,1999.

18. Martinez,J.etal.,ExtractionofvolatileoilfromPiper aduncum L. leaveswithsuper-criticalcarbondioxide,inProceedings of the Sixth International Symposium on Super-critical Fluids,Brunner,G.,Kikic,I.andPerrut,M.,Eds.,ISASF,Versailles,France,65–70,2003.

taBle 8.8 (continued)technical-economical analysis report: results

Waste treatment Cost (continued)

Gaswaste(US$) 0.00

Total(US$) 0.00

Cost of manufacturing

variable value (us$) value in Com (us$) % of Com

Investment(US$)-IT 2,000,000.00 607,407.40 47.53

Rawmaterial(US$)-CRM

442,094.40 545,795.55 42.71

Operationlabor(US$)-COL

43,956.00 120,200.67 9.41

Utilities(US$)-CUT 3,592.63 4,435.34 0.35

Wastetreatment(US$)-CWT

0.00 0.00 0.00

Costofmanufacturing(US$)-COM

1,277,838.95

Massofextract(kg) 116,468.30

Specificcost(US$/kg) 10.97

7089_C008.indd 265 10/15/07 5:46:13 PM


19. Leal, P.F. et al., Global yields, chemical compositions, and antioxidant activities ofclovebasil(Ocimum gratissimum L.)extractsobtainedbysupercriticalfluidextraction,J. Food Proc. Eng., 29,547–559,2006.

20. Ferreira,S.R.S.,Meireles,M.A.A.andCabral,F.A.,Extractionofessentialoilofblackpepperwithliquidcarbondioxide,J. Food. Eng., 20,121–133,1993.

21. Ferreira,S.R.S.etal.,Supercriticalfluidextractionofblackpepper(Piper nigrum L.)essentialoil,J. Supercrit. Fluids, 14,235–245,1999.

22. Perakis,C.,Louli,V.,andMagoulas,K.,Supercriticalfluidextractionofblackpepperoil,J. Food Eng., 71,386–393,2005.

23. Braga,M.E.M.,Ehlert,P.A.D.,Ming,L.C.andMeireles,M.A.A.,SupercriticalfluidextractionfromLippia alba: Globalyields,kineticdata,andextractchemicalcomposi-tion,J. Supercrit. Fluids, 34,149–156,2005.

24. Povh,N.P.,Marques,M.O.M.andMeireles,M.A.A.,SupercriticalCO2extractionofessential oil and oleoresin from chamomile (Chamomilla recutita [L.] Rauschert),J. Supercrit. Fluids,21,245–256,2001.

25. Muller, C., et al., Supercritical extraction of essential oils of Cymbopogon martini, Cymbopogon winterianus, and Eycalyptus citriodora with carbon dioxide, Chem.PreprintArchiveVolume2001, Issue5,May2001,pp.158–177.Availableathttp://www.sciencedirect.com/preprintarchive

26. Meireles,M.A.A.andNikolov,Z.L.,Extractionandfractionationofessentialoilswithliquid carbon dioxide, in Spices, Herbs and Edible Fungi, Charalambous, G., Ed.,ElsevierScience,Amsterdam,1994,chap.6,pp.171–199.

27. Rodrigues,V.M.etal.,DeterminationofthesolubilityofextractsfromvegetablerawmaterialinpressurizedCO2:Apseudo-ternarymixtureformedbycellulosicstructure+solute+solvent,J. Supercrit. Fluids, 22,21–36,2002.

28. Oliveira,A.L.,Eberlin,M.N.andCabral,F.A.,Aromaticoil fromBrazilianroastedcoffeeextractedbysupercriticalcarbondioxide:CompositionanalysisbyGC/MS,inV Brazillian Meeting on Supercritical Fluids,Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

29. Yepez, B., Espinosa, M. and Bolaños, G., Producing antioxidant fractions fromherbaceousmatricesbysupercriticalfluidextraction,Fluid Phase Equilibria, 194–197,879–884,2002.

30. Kraut,S.,Braga,M.E.M.andMeireles,M.A.A.,Extractionofcoriander(Coriandrum sativum L.) leavesoilbySFE:Totalphenoliccontent, inProceedings of the Eighth Conference on Supercritical Fluids Application, Reverchon,E.,Ed., ISASF, Ischia,Italy,2006,143–146.

31. Sousa,E.M.B.D.etal.,Extractionofvolatileoil fromCroton zehntneri PaxetHoffwithpressurizedCO2:Solubility,compositionandkinetics,J. Food Eng., 69,325–333,2005.

32. Quispe-Condori, S., Rosa, P.T.V., Foglio, M.A. and Meireles, M.A.A., Supercriticalextractionof essential oil fromCordia curassavica (Jacq.)Roemer andSchultes, inProceedings of the Sixth International Symposium on Supercritical Fluids, Brunner,G.,Kikic,I.andPerrut,M.,Eds.ISASF,Versailles,France,2003,285–290.

33. Sousa,E.M.B.D.,Construçãoeutilizaçãodeumdispositivodeextraçãocomfluidopressurizado, aplicado a produtos naturais, Ph.D. thesis, Federal University of RioGrandedoNorte(UFRN),Natal,Brazil.

34. Moura, L.S. et al., Supercritical fluid extraction from fennel (Foeniculum vulgare):Globalyield,compositionandkineticdata,J. Supercrit. Fluids, 35,212–219,2005.

35. Martinez,J.etal.,Multicomponentmodel todescribeextractionofgingeroleoresinwithsupercriticalcarbondioxide,Ind. Eng. Chem. Res., 42,1057–1063,2003.

7089_C008.indd 266 10/15/07 5:46:14 PM


36. Monteiro,A.R.,Extraçãodoóleoessencial/oleoresinadegengibre(Zingiber officinale Roscoe) com CO2 supercrítico: uma avaliação do pré-tratamento e das variáveis deprocesso,Ph.D.thesis,StateUniversityofCampinas,Campinas,Brazil,1999.

37. Zancan,K.C.,Marques,M.O.M.,Petenate,A.J.andMeireles,M.A.A.,Extractionofginger(Zingiber officinale Roscoe)oleoresinwithCO2andco-solvents:Astudyoftheantioxidantactionoftheextracts,J. Supercrit. Fluids, 24,57–76,2002.

38. Quispe-Condori, S., Determinação de parâmetros de processo nas diferentes etapasdoprocessode extração supercríticadeprodutosnaturais:Artemisia annua,Cordiaverbenacea, Ocimum selloi e Foeniculum vulgare, Ph.D. thesis, State University ofCampinas,Campinas,Brazil,2005.

39. Michielin,E.M.Z.etal.,Compositionprofileofhorsetail (Equisetum giganteum L.)oleoresin:ComparingSFEandorganic solvents extraction,J. Supercrit. Fluids, 33,131–138,2005.

40. Portillo,R.,Extraçãodoóleoessencialdekhoa(Satureja bolivianaBenthBriq)pordiferentesprocessos:destilaçãoporarrasteavapor,solventesorgânicosedióxidodecarbonopressurizado,Ph.D.thesis,StateUniversityofCampinas,Campinas,Brazil,1999.

41. Pereira, C.G. and Meireles, M.A.A., Evaluation of global yield, composition, anti-oxidant activity, cost of manufacturing of extracts from lemon verbena (Aloysia triphylla L’HeritBritton)andmango(Mangifera indica L.)leaves,J. Food Proc. Eng., 30,150–173,2007.

42. Ferrua,F.Q.,Marques,M.O.M.andMeireles,M.A.A.,Óleoessencialdecapim-limãoobtido por extração com dióxido de carbono líquido, Ciênc. Tecnol. Aliment., 14,83–92,1994.

43. Sousa,E.M.B.D. et al.,Experimental results for the extractionof essential oil fromLippia sidoides Cham. using pressurized carbon dioxide. Braz. J. Chem. Eng., 19,229–241,2002.

44. Leal, P.F. et al., Global yields, chemical compositions, and antioxidant activities ofextractsfromAchyrocline alata andAchyrocline satureioides,Phcog. Mag., 2,153–159,2006.

45. Campos, L.M.A.S., Michielin, E.M.Z., Danielski, L. and Ferreira, S.R.S., Experi-mental data and modeling the supercritical fluid extraction of marigold (Calendula officinalis)oleoresin,J. Supercrit. Fluids, 34,163–170,2005.

46. Chacin, J.,Marquina-Chidsey,G.andFigueroa,Y.,Extractionofmastranto (Hyptis suaveolens)essentialoilusingsupercriticalcarbondioxide, inV Braz. Meet. Super-critical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

47. Berna,A.,Tarrega,A.,Blasco,M. andSubirats,S.,SupercriticalCO2 extractionofessentialoilfromorangepeel:Effectoftheheightofthebed,J. Supercrit. Fluids, 18,227–237,2000.

48. Fernandes,J.B.etal.,CitrusseedoilextractionsandtheiractivityagainstleafcuttingantAttasexdensanditssymbioticfungus,Quim. Nova, 25,1091–1095,2002.

49. Rodrigues,M.R.A.etal.,ChemicalcompositionandextractionyieldoftheextractofOriganum vulgare obtainedfromsub-andsupercriticalCO2,J. Agri. Food Chem.,52,3042–3047,2004.

50. Costa, T.S., Corrêa, N.C.F., Machado, N.T. and França, L.F., Extração de óleosessenciaisdevetiver(Vetiveria zizaniodes)epiprioca(Cyperus sesquiflorus),V Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

51. Leal,P.F.etal.,Functionalpropertiesofspiceextractsobtainedviasupercriticalfluidextraction,J. Agri. Food Chem., 51,2520–2525,2003.

7089_C008.indd 267 10/15/07 5:46:15 PM


52. Carvalho, R.N., Moura, L.S., Rosa, P.T.V. and Meireles, M.A.A., Supercritical fluidextractionfromrosemary(Rosmarinus officinalis):Kineticdata,extract’sglobalyield,composition,andantioxidantactivity,J. Supercrit. Fluids, 35,197–204,2005.

53. Yoda,S.K.,Marques,M.O.M.,Petenate,A.J.andMeireles,M.A.A.,SupercriticalfluidextractionfromStevia rebaudiana BertoniusingCO2andCO2pluswater:Extractionkineticsandidentificationofextractedcomponents,J. Food Eng., 57,125–134,2003.

54. Pasquel, A., Meireles, M.A.A., Marques, M.O.M. and Petenate, A.J., Extraction ofsteviaglycosideswithCO2+water,CO2+ethanol,andCO2+water+ethanol,Braz. J. Chem. Eng., 17,271–282,2000.

55. Martinez,J.etal.,ValorizationofBrazilianvetiver(Vetiveria zizanioides (L.)NashexSmall)oil,J. Agri. Food Chem., 52,6578–6584,2004.

56. Talansier,E.etal.,Supercriticalfluidextractionfromvetiver roots:AstudyofSFEkinetics, 10th European Meeting on Supercritical Fluids, Perrut, M., Ed., ISASF,Colmar,France,2005,CD-ROM.

57. Stashenko, E.E., Jaramillo, B.E. and Martinez, J.R., Analysis of volatile secondarymetabolitesfromColombianXylopia aromatica (Lamarck)bydifferentextractionandheadspacemethodsandgaschromatography,J. Chromatog. A, 1025,105–113,2004.

58. Monteiro, A.R.,Meireles,M.A.A.,Marques,M.O.M. andPetenate,A.J.,Extractionofthesolublematerialfromtheshellsofthebacurifruit(Platonia insignisMart)withpressurizedCO2andothersolvents,J. Supercrit. Fluids, 11,91–102,1997.

59. França,L.F.etal.,Supercriticalextractionofcarotenoidsandlipidsfromburiti(Mauritia flexuosa),afruitfromtheAmazonregion,J. Supercrit. Fluids, 14,247–256,1999.

60. Azevedo,A.B.A.,Kopcak,U.andMohamed,R.S.,Extractionoffatfromfermentedcupuaçuseedswithsupercriticalsolvents,J. Supercrit. Fluids, 27,223–237,2003.

61. Tobares, L. et al., Chemical characteristics of jojoba wax obtained by supercriticalfluidextraction, inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

62. del Valle, J.M. et al., Microstructural effects on internal mass transfer of lipids inprepressedandflakedvegetablesubstrates,J. Supercrit. Fluids, 37,178–190,2006.

63. França, L.F. and Meireles, M.A.A., Extraction of oil from pressed palm oil (Elaes guineensis)fibersusingsupercriticalCO2,Ciênc. Tecnol. Aliment., 17,384–388,1997.

64. Franca,L.F.andMeireles,M.A.A.,Modelingtheextractionofcaroteneandlipidsfrompressedpalmoil(Elaes guineensis)fibersusingsupercriticalCO2,J. Supercrit. Fluids, 18,35–47,2000.

65. deDeus,G.A.,Corrêa,N.C.F.,Machado,N.T.andFrança,L.F.,Effectofthesolventflowrateonthesupercriticalextractionoflipidsandvitaminsfrompressedpalmoil(Elaes guineensis)fibers,inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

66. Corrêa,N.C.F.,Araújo,M.E.,Machado,N.T.andFrança,L.F.,Supercriticalfluidextrac-tionofpalmkerneloilfromdendê(Elaeis guineensis)withCO2,inENPROMER’99–II Congresso de Engenharia de Processos do MERCOSUL, CD-ROM.

67. Ambrogi,A.,Cardarelli,D.A.andEggers,R.,Separationofnaturalcolorantsusinga combined high pressure extraction-adsorption process, Lat. Am. App. Res., 33, 3,323–326,2003.

68. Corrêa,N.C.F.,Meireles,M.A.A.,França,L.F.andAraújo,M.E.,Extraçãodeóleodasementedemaracujá(Passiflora edulis)comCO2supercrítico,Ciênc. Tecnol. Aliment., 14,29–37,1994.

69. Araújo,M.E.,Machado,N.T.,França,L.F.andMeireles,M.A.A.,Supercriticalextrac-tionofpupunha (Guilielma speciosa)oil inafixedbedusingcarbondioxide,Braz. J. Chem. Eng., 17,297–306,2000.

7089_C008.indd 268 10/15/07 5:46:16 PM


70. Pasquel,A.,Castillo,A.andSotero,V.,OilextractionfromshellofBactris gasipaes HBKfruitsusingcompressedcarbondioxide,inProceedings of the Sixth International Symposium on Supercritical Fluids,Brunner,G.,Kikic,I.andPerrut,M.,Eds.,ISASF,Versailles,France,2003,77–82.

71. Sarmento,C.M.P.,Ferreira,S.R.S.andHense,H.,Supercriticalfluidextraction(SFE)ofricebranoil toobtainfractionsenrichedwithtocopherolsandtocotrienols,Braz. J. Chem. Eng., 23,2,243–249,2006.

72. Danielski,L.,Zetzl,C.,Hense,H.andBrunner,G.,Aprocesslinefortheproductionofraffinatedriceoilfromricebran,J. Supercrit. Fluids, 34,133–141,2005.

73. Christensen,T.,Machado,N.T.,França,L.F.andBrunner,G.,Extraçãodevitaminase lipídeosdo tucumã(Astrocaryum vulgare,Mart.)emleitofixousandoCO2super-crítico,inProceedings of the 13th Brazilian Congress of Chemical Engineering, ÁguasdeSãoPedro,Brazil,2000,CD-ROM.

74. Morais,J.L.C.,Machado,N.T.andBayma,J.C.,Extractionoftrimirystinfromucuúba(Virola surinamensis,Miristicaceae)infixedbedwithsupercriticalCO2,inProceedings of the Fifth International Symposium on Supercritical Fluids, ISSF,Atlanta,2000.

75. Santana, L.L.B. et al., Selectivity in the extraction of 2-quinolones alkaloids withsupercriticalCO2,inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

76. Takeuchi,T.M.etal.,inProceedings of the Eighth Conference on Supercritical Fluids Application, Reverchon,E.,Ed.,ISASF,Ischia,Italy,2006,147–150.

77. Cardoso, L.A. et al., Preliminary study of Schinus therebinthifolius extraction bysupercriticalcarbondioxide, inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

78. Cardoso, L.A. et al., Supercritical extraction of isoflavones and monoterpenoid ofPoiretiabahiana,inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

79. Quispe-Condori,S.etal.,GlobalyieldisothermsandkineticofartemisininextractionfromArtemisia annua Lleavesusingsupercriticalcarbondioxide,J. Supercrit. Fluids, 36,40–48,2005.

80. Cassel,E.etal.,ExtractionofbaccharisoilbysupercriticalCO2,Ind. Eng. Chem. Res., 39,4803–4805,2000.

81. Pansera,M.R.etal.,ExtractionoftanninbyAcacia mearnsii withsupercriticalfluids,Braz. Arch. Biol. Technol., 47,995–998,2004.

82. Leal,P.F.,Maia,N.B.,Carmello,Q.A.C.andMeireles,M.A.A.,Supercriticalextrac-tionfromOcimum basilicum usingCO2andCO2+H2O:Globalyieldsandantioxidantactivityof the extracts, inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

83. Leal,P.F.,Alexandre,F.C.,Kfouri,M.B.andMeireles,M.A.A.,inProceedings of the Eighth Conference on Supercritical Fluids Application, Reverchon,E.,Ed., ISASF,Ischia,Italy,2006,157–160.

84. delValle,J.M.,Rogalinski,T.,Zetzl,C.andBrunner,G.,Extractionofboldo(Peumus boldusM.)leaveswithsupercriticalCO2andhotpressurizedwater,Food Res. Int., 38,203–213,2005.

85. Saldaña,M.D.A.,Mohamed,R.S.andMazzafera,P.,ExtractionofcocoabutterfromBrazilian cocoa beans using supercritical CO2 and ethane, Fluid Phase Equilibria, 194–197,885–894,2002.

86. Patel,R.N.,Bandyopadhyay,S. andGanesh,A.,Extractionof cashew (Anacardium occidentale)nutshell liquidusingsupercriticalcarbondioxide,Bioresour. Technol., 97,847–853,2006.

87. Smith, R.L., Jr. et al., Separation of cashew (Anacardium occidentale L.) nut shellliquidwithsupercriticalcarbondioxide,Bioresour. Technol.,88,1–7,2003.

7089_C008.indd 269 10/15/07 5:46:18 PM


88. delValle,J.M.,Rivera,O.,Teuber,O.andPalma,M.T.,SupercriticalCO2extractionofChileanhop(Humulus lupulus)ecotypes,J. Sci. Food Agric., 83,1349–1356,2003.

89. Brachet,A.etal.,Experimentaldesigninsupercriticalfluidextractionofcocainefromcocaleaves,J. Biochem. Biophys. Methods, 43,353–366,2000.

90. Carvalho,R.N.,Jr.,Braga,M.E.M.andMeireles,M.A.A.,Determinationoftheglobalyieldsisothermsforthesystemcopaiba(Copaiferasp.)+CO2,inProceedings of the Eighth Conference on Supercritical Fluids Application, Reverchon,E.,Ed., ISASF,Ischia,Italy,2006,589–592.

91. Pascual-Martý, M.C., Salvador, A., Chafer, A. and Berna, A., Supercritical fluidextractionofresveratrolfromgrapeskinofVitis vinifera anddeterminationbyHPLC,Talanta, 54,735–740,2001.

92. Giannuzzo, A.N., Boggetti, H.J., Nazareno, M.A. and Mishima, H.T., Supercriticalfluidextractionofnaringin from thepeelofCitrusparadise,Phytochem. Anal., 14,221–223,2003.

93. Saldaña,M.D.A.,Zetzl,C.,Mohamed,R.S. andBrunner,G.,Extractionofmethyl-xanthinesfromguaranáseeds,mateleaves,andcocoabeansusingsupercriticalcarbondioxideandethanol,J. Agr. Food Chem.,50,4820–4826,2002.

94. Saldaña,M.D.A.,Zetzl,C.,Mohamed,R.S.andBrunner,G.,DecaffeinationofguaranáseedsinamicroextractioncolumnusingwatersaturatedCO2,J. Supercrit. Fluids, 22,119–127,2002.

95. delValle,J.M.etal.,Supercriticalcarbondioxideextractionofpelletizedjalapenopep-pers,J. Sci. Food Agri., 83,550–556,2003.

96. delValle,J.M.,Jimenez,M.anddelaFuente,J.C.,Extractionkineticsofpre-pelletizedjalapenopepperswithsupercriticalCO2,J. Supercrit. Fluids, 25,33–44,2003.

97. Uquiche, E., del Valle, J.M. and Ihl, M., Microstructure-extractability relationshipsintheextractionofprepelletizedjalapenopepperswithsupercriticalcarbondioxide,J. Food Sci., 70,E379–E386,2005.

98. Uquiche,E.,delValle,J.M.andOrtiz,J.,Supercriticalcarbondioxideextractionofredpepper(Capsicum annuum L.)oleoresin.J. Food Eng., 65,55–66,2004.

99. Pasquini,D.,Pimenta,M.T.B.,Ferreira,L.H.andCurvelo,A.A.D.,ExtractionofligninfromsugarcanebagasseandPinustaedawoodchipsusingethanol-watermixturesandcarbondioxideathighpressures,J. Supercrit. Fluids, 36,31–39,2005.

100. Pereira, C.G., Obtenção de extratos de leiteira de dois irmãos (Tabernaemontana catharinensisA.DC.),cidrão(Aloysia triphylla L’Herit.Britton)emanga(Mangifera indica L.)porextraçãosupercrítica:Estudodosparâmetrosdeprocesso,caracterizaçãoe atividade antioxidante dos extratos. PhD thesis, State University of Campinas,Campinas,Brazil,2005.

101. Hamburger,M.etal.,Preparativepurificationofthemajoranti-inflammatorytriterpe-noidestersfrommarigold(Calendula officinalis),Fitoterapia,74,328–338,2003.

102. Saldana,M.D.A.,Zetzl,C.,Mohamed,R.S. andBrunner,G.,Extractionofmethyl-xanthinesfromguaranáseeds,matéleaves,andcocoabeansusingsupercriticalcarbondioxideandethanol,J. Agric. Food Chem., 50,4820–4826,2002.

103. Viana,L.M.,Freitas,M.R.,Rodrigues,S.V.andBaumann,W.,Extractionoflapacholfrom Tabebuia avellanedae wood with supercritical CO2: An alternative to Soxhletextraction?Braz. J. Chem. Eng., 20,317–325,2003.

104. Pasquini,D.,Pimenta,M.T.B.,Ferreira,L.H.andCurvelo,A.A.S.,SugarcanebagassepulpingusingsupercriticalCO2associatedwithco-solvent1-butanol/water,J. Super-crit. Fluids, 34,125–131,2005.

105. Pereira,C.G.etal.,ExtractionofindolealkaloidsfromTabernaemontana catharinensis usingsupercriticalCO2+ethanol:Anevaluationoftheprocessvariablesandtherawmaterialorigin, J. Supercrit. Fluids, 30,51–61,2004.

7089_C008.indd 270 10/15/07 5:46:19 PM


106. Pereira,C.G.,Leal,P.F.,Sato,D.N.andMeireles,M.A.A.,Antioxidantandantimyco-bacterial activities of Tabernaemontana catharinensis extracts obtained by super-criticalCO2+cosolvent.J. Med. Food, 8,533–538,2005.

107. Chassagnez, A.L.M., Corrêa, N.C. and Meireles, M.A.A., Extração de oleoresinadecúrcuma (Curcuma longa L.)comCO2 supercrítico,Ciênc. Tecnol. Aliment., 17,399–404,1997.

108. Chassagnez-Méndez, A.L. et al., Supercritical CO2 extraction of curcuminoids andessentialoilfromrhizomesofturmeric(Curcuma longa L.),Ind. Eng. Chem. Res., 39,4729–4733,2000.

109. Braga,M.E.M.,Leal,P.F.,Carvalho,J.E.andMeireles,M.A.A.,Comparisonofyield,composition,andantioxidantactivityofturmeric(Curcuma longa L.)extractsobtainedusingvarioustechniques,J. Agric. Food Chem., 51,6604–6611,2003.

110. Braga,M.E.M.,Moreschi,S.R.M.andMeireles,M.A.A.,Effectsofsupercriticalfluidextraction on Curcuma longa L. and Zingiber officinale R. starches, Carbohydrate Polymers, 63,340–346,2006.

111. Moreschi, S.R.M., Leal, J.C., Braga, M.E.M. and Meireles, M.A.A., Ginger andturmericstarcheshydrolysisusingsubcriticalwater+CO2:TheeffectoftheSFEpre-treatment,Braz. J. Chem. Eng., 23,235–242,2006.

112. Marques, D.S., Desterpenação de Óleo Essencial de Laranja por CromatografiaPreparativadeFluidoSupercrítico,MScthesis,StateUniversityofCampinas,Campinas,Brazil,1996.

113. Coimbra,P.etal.,Experimentaldeterminationandcorrelationofartemisinin’ssolubil-ityinsupercriticalcarbondioxide,J. Chem.Eng. Data, 51,1097–1104,2006.

114. delaFuente,J.C.,Quezada,N.anddelValle,J.M.,Solubilityofboldoleafantioxidantcomponents(Boldine)inhigh-pressurecarbondioxide,Fluid Phase Equilibria, 235,196–200,2005.

115. Kopcak,U.andMohamed,R.S.,Caffeinesolubility insupercriticalcarbondioxide/co-solventmixtures,J. Supercrit. Fluids, 34,209–214,2005.

116. Carvalho,R.N., Jr.,Corazza,M.L.,Cardozo-Filho,L. andMeireles,M.A.A.,Phaseequilibriumfor(camphor+CO2),(camphor+propane),and(camphor+CO2+propane),J. Chem. Eng. Data, 51,997–1000,2006.

117. de laFuente, J.C.,Valderrama, J.O.,Bottini,S.B.anddelValle, J.M.,Measurementandmodelingofsolubilitiesofcapsaicininhigh-pressureCO2,J. Supercrit. Fluids, 34,195–201,2005.

118. Ndiaye,P.M.etal.,Phasebehaviorofsoybeanoil,castoroilandtheirfattyacidethylestersincarbondioxideathighpressures,J. Supercrit. Fluids, 37,29–37,2006.

119. Souza, A.T. et al., Phase equilibrium measurements for the system clove (Eugenia caryophyllus)oil+CO2.J. Chem. Eng. Data, 49,352–356,2004.

120. Moura,L.S.,Corazza,M.L.,Cardozo-Filho,L.andMeireles,M.A.A.,Phaseequilib-riummeasurementsforthesystemfennel(Foeniculum vulgare)extract+CO2.J. Chem. Eng. Data, 50,1657–1661,2005.

121. Corrêa, A.P.A., Gonçalves, L.A.G. and Cabral, F.A., Fractionation of fish oil withsupercriticalcarbondioxide, inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

122. VieiradeMelo,S.A.B.,Melo,R.L.F.V.,Costa,G.M.N.andAlves,T.L.M.,SolubilityofL-dopainsupercriticalcarbondioxide:Predictionusingacubicequationofstate,J. Supercrit. Fluids, 34,231–236,2005.

123. Corazza,M.L.,Cardozo,L.,Antunes,O.A.C.andDariva,C.,Phasebehaviorof thereaction medium of limonene oxidation in supercritical carbon dioxide, Ind. Eng. Chem. Res., 42,3150–3155,2003.

7089_C008.indd 271 10/15/07 5:46:20 PM


124. Corazza, M.L., Filho, L.C., Antunes, O.A.C. and Dariva, C., High pressure phaseequilibria of the related substances in the limonene oxidation in supercritical CO2,J. Chem. Eng. Data, 48,354–358,2003.

125. Diaz,M.S.,Espinosa,S.andBrignole,E.A.,Optimal solventcycledesign in super-criticalfluidprocesses,Lat. Am. Appl. Res., 33,161–165,2003.

126. Diaz, S., Espinosa, S. and Brignole, E.A., Citrus peel oil deterpenation with super-criticalfluids:Optimalprocessandsolventcycledesign,J. Supercrit. Fluids, 35,49–61,2005.

127. Espinosa, S., Diaz, M.S. and Brignole, E.A., Process optimization for supercriticalconcentrationoforangepeeloil,Latin Am. Appl. Res., 35,321–326,2005.

128. Saito,A.M.,Peixoto,C.A.andCabral,F.A.,Phaseequilibriamodellingofpalmfattyaciddistillate(PFAD)andsupercriticalcarbondioxide,inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

129. Peixoto,C.A.,França,L.F.andCabral,F.A.,Phaseequilibriaofpalmfattyaciddistil-late(PFAD)andsupercriticalcarbondioxide,inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

130. Favero, F.W. and Skaf, M.S., Solvation of purine alkaloids in supercritical CO2 bymoleculardynamicssimulations,J. Supercrit. Fluids, 34,237–241,2005.

131. Chafer,A.,Fornari,T.,Berna,A.andStateva,R.P.,Solubilityofquercetininsuper-criticalCO2plusethanolasamodifier:Measurementsandthermodynamicmodelling,J. Supercrit. Fluids, 32,89–96,2004.

132. Florusse, L.J., Fornari, I., Bottini, S.B. and Peters, C.J., Phase behavior of carbondioxide–low-molecularweighttriglyceridesbinarysystems:Measurementsandthermo-dynamicmodeling.J. Supercrit. Fluids, 31,123–132,2004.

133. Favareto,R. et al.,Supercriticalfluid extraction fromvetiver (Vetiveria zizanioides)roots: Kinetics and phase equilibrium, in Proc. VII Iberoamerican Conference on Phase Equilibria and Fluid Properties for Process Design - EQUIFASE, Galicia-Luna,L.A.,Ed.,Morelia,Mexico,2006,CD-ROM.

134. Rissato, S.R., Galhiane, M.S., Knoll, F.R.N. and Apon, B.M., Supercritical fluidextractionforpesticidemultiresidueanalysisinhoney:Determinationbygaschroma-tographywithelectron-captureandmass spectrometrydetection,J. Chromotogr. A, 1048,153–159,2004.

135. Grinberg, P., Campos, R.C., Mester, Z. and Sturgeon, R.E., Solid phase micro-extractioncapillarygaschromatographycombinedwithfurnaceatomizationplasmaemissionspectrometryforspeciationofmercuryinfishtissues,Spectrochim. Acta B, 58,427–441,2003.

136. Paixao,J.A.andCampos,J.M.,Determinationoffatsolublevitaminsbyreversed-phaseHPLCcoupledwithUVdetection:Aguidetotheexplanationofintrinsicvariability,J. Liq. Chromatogr., 26,641–663,2003.

137. Godoi,A.F.L.,Favoreto,R.andSantiago-Silva,M.,Environmentalcontaminationfororganotincompounds,Quim. Nova,26,708–716,2003.

138. Andrade,R.,Reyes,F.G.R.andRath,S.,AmethodforthedeterminationofvolatileN-nitrosaminesinfoodbyHS-SPME-GC-TEA,Food Chem.,91,173–179,2005.

139. Godoi, A.F.L., Montone, R.C. and Santiago-Silva, M., Determination of butyltincompoundsinsurfacesedimentsfromtheSaoPauloStatecoast(Brazil)bygaschroma-tography-pulsedflamephotometricdetection,J. Chromatogr. A, 985,205–210,2003.

140. Rissato,S.R.,Galhiane,M.S.,Apon,B.M.andArruda,M.S.P.,Multiresidueanalysisofpesticidesinsoilbysupercriticalfluidextraction/gaschromatographywithelectron-capturedetectionandconfirmationbygaschromatography-massspectrometry,J. Agr. Food Chem., 53,62–69,2005.

7089_C008.indd 272 10/15/07 5:46:21 PM


141. Rissato,S.R.,Galhiane,M.S.,deSouza,A.G.andApon,B.M.,Developmentofasuper-criticalfluidextractionmethodforsimultaneousdeterminationoforganophosphorus,organohalogen,organonitrogenandpyretroidspesticidesinfruitandvegetablesanditscomparisonwithaconventionalmethodbyGCECDandGCMS,J. Braz. Chem. Soc., 16,1038–1047,2005.

142. Richter,P.etal.,Screeninganddeterminationofpesticidesinsoilusingcontinuoussub-criticalwaterextractionandgaschromatography-massspectrometry,J. Chromatogr. A, 994,169–177,2003.

143. Zougagh,M.,Redigolo,H.,Rios,A.andValcarcel,M.,ScreeningandconfirmationofPAHsinvegetableoilsamplesbyuseofsupercriticalfluidextractioninconjunctionwith liquid chromatography and fluorimetric detection, Anal. Chimica Acta, 525,265–271,2004.

144. Arancibia,V.etal.,Quantitativeextractionofsulfonamidesinmeatsbysupercriticalmethanol-modifiedcarbondioxide:Aforayintoreal-worldsampling,J. Sep. Sci., 26,1710–1716,2003.

145. Arancibia,V.,Valderrama,M.,Silva,K.andTapia,T.,Determinationofchromiuminurinesamplesbycomplexation-supercriticalfluidextractionandliquidorgaschroma-tography,J. Chromatogr. B, 785,303–309,2003.

146. Arancibia,V.etal.,Extractionofnitrofurantoinanditstoxicmetabolitefromurinebysupercriticalfluids.QuantitationbyhighperformanceliquidchromatographywithUVdetection,Talanta, 61,377–383,2003.

147. Rosa, P.T.V. and Meireles, M.A.A., Rapid estimation of the manufacturing cost ofextractsobtainedbysupercriticalfluidextraction,J. Food Eng., 67,235–240,2005.

148. Turton,R.C.Bailie,Whiting,W.B.andShaeiwitz,J.A.,Analysis, Synthesis, and Design of Chemical Process, PrenticeHall,UpperSaddleRiver,1998.

149. Sovová,H.,RateofthevegetableoilextractionwithsupercriticalCO2I.Modelingofextractioncurves.Chem. Eng. Sci., 49,409–414,1994.

150. delValle,J.M.anddelaFuente,J.C.,SupercriticalCO2extractionofoilseeds:Reviewofkineticandequilibriummodels,Crit. Rev. Food Sci. Nutrit., 46,131–160,2006.

151. Ruetsch, L., Daghero, J. and Mattea, M., Supercritical extraction of solid matrices.Modelformulationandexperiments,Latin Ame. Appl. Res., 33,103–107,2003.

152. Hammerschmidt,P.A.andPratt,D.E.,Phenolicantioxidantsofdriedsoybeans,J. Food Sci., 43,556–559,1978.

153. Freud,R.J.andLittle,R.C.,SAS System for Regression, SAS Series in Statistical Appli-cations, 2nd Ed.,SASInstitute,Cary,NC,1995,211.

154. Rodrigues,V.M.etal.,Supercriticalextractionofessentialoilfromaniseed(Pimpinella anisum L)usingCO2:Solubility,kinetics,andcompositiondata,J. Agric. Food Chem., 51,1518–1523,2003.

155. Germain,J.C.,delValle,J.M.anddelaFuente,J.C.,Naturalconvectionretardssuper-criticalCO2extractionofessentialoilsandlipidsfromvegetablesubstrates,Ind. Eng. Chem. Res., 44,2879–2886,2005.

156. Herek, L.C.S., Oliveira, R.C., Rubira, A.F. and Pinheiro, N., Impregnation of PETfilmsandPHBgranuleswithcurcumininsupercriticalCO2,Braz. J. Chem. Eng., 23,227–234,2006.

157. Moreschi,S.R.M.,Petenate,A.J.andMeireles,M.A.A.,Hydrolysisofgingerbagassestarch in subcritical water and carbon dioxide, J. Agr. Food Chem., 52, 1753–1758,2004.

158. Castelan,L.H.etal.,Extractionoflemongrassessentialoilwithdensecarbondioxide,J. Supercrit. Fluids, 21,33–39,2001.

7089_C008.indd 273 10/15/07 5:46:21 PM


159. Pokrywiecki, J.C. et al., Separation of active principles from the essential oil ofmedicinalplantswithsupercriticalcarbondioxideandreverseosmosismembrane,inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.

160. Pereira,C.G.andMeireles,M.A.A.,Manufacturingcostofessentialoilsobtainedbysupercriticalfluidextraction,inProceedings of the Eighth Conference on Supercritical Fluids Application, Reverchon,E.,Ed.,ISASF,Ischia,Italy,2006,77–82.

161. Pereira,C.G.,Rosa,P.T.V. andMeireles,M.A.A.,Extraction and isolationof indolealkaloids from Tabernaemontana catharinensis A.DC: Technical and economicalanalysis,J. Supercrit. Fluids, 40,232–238,2006.

7089_C008.indd 274 10/15/07 5:46:22 PM

275

9 Antioxidant Extraction by Supercritical Fluids

Beatriz Díaz-Reinoso, Andrés Moure, Herminia Domínguez, and Juan Carlos Parajó

Contents

9.1 Introduction................................................................................................. 2759.2 TypesofAntioxidantsandRegulationAspects.......................................... 2769.3 NaturalAntioxidantsandSources.............................................................. 277

9.3.1 PhenolicCompounds.......................................................................2809.3.2 Terpenoids........................................................................................2809.3.3 Carotenoids...................................................................................... 2819.3.4 VitaminE......................................................................................... 2819.3.5 OtherNaturalAntioxidants.............................................................282

9.4 BiologicalPropertiesofAntioxidantCompounds......................................2829.4.1 PhenolicCompounds.......................................................................2829.4.2 Terpenoids........................................................................................ 2839.4.3 Carotenoids......................................................................................2849.4.4 VitaminE.........................................................................................2849.4.5 AntioxidantPropertiesofSC-CO2Extracts....................................284

9.5 DeterminationofAntioxidantActivity.......................................................2859.6 Supercritical-CO2ExtractionofAntioxidants............................................286

9.6.1 ProcessingSchemes.........................................................................2879.6.2 EffectsoftheMostInfluentialOperationalVariables.....................288

9.6.2.1 PressureandTemperature..................................................2899.6.2.2 Modifier..............................................................................290

9.6.3 SC-CO2ExtractsversusConventionalSolventExtracts.................292References.............................................................................................................. 293

9.1 IntroduCtIon

According toawidelyuseddefinition,anantioxidant isanysubstance that,whenpresentatlowerconcentrationsthanthoseofanoxidizablesubstrate(suchaslipids,proteins,deoxyribonucleicacid[DNA]orcarbohydrates),significantlydelaysorpre-ventsoxidationof thatsubstrate [1,2].Neither thisdefinitionnorotherdefinitions[3]restrictantioxidantactivitytoaspecificgroupofcompoundsortoanyparticularmechanismofaction.Naturalantioxidantsplayadecisiveroleindifferentsystems:i)inplants,theyactasprotectingagentsagainstradiationormicrobialinfections,ii)in

7089_C009.indd 275 10/8/07 12:13:02 PM


foods,theydelayorinhibittheformationoftoxiclipidoxidationproducts,maintain-ingnutritionalqualityandincreasingshelflife,andiii)inbiologicalsystems,alongwithendogenousdefenses(enzymes,vitamins,proteins,andothers),dietaryantioxi-dantsmayhelppreventorslowtheoxidativestressinducedbyfreeradicals[4].Sinceconsiderableevidenceindicatesthatoxidativedamagemaycontributetothedevel-opmentofage-relatedanddegenerativediseases,theprotectiveeffectsofbeneficialcompoundshavebeenascribedtotheirantioxidantactivity,althoughmanyantioxi-dantsin vivoprobablyactbyothermechanismsthanin vitro assaysorareunlikelytohavesucheffectsattheconcentrationsavailableinplasma[5,6].

Duetoanincreasingconsumerdemandtoreplacecontroversialsyntheticanti-oxidants,suchasbutylatedhydroxytoluene(BHT),butylatedhydroxyanisole(BHA),tertiary butyl hydroquinone (TBHQ), and gallates, the preservation of foods is apromisingapplicationofnaturalantioxidants,whichcouldconferadditionalbiologi-calactivitiestotheproducts.Althoughnaturalantioxidantsareassumedtobesafeandinnocuous,theirlackoftoxicityshouldbeconfirmed.

Greateffortisbeingdevotedtothesearchforalternativeandcheapsourcesofnaturalantioxidants,aswellastothedevelopmentofefficientandselectiveextrac-tiontechniques.Extractionwithconventionalsolvents issometimescharacterizedbypoorselectivityandrequireshightemperatures,whichcouldresultindegradationofthedesiredcompounds.Supercriticalfluidextraction(SFE)ismoreselectivethanconventionalextractionandisoptimalwhenproductsfreefromresidualsolventsarerequired (for example, for food, cosmetic, and pharmaceutical purposes). Carbondioxide(CO2)isthemostsuitedsolventforSFEofthermolabilecompounds,owingto its nontoxic andnonflammable character andhigh availability at lowcost andhighpurity, allowinganoptimal reproductionof thephysicochemical,biological,and therapeutic properties of the target compounds. Supercritical CO2 (SC-CO2)extracts are regarded as “natural”; are free from pathogenic and spoilage micro-organisms,spores,andenzymes;theabsenceoflightandoxygenpreventsoxidationreactions.FuturedevelopmentsinextractionofantioxidantswillprobablyberelatedtoSFE[7],whichiswellpositionedwithrespecttoincreasinglyrestrictiveenviron-mental,toxicological,andhealthregulations.

Theoretical and practical aspects of the SFE of compounds with recognizedantioxidantactivityhavebeenrevised[8–11]andparticularizedfortheextractionofantioxidants[12–14].

9.2 types of AntIoxIdAnts And regulAtIon AspeCts

Lipid oxidation is important in food deterioration—for example when oxygenreactswith lipids ina seriesof free radicalchain reactions [15]or in theoxida-tivemodificationoflow-densitylipoproteins(LDLs).Accordingtothefreeradicaltheoryofaging,variousoxidativereactionsoccurringintheorganism(mainlyinmitochondria)generatefreeradicalsasby-products,whichdamagenucleicacids,proteins,andlipidsandresultinagingandage-associatedpathologies.Thestagesoftheclassicalnonenzymaticfreeradical–mediatedchainreactionsare:1)initiation(byheat,light,ionizingradiation,metalions,ormetalloproteins),2)propagation,

7089_C009.indd 276 10/8/07 12:13:03 PM

Antioxidant Extraction by Supercritical Fluids 277

3)branching, and 4)termination. The main features of the mechanisms of lipidoxidationandantioxidantactionhavebeendetailedintheliterature[3,4,15–19].

Antioxidants have traditionally been divided into two groups: primary andsecondary. Primary antioxidants (such as phenolic compounds or vitamin E) aredestroyedduringtheinductionperiod,whentheydelayorinhibittheinitiationstepbyreactingwithradicals.Secondary,orpreventative,antioxidantsslowtheoxida-tionrate,removingsubstratebybindingoxygenfromair,complexingwithtransitionmetalions(acetates,citrates,tartrates,andphosphates),quenchingsingletoxygen,bindingcertainproteinswithprooxidanteffects,absorbingultraviolet(UV)radia-tion or (in the case of phospholipids) creating a protective layer between oil andairsurface.Antioxidantscanactaccordingtoseveralmechanisms,andsynergismamongdifferentoxidationinhibitorscanoccur[15,17].

Othernonmechanisticclassificationshavebeenestablishedforantioxidants.Forexample,accordingtotheirorigin,theycanbeclassifiedasnaturalproducts,naturalidentical(α-tocopherol),orartificial.Dependingontheirchemicalstructure,anti-oxidantshavebeengroupedintophenolics(BHA,BHT,TBHQ,gallates),quinones(hydroquinone,tocopherols,hydroxychromanes,hydroxycoumarins),organicacids(ascorbic, citric, tartaric, and lactic acids and their salts and ethylenediaminetetraacetic acid and its salts), sulfur compounds (inorganic: sulfites, bisulfites, andmetasulfites;organic:methionine,cisteine),andenzymes(catalases,peroxidases,superoxidedismutase).Thenatural antioxidants foundwithinbiological systemsinclude four general groups: enzymes, large molecules (albumin, ceruloplasmin,ferritin, other proteins), small molecules (ascorbic acid, glutathione, uric acid,tocopherol,carotenoids,polyphenols),andsomehormones(estrogen,angiotensin,melatonin)[20].

Technological requirements for food antioxidants include low volatility andstability (to avoid losses during processing and storage), ability to protect fromoxidation at low concentrations, solubility and compatibility with other compo-nentsoftheoxidizablesubstrate,nontoxicandnonirritantcharacterattheeffectiveconcentration, and ability to not confer color, odor, or taste to the final product.Foodutilizationof syntheticantioxidants suchasBHT,BHA,andgallates isnotpermitted in theEuropeanUnion (EU) for some special foods, suchas those forinfants and young children [21, 22], and it is generally restricted to levels thatdependon theconsideredapplication.However,antioxidants fromnaturalorigins(suchasspices)donotneedtobedeclaredandareallowedathigherdoses[12,17].Table9.1summarizesdataonthemajorfoodantioxidantsaccordingtotheEUandU.S.regulations,aswellasmaximumlevelsofacceptabledailyintake(ADI)estab-lishedbytheCodex Alimentarius.

9.3 nAturAl AntIoxIdAnts And sourCes

ThemoststudiedantioxidantsextractablefromvegetalbiomassbySFEwithCO2arephenolics,terpenoids,carotenoids,andtocopherols.Astheavailabledatashowacomparativelyhigherantioxidantactivityforphenolics,amoredetaileddiscussionisprovidedforthesecompounds.

7089_C009.indd 277 10/8/07 12:13:03 PM


tAb

le 9

.1M

ajor

foo

d A

ntio

xida

nts

and

thei

r M

axim

um l

evel

in d

iffer

ent

prod

ucts

Acc

ordi

ng t

o eu

rope

an a

nd u

.s. r

egul

atio

ns

nam

eA

dI

(mg/

kg)

eu r

egu

lAtI

on

u.s

. reg

ulA

tIo

n

food

Max

imum

lev

elst

anda

rdiz

ed f

ood

Max

imum

lev

el

(mg/

kg)

Toco

pher

ols

(Toc

)0.

15–0

.2Q

uant

um s

atis

Ani

mal

fato

rani

mal

and

ve

geta

ble

fatm

ixtu

re30

0

Toco

pher

olri

ch–e

xtra

ct,

α-to

coph

erol

,γ-

toco

pher

ol,

β-to

coph

erol

Non

emul

sifie

doi

lsa

ndf

at(

exce

pt

virg

ino

ilsa

ndo

live

oils

)Q

uant

um s

atis

Saus

ages

and

rel

ated

pro

duct

s,

mea

tpro

duct

s30

0

Refi

ned

live

oil,

exce

pto

live

pom

ace

oil

200

mg/

l(α-

toc)

Froz

enr

awb

read

eds

hrim

p20

0

Non

emul

sifie

doi

lsa

ndf

at(

exce

pt

virg

ino

ilsa

ndo

live

oils

)Q

uant

um s

atis

Bac

on(

pum

p-cu

red)

Poul

try

and

poul

try

prod

ucts

500

(α-t

ocop

hero

l)30

0(2

00+

oth

ers

exce

ptT

BH

Q)

BH

TB

HA

Gal

late

s(G

’s)

0.12

50.

5Fa

ts a

ndo

ils (

for

heat

trea

ted

food

stuf

fs)

200

mg/

kg(

G’s

and

BH

A

sing

lyo

rco

mbi

ned)

Deh

ydra

ted

pota

tos

hred

s50

(B

HA

,BH

T)

Act

ive

dry

yeas

t10

00(

BH

A)

Dry

mix

esf

orb

ever

ages

and

de

sser

ts2

(BH

A)

-Pr

opyl

gal

late

(PG

)2.

5O

ilan

dfa

tto

fry

(exc

epto

live

pom

ace

oil)

;lar

d;fi

sho

il;b

eef,

po

ultr

yan

dsh

eep

fat

100

mg/

kg (B

HT

)D

ryb

reak

fast

cer

eals

50(

BH

A,B

HT

)

-O

ctyl

gal

late

(O

G)

—D

ryd

iced

gla

zed

frui

t32

(B

HA

)

-D

odec

ylg

alla

te(

DG

)—

Dry

mix

esf

orb

ever

ages

and

de

sser

ts90

(B

HA

)

7089_C009.indd 278 10/8/07 12:13:04 PM


Cak

em

ixes

,cer

eal-

base

dsn

acks

,milk

po

wde

r,de

hydr

ated

mea

t,se

ason

ings

,pr

oces

sed

nuts

,pre

cook

edc

erea

ls

200

mg/

kg(

G’s

and

BH

A

sing

lyo

rco

mbi

ned)

Shor

teni

ngs

emul

sion

sta

biliz

ers

200

(BH

A,B

HT

)

Pota

toa

nds

wee

tpot

ato

flake

s50

(B

HA

,BH

T)

Pota

tog

ranu

les

10(

BH

A,B

HT

)

Swee

tpot

ato

flake

s50

(B

HA

,BH

T)

Che

win

ggu

m10

00(

BH

A,B

HT,

PG

)

Deh

ydra

ted

pota

toes

25m

g/kg

(G

’sa

ndB

HA

,si

ngly

or

com

bine

d)A

nim

alf

ato

ran

imal

and

ve

geta

ble

fatm

ixtu

re10

0(B

HA

,BH

T,P

G)

or2

00

Mar

gari

ne20

0

Froz

enr

awb

read

eds

hrim

p20

0(B

HA

,BH

T)

Dri

edm

eats

100

(BH

A,B

HT,

PG

)

Dry

sau

sage

30s

ingl

yor

60

(BH

A,B

HT,

PG

)

Fres

hpo

rk,s

ausa

ges

and

saus

age

prod

ucts

,mea

tpro

duct

s10

0si

ngly

or

200

(BH

A,B

HT,

PG

)

Var

ious

pou

ltry

prod

ucts

100

(BH

A,B

HT

)or

200

Flav

orin

gsu

bsta

nces

5000

(B

HA

)

7089_C009.indd 279 10/8/07 12:13:04 PM


9.3.1 Phenolic comPounds

Phenoliccompoundsareoneofthemainclassesofsecondarymetabolitesinplants,responsibleforcolordevelopment,pollination,andprotectionagainstUVradiationandpathogens.Infoods,thesecompoundscontributetosensoryproperties(color,astringency). Phenolics refer to monomeric, oligomeric, or polymeric compoundswith an aromatic ring bearing one or more hydroxyl substituents and functionalderivatives(esters,methylethers,glycosides,etc.).

Phenolicsincludesimplephenols,coumarins,flavonoids,stilbenes,lignans,andhydrolyzableandcondensedtannins.Flavonoids(alargeandcomplexgroupofcom-poundscontaininga three-ring structurewith twoaromaticcenters andacentraloxygenated heterocycle) are common antioxidants. The six major subclasses offlavonoidsareflavones,flavonols,flavanones,catechinsorflavanols,anthocyanidins,and isoflavones. Most flavonoids present in plants are conjugated with sugars,althoughoccasionally theyare foundasaglycons [23].More than4,000differentnaturallyoccurringflavonoidshavebeendiscovered,andmorethan36,000differentflavonestructuresarepossible.

Phenoliccompoundshavepowerfulantioxidantactivitiesin vitro[24],basedontheirstructure,hydrogen-donatingpotential,andabilitytochelatemetalions.Theymay show higher efficacy than endogenous or synthetic antioxidants [25]. Theirantioxidant activity [26–28] and their structure-activity relationships have beenexamined[17,29–32].

The most-studied sources of phenolic antioxidants are fruits and vegetables[33–36],grainsandcereals[37],andteas[38,39].Agriculturalandindustrialwastesarerenewable,cheap,andhighlyavailablesourcesofphenolicantioxidants.

9.3.2 TerPenoids

Terpenoids,alsoknownasisoprenoids, aresecondaryplantmetabolitesaccountingforthelargestfamilyofnaturalcompounds,widespreadinplantsandlowerinverte-brates. The isoprenoid biosynthetic pathway generates primary and secondarymetabolitesofecologicalrelevancetoplantgrowthandsurvival.Thesecompoundsare involved in interactions between plants, between plants and microorganisms,and between plants and insects, acting as allelopathic agents and attractants orrepellants in plants [40]. They are involved in the defense, wound sealing, andthermotoleranceoftheplantsaswellasinthepollinationofseedcrops,theflavoroffruits,andthefragranceofflowers,determiningthequalityofagriculturalproducts.Some terpenoids or their precursors act as scavengers for external aggressivemoleculesinthegaseousphase(i.e.,ozone).Thetermterpenesisusedforagroupofcompoundswith thebasicC5 isopreneunit.According to thenumberof theseunits (1 to6), terpenoidsareclassified intohemiterpenoids,monoterpenoids (C10)(limonene,carvone,carveol);sesquiterpenoids(C15);diterpenoids(C20)(retinoids);sesterterpenoids(C25);tri-(C30);andtetraterpenoids(carotenoids),havingeightiso-prenoidC5residues.

Terpenoid compounds (monoterpenes, sesquiterpenes, andditerpenes) are themaincomponentsofessentialoils,whichalsocontainoxygenatedderivativesandother compounds (including aldehydes, ketones, phenolic, acetates, and oxides).

7089_C009.indd 280 10/8/07 12:13:04 PM


Theantioxidantactivityofdifferentessentialoilsindifferentmodelsystemsiswellknown[41,42],andsynergisticeffectswithphenolicshavebeenreported[40,43].Essential oils are the commercial sources of terpenoids, whereas enzymes andextractsfrombacteria,cyanobacteria,yeasts,microalgae,fungi,plants,andanimalcellshavealsobeenusedfor theproductionandbioconversionof terpenes.Theirbiotechnologicaltransformationsappearespeciallypromisingbecauseapplicationssuchasfragrancesandflavorsincosmeticsandfoodsdependontheabsolutecon-figuration(differentenantiomerspresentdifferentproperties).

9.3.3 caroTenoids

Carotenoids are a group of more than 600 different compounds, with isoprenoid(tetraterpenoid) structure, synthesized by plants, photosynthetic organisms, andsomenonphotosyntheticbacteria,yeasts,andmolds.Theycanbefoundaspigmentsinfruits,flowers,andanimalspecies(birds,insects,fish,andcrustaceans)andplayanimportantroleintheprotectionagainstphotooxidativedamage.Mostcarotenoidsarecomposedofacentralcarbonchainofalternatingsingleanddoublebonds(3to15conjugateddoublebonds)withdifferentcyclicoracyclicendgroups.Theyareclassified as carotenes (α- and β-carotene, lycopene), composed only of carbonandhydrogenatoms,orxanthophylls(zeaxanthin,lutein,α-andβ-cryptoxanthin,canthaxanthin,astaxanthin),withatleastoneoxygenatom.Carotenoidspredomi-nantly occur in their all-trans configuration, although cis-isomers can be formedduringfoodprocessing[44].Lycopeneexhibitsthehighestantioxidantactivity,anditsplasmalevelisslightlyhigherthanthatofβ-carotene[45].Theresultsreportedfortheantioxidantactivityofβ-carotenedifferwidelyduetothevarioustestsystemsandtheexperimentalconditionsused[46].Theconjugateddouble-bondsystemisresponsiblefortheantioxidantpropertiesofcarotenoids,whichcanactbyquenchingsingletoxygenformedduetotheeffectsofUVlight,scavengingperoxylradicals,hydrogentransfer,orelectrontransfer[47–49].

Major sourcesof lycopene include tomatoes, rosehip,apricots,guavas,water-melons,papayas,andpinkgrapefruits;α-caroteneisfoundincarrots,tomatoes,andgreenvegetables;β-caroteneispresentinthesamematerialsasα-caroteneaswellas in paprika and sweet potatoes;β-cryptoxanthin is present in mangos, papaya,peaches, paprika, oranges, lutein in bananas, egg yolks, spinach, parsley, andmarigoldflowers;zeaxanthininpaprika;astaxanthininsalmon, theyeastPhaffia rhodozyma,andthealgaeHaematococcus pluvialis;andcanthaxanthinincarrots.

9.3.4 ViTamin e

VitaminEincludesafamilyoftocopherols(havingaphytyltailattachedtotheirchromanolnuclei), tocotrienols (withanunsaturated tail),andsomeof theiresterderivatives(suchassuccinateandacetate).VitaminEeffectivelyinhibitstheperoxida-tionoflipidsbecauseitcanscavengetheperoxylradicals.Theradical-scavengingcapacityofα-tocopherolandα-tocotrienol issimilar inhexane,butα-tocotrienolis more active in membrane systems andα-tocopherol shows higher bioactivity.Themajor sourcesofvitaminEareplant species,and itscontentvariesbetweentissues,withpreferentialaccumulation inseeds.Due to theiramphipathicnature,

7089_C009.indd 281 10/8/07 12:13:05 PM


tocopherolsareassociatedwithmembranelipidsorlipidstoragestructures.VitaminEisthemostimportantnaturalantioxidantinvegetableoil–derivedfoods,foundinricebran,palmoil,andwheatgerm[50].Therichestsourceisaby-productofsoy-beanprocessing(theoildeodorizerdistillate).

9.3.5 oTher naTural anTioxidanTs

Othercompoundswithantioxidantactivitysuitableasfoodadditivesarepeptidesandproteins[51,52],Maillardproducts[53,54],oligosaccharides,sugarsandpolyols[55],andmicrobialmetabolites[56].

9.4 bIologICAl propertIes of AntIoxIdAnt CoMpounds

9.4.1 Phenolic comPounds

Avarietyofbiologicaleffectshavebeenreportedforphenolicacids,includingalleviationofhyperuricemiaandprotectionagainstLDLoxidation,anti-inflammatory,antitumor,andautoimmune-relatedeffects[57–61].Caffeicandferulicacidsprovideprotectionagainstcarcinomas[62],ferulicacidestersprotectagainstUVradiation[63],andtrans-cinnamicacidcanbeusedinthepreventionortreatmentofdiabetes[64].

Research in flavonoids has increased since the discovery of the low cardio-vascularmortalityrateinMediterraneanpopulationsthatisassociatedwithredwineconsumptionandhighdietarysaturatedfatintake(“Frenchparadox”).Theirstrongantioxidantpowermakesflavonoidsabletoquenchfreeradicalsandtoactagainstthe oxidation of LDLs, attenuating the development of atherosclerosis, reducingthrombosis, and promoting normal endothelial function [65–68]. Flavonoids areexcellentcandidatesashealth-promoting,disease-preventing,andchemopreventiveagentsbecause theyare extremely safe andassociatedwith low toxicity [69,70].Protectiveactionhasbeenpostulatedforchronicdiseases[71,72],cardiovasculardiseases[71,73–77],stroke[77],hyperlipidemia[71],diabetes[74], inflammation[74,78–81],allergies[74,78,79],immunesystemdisorders[72,82],mutagenesis[74,83],andcataracts[72]aswellasforneurologicaldisorders[72,73,84],particularlythoserelatedtoaging,suchascognitive,motoric,andmooddecline[85].Flavonoidshavebeenrecognizedtoexertavarietyofbiologicalactivities(includingestrogenic,antimicrobial,antiviral,andanalgesic)[78,79]andtohavehepatoprotective,cyto-static, and apoptotic properties [79]. Some of these protective effects have beenconfirmedbyepidemiologicalstudies[75,76,83,86].Allinflammatoryprocessesincludeoxygen-activatingprocessesthatproducereactiveoxygenspecies,andfreeradicalscavengersorquenchersofactivatedstateswarrantmetaboliccontrolwithincertainlimits.Cardiovasculardiseaseisrelatedtoinflammationand,consequently,isamenabletointerventionviamoleculeswithanti-inflammatoryeffects[67].Withregardtotheimmunesystem,flavonoidsmaypreserveTcell–mediatedimmunity[82].Flavonoidsinthehumandietmayreducetheriskofvariouscancers,includinghormone-dependentbreastandprostatecancers[79],intestinalneoplasia[83],andskincancer[87].

In vitro flavonoids can bind electrophils, inactivate oxygen radicals, preventlipidperoxidation,andinhibitDNAoxidation.Incellcultures,theyincreasetherate

7089_C009.indd 282 10/8/07 12:13:05 PM


ofapoptosis,and inhibitbotcellproliferation,andangiogenesis [83],butadirectextrapolationtohumanscannotbemadeonthebasisofthesedata.Flavonoidsarepresentinthedietasglycosylated,esterified,orpolymerizedderivatives,andhumaninterventionstudieshaveprovidedevidencethatflavonoidsarepartlyabsorbed.Duetothelowassimilationrateandthehighconcentrationspresent,significantflavonoidintakemightresultindirecteffectswithinthegastrointestinaltract,suchasbindingofprooxidant iron;scavengingof reactivenitrogen,chlorine,andoxygenspecies;andperhapsinhibitionofcyclooxygenasesandlipoxygenases[6].

A growing body of in vivo studies is beginning to provide insight into thebiologicalmechanismsofflavonoidaction[77].Thenatureofpolyphenolconjugatesin vivohasbeenidentified,showingthatthebiologicalfateofflavonoids,includingtheirdietaryforms,ishighlycomplexanddependentonalargenumberofprocesses[88]. The forms reaching the blood and tissues are, in general, neither aglycons(exceptforgreenteacatechins)northesameasthedietarysource.Asaconsequence,thepolyphenolconjugatesarelikelytopossessdifferentbiologicalpropertiesanddistributionpatternswithintissuesandcellsthanpolyphenolaglycons.Ontheotherhand,polyphenolconcentrationstestedshouldbeofthesameorderasthemaximumplasma concentrations achieved after a polyphenol-rich meal [89]. The biologicaleffectsofthesepolyphenolsdependontheextentandwayinwhichthecirculatingmetabolitesinteractwithandassociatewithcells[73].

Antioxidantpropertiesalonearenotsufficienttoexplainthebiologicalpropertiesofflavonoids.Withinthelastdecade,reportsonflavonoidactivitieshavebeenlargelyassociatedwithenzymeinhibitionandantiproliferativeactivity,whicharedependentonparticularstructures[75].Althoughtheactionmechanismsarenotfullyunder-stood,recentstudieshaveclearlyshownthattheroleofflavonoidsasmodulatorsofcellsignallingmaybeattributedtotheireffectsasanticanceragents,cardioprotec-tants,andinhibitorsofneurodegeneration[90].Certainflavonoids,especiallyflavonederivatives,expresstheiranti-inflammatoryactivityatleastinpartbymodulationofproinflammatorygeneexpression[80,81].Thepotentialneuroprotectiveeffectsofdietaryflavonoidsandtheirroleinmodulatingoxidativestressmayberelatedtocellsignallingcascades,geneexpression,anddown-regulationofpathwaysleadingtocelldeathandneuronalapoptosis[85,91].

9.4.2 TerPenoids

Someterpenoidsarethebioactivecompoundsoftraditionalherbalremediesusedinthetreatmentofpain,colds,bronchitis,andgastrointestinaldiseases.Terpenoidsarepresentinalmosteverynaturalfoodandhavebeenassociatedwithprotectionfromoxidative stress and chronic diseases [92]. Some exhibit cardioprotective action,such as ginkgolides A and B and bilobalide from G. biloba [93]. Other relevantpropertieshavebeenreported,includingantibacterial[94],anti-inflammatory[95],anticarcinogenic[40],antimalarial,antiulcer,antimicrobial,anddiureticactivities.Protectionagainstavarietyofinfectiousdiseases(viralandbacterial)andacaricidalactivityhavebeenreportedformonoterpenes[96].Thepresentcommercialimpor-tanceofterpene-basedpharmaceuticalsisexpectedtoplayamoresignificantroleinhumandiseasetreatmentinthefuture[97].

7089_C009.indd 283 10/8/07 12:13:05 PM


9.4.3 caroTenoids

Themajorbiologicalfunctionsofcarotenoidsarerelatedtointercellulargapjunctioncommunication,celldifferentiation,immunoenhancement,andinhibitionofmuta-genesis.Somecarotenoids(α-andβ-carotene,β-cryptoxanthin)areprecursorsofvitaminAandprotectagainstchemicaloxidativedamage,severalkindsofcancer,andage-relatedmaculardegeneration.Noconvincingevidenceexistsoftheirprotec-tiveactionagainstcardiovasculardisease[47,48,98–100].In vitrostudiesevidencedthatcarotenoidscaninteractwithseveralreactivespeciesandcanactasprooxidants,althoughnodocumentedevidencetodateindicatestrueprooxidantactivityin vivo[101]. The maximum antioxidant effectiveness of carotenoids in human cells isrelatedtoanoptimaldose,becausehigherdosescanbelesseffectiveorresult incelldamage.Therelationshipbetweencarotenoidintakeandcancerhasbeenevalu-ated, showing an inverse association for lung, colon, breast, and prostate cancer,althoughnegativeeffectsofsupplementationshavebeenfound[49]anditisnotcleariftheassociationbetweendietanddiseaseisduetothespecificcarotenoid,othermicronutrientspresentinthespecificdiet,orthecombinedeffectofseveraloftheseactive ingredients.Studieson themechanismof cancer cell growth inhibitionbycarotenoidsattheproteinexpressionlevelmayinvolvechangesinpathwaysleadingtocellgrowthorcelldeath,includinghormoneandgrowthfactorsignaling,regula-torymechanismsofcellcycleprogression,celldifferentiation,andapoptosis[102].

9.4.4 ViTamin e

Theactionsoftocopherolsandtocotrienolshavebeenextensivelystudied.VitaminEprotectsvitaminA,sparesseleniumandvitaminC,andisthemosteffectivelipid-solubleantioxidant,whichprotectsunsaturatedfattyacidsinmembranes.Othernon-antioxidantfunctionsincludeenhancedimmuneresponseandregulationofplateletaggregation [50, 103]. The effects of Vitamin E have been observed at the levelofmessenger ribonucleicacid (mRNA)orproteinandcouldbe related to regula-tionofgenetranscription,mRNAstability,proteintranslation,andproteinstability.Landviketal.[103]publishedacompilationofhumanepidemiologicalstudiesonvitaminE,carotenoids,andcancerrisk.Thisvitaminalsoprotectsagainstcoronaryheartdisease[104],aging,cataracts,UVradiation,airpollution,andlipidperoxida-tionassociatedwithstrenuousexercise.VitaminEbioavailabilityandmetabolismis influenced by intestinal absorption, plasma lipoprotein transport, and hepaticmetabolism[105].DifferentdistributionofvitaminEisoformsintissueshasbeenreported, being an essential part of the antioxidant defense systems, particularlyintheskin,wheretocotrienolsarepreferentiallydistributed.Tocotrienolsaremoreeffective than tocopherolsat inhibitingneuronalcelldeath. Ithasbeensuggestedthatneithertheanticarcinogeniceffectsoftocotrienolsnortheneuroprotectionarerelatedtotheantioxidantpropertiesoftocopherolsandtocotrienols[50].

9.4.5 anTioxidanT ProPerTies of sc-co2 exTracTs

SinceSFEisarelativelynovelapplication,studiesonthebiologicalpropertiesoftheseextractswillprobablyincreaseinthefuture.Antimicrobialactivityofseveral

7089_C009.indd 284 10/8/07 12:13:06 PM


extractshasbeenobservedforwhitegrapeseedfractions[106],spices[107],andmarjoram [108]. Protection from ischemic damage was reported for cocoa hullextracts [109]. Antimutagenic and antineoplastic properties have been claimedforSC-CO2extractsofplantandspices[107,110]aswellasantimutagenicityforTerminalia catappaleaveextracts[111].

9.5 deterMInAtIon of AntIoxIdAnt ACtIvIty

Theactivityofnaturalantioxidants,whicharemixturesormultifunctionalsystemsacting in complex media, cannot be evaluated satisfactorily by a simple test, andcontradictoryresultsusingdifferentassayshavebeenreported.Acomparativeevalu-ationofantioxidants isdifficultbecause, in foodstuffsandbiological systems, theactivitydependsonthesubstrate,themedium,theoxidationconditions,interfacialphenomena,andthepartitioningpropertiesoftheantioxidantbetweenphases.Theaffinity of antioxidants toward air, oil, water, and interfaces explains why polarantioxidantsaremoreactiveinbulkoilsandnonpolarantioxidantsaremoreactivein emulsions, a behavior known as the “polar paradox.” The need for approved,standardizedmethodsisespeciallyimportantforcomparingfoodornutraceuticalsinordertoprovidequalitycriteriaforregulatoryissuesandhealthclaims.Evalua-tionoftheantioxidantactivityatdifferentlevelshasbeensuggested[112],including:i)quantificationandidentificationoftheactivecompounds,ii)evaluationoftheradicalscavengingactivitywithmorethanonemethodindifferentsolvents,iii)evaluationofprotectionagainstlipidoxidationinmodelsystems,andiv)studiesofrelevanceforfoodapplicationsandhumanstudieswithmarkersforoxidativestress.

Many in vitro methods are performed in the absence of lipids and the parti-tioningofantioxidantsisnotevaluated,orthesemethodsdonotpredicttheabilityto inhibit oxidation of foods or in biological systems. More realistic informationcanbeachievedbyperformingseveraltestsandfollowingsomegeneralrecommen-dations:i)substratesandoxidationconditionsshouldsimulatechemical,physical,andenvironmentalconditionsinfoodorbiologicalsystems;ii)lowlevelsofoxida-tionshouldalsobeconsidered; iii)both initialandsecondaryproductsshouldbemeasured;andiv)theconcentrationsofcatalyst,antioxidants,andsubstratesshouldbecarefullyestablishedand thecompositionaldata shouldbeknown tocomparesamples[3,16,18,20,113,114].Theresultsarealsoinfluencedbythespecificityandmethodsusedtoanalyzetheprogressofoxidationandbythedegreeofoxida-tionchosenasend-pointfortesting[16,17,113,114].Acceleratedoxidationofoils,fats,oil-wateremulsions,andmusclefoodsarerelevantduringfoodprocessingordomestic use [114]. However, under some testing conditions (temperature, partialpressureofoxygen,metalcatalystsandotherinitiators,lightorUVradiation),theoxidationmechanismsmaychange[18].Methodsofexpressingantioxidantactivity,summarized by Antolovich et al. [18], include the induction period, percentageinhibitionofrates,IC50(concentrationtoachieve50%inhibition),andscalereadings(absorbance,conductivity).

Asfreeradicalgenerationisdirectlyrelatedtooxidation,variousmethodshavebeendevelopedbasedontheabilitytoscavengefreeradicals[5,19,115,116].Huangetal.[3]andPrioretal.[20]comparedtheperformanceandbiologicalrelevanceof

7089_C009.indd 285 10/8/07 12:13:06 PM


differentmethodsandestablishedaclassificationbasedonthemechanisms:hydrogenatom transfer (HAT, measuring the ability to quench free radicals by hydrogendonation),singleelectrontransfer(SET, measuringtheabilitytotransferoneelectrontoreduceanycompound),oracombinationofHATandSET.AmongthemethodsbasedonHAT,themostfrequentlyemployedaretheOxygenRadicalAbsorbanceCapacity (ORAC)and theTotalPeroxylRadical-TrappingAntioxidantParameter(TRAP).Bychangingtheoxidantsourcesofperoxylradicals,thereactioncandif-ferentiate quenching of specific oxidants (O2

·-, HO·, HOCl, LO(O)·, ·OONO, and·O2).OtherHATtestsandtheirtargetapplicationsaretheTotalOxidantScavengingCapacitytest(TOSC,towardhydroxylradicals,peroxylradicals,andperoxynitrite);carotenoidsbleachingviaautoxidation (towardoxidation inducedby lightorheatoroxidationinducedbyperoxylradicals);andLDLOxidationinitiatedbyCu(II)orAAPH(withrelevancetooxidativereactionsthatmightoccurin vivo).Methodsbased on SET reactions are the Ferric Reducing Antioxidant Power (FRAP) andCopperReductionAssay(CUPRAC).Themostfrequentlyemployedmethodsbasedon both HAT and SET mechanisms are Trolox Equivalent Antioxidant Capacity(TEAC)andDPPH(2,2-diphenyl-1-picrylhydrazylradical).Bothofthemareoper-ationally simple andwidelyused, although the radical anionABTS·+ used in thefirstisnotfoundinhumanbiology,andthesecondhasseveraldrawbacks[20].TheFolin-Ciocalteutest,usedtoquantifyphenoliccontent,alsomeasurestheeffectiveoxidation/reductionefficiencyofalltheantioxidantspresentinthemedium.

Extrapolationofantioxidantmechanismsestablishedinfoodormodelsystemsto in vivo situations isnotdirect.Bioavailabilityandmetabolismofantioxidantsmustbeaddressedtoknowifthesecompoundsreachtargettissuesbecausetheirbiologicaleffectsmaybeaffectedbyavarietyoffactors,includingdigestion,absorp-tion,metabolism,andthepresenceofcompetitiveenzymesandotherantioxidantsor prooxidants. Although in vitro assays do not reflect the cellular physiology,metabolismandin vivoassays(withanimalsorhumans)arelesssuitedforinitialscreeningofantioxidantsthancellculturemodelsbecausetheyareexpensiveandtime-consuming [117]. However, cells in culture behave differently from thosein vivoduetothe“cultureshock”andtotheoxidativestresscausedbytheprocess[118].Apartfromthecriticalgeneralworksontheanalyticalmethodstodetermineantioxidant activity [3, 16, 17, 18, 20, 112, 113, 116], specific revisions concern-ingfoodapplications[19,114,115]havebeenpublished.Theavailablemethodstomeasurefreeradicalsandotherreactive(oxygen[ROS]/nitrogen/chlorine)speciescontributingtothedevelopmentofseveraldiseasesbyoxidativedamagehavebeenrevised[4,119].

9.6 superCrItICAl-Co2 extrACtIon of AntIoxIdAnts

Dependingonthephysicalstate(solidorliquid)ofthephasecontainingthetargetcompounds,SFEcaninvolvesolid-liquidorliquid-liquidmasstransfer.Solid-liquidextraction is a heterogenous operation involving the transfer of solutes from thevegetalmatrixtoafluid.Theextractionratedependsontheexternalmasstransfer,effectivesolutediffusivity in thesolid, solute solubility in thesolvent,andsolutebindingtothesolidmatrix.Batchextractionandsemicontinuousextractionarethe

7089_C009.indd 286 10/8/07 12:13:07 PM


most commonly used experimental methods. Extraction by solvent flow throughafixedbedof solid particles allows the recoveryof fractionsobtained along theextractionperiod.WhenaliquidstreamhastobeprocessedbySFE,bothsolubilityand interphasemass transfer are relevant.Operation is similar to extractionwithconventionalsolvents,andcontinuousoperationcanbecarriedoutinsingle-stageormultistagecontact(cross-floworcountercurrent).

9.6.1 Processing schemes

Different processing schemes have been proposed for SFE of compounds fromnatural sources.Figures9.1a to9.1dpresent simplifiedflowdiagramsof themostusualalternatives,including:

1.Singleextractionstageandfractionalseparationinseveralseparators.Theextractobtainedinasingleextractionstepcanbefractionatedbyreleasingpressure in theseparators.Thisdispositioniswidelyusedforprocessingsolidsandforanalyticalpurposes[120–122].

2.Stagewiseextractionatprogressivelyincreasedseverity.Afterafirststageat low severity (< 15 MPa, no modifier) to extract nonpolar compounds(essentialoilandwaxes),furtherSFEofthesolidresidueisperformedatincreasedseverity(upto50MPa,40%modifier)toextractmorepolaranti-oxidants [123, 124]. Stepwise extraction needs more solvent than simpleextractionwithstagewisefractionationofextracts[12,125],althoughtheextractionyieldscanbesimilar.

3.CombinationofconventionalsolventandSFEofsolidsamples.AfirstSFEstageunder lowseverityconditionscanbeperformed to removevolatilecompoundsandwaxesfromthesolidsubstrate[126,127]beforeextractionwithconventional solvents.Ahydrothermal treatment,withenvironmen-talandoperationaladvantagesderivedfromthenontoxiccharacterofthesolvent,hasbeenusedforextractingbiologicallyactivecompoundsfromSFE-extractedbamboo[128].

4.SFEofdryextractsorsolidresidues.Solid-liquidSC-CO2extractioncanbeemployedtopurifycommercialextracts,driedextractsfromconventionalsolvent extraction (CSE), or compounds remaining in the solid residuefromCSE.The twofirst schemeshavebeenproposed forenhancing theantioxidantactivityand improving theorganolepticproperties (dearoma-tization)ofextracts [129,130]. Improvedbenefitshavebeenreportedforhigh-molecular-weightcompounds,probablyduetotheirlowerconcentra-tionandinteractionswiththematrix[131].

AntioxidantshavebeenalsoobtainedbySFEofliquidfeedstreams,includingoilsanddistillates[132]andjuices[133].

Usually,naturalrawmaterialsforSFEshowbothlimitedcontentsofthetargetcompoundsandlowbulkdensity,makingtheutilizationoflargevolumeextractorsnecessary[134].Becauseofthis,processes involvingCSEandfurtherpurificationof

7089_C009.indd 287 10/8/07 12:13:07 PM


thecrudebySFEarecomparativelyadvantageous,astheyprovidehigheryieldsand/orlowerspecificCO2consumptionthandirectextractionofthevegetablefeedstock.

9.6.2 effecTs of The mosT influenTial oPeraTional Variables

Previous conditioning of the starting material and the experimental conditionsemployedinextractionandseparationinfluenceSFEperformance.Thenatureandpropertiesofvegetablefeedstocksor theirprocessingstreams(includingmaturitystage,cultivar,variety,edaphoclimaticconditions)stronglyinfluencetheextractionofterpenoidsandphenolics[135,136],carotenoids[137–139],andtocopherolsfrom

RawMaterial

SCFE10–45 MPa

Antioxidant Compounds

R2R1

(a) E1

Crustacean, Micro-algae, TomatoRaw Material Flowers, Fruits, Leaves, Spices, Medicinal

Plants, Seeds, Hulls, Roots

Phenolics and Terpenoids

Olive Leaves, MedicinalPlants, Wheat Germ, Seeds

FRACTIONA-TION (1–3 separators)

RawMaterial

RawMaterial

RawMaterial

SCFE9–15 MPa

SCFE9–15 MPa

SCFE20–50 MPa


R1

(b)

PaprikaRaw materialCarotenoids

FRACTIONATION(1–3 separators)

E1Oil

E2

R2

R1

(c)

E1

HydrothermalTreatment

Conventional SolventExtraction

ConventionalSolvent Extraction

R2

R3

(d)

DryingSCFE

10–35 MPa35–80°C

R1

Antioxid. CompoundsR2

Raw Material

E1Aroma

AntioxidantCompounds


Raw MaterialAntioxidant Compounds




Carotenoids

Vitamin E

Leaves, Medicinal Plants, SeedsPhenolics and Terpenoids

Medicinal Plants, StalksPhenolics and Terpenoids

Grape Seeds, Grape PomacePhenolics and Terpenoids

fIgure 9.1 ProcessingschemesforextractionofantioxidantcompoundsinvolvingSFEstages.Nomenclature:E1,E2:extracts;R1,R2,R3:solidresidues.

7089_C009.indd 288 10/8/07 12:13:09 PM


solidsamples.Whenprocessingsolids,mechanical-thermalconditioningisdecisivetofacilitatetheextractionofintracellularsolutes.Reducedparticlesizefavorsmasstransfer,buttoo-smallparticlescouldlimittheperformanceoffixedbedsandgrind-ingmayresultinlossesbyvolatilizationanddegradationofactivecompounds.ThemajorvariablesinfluencingSFEofantioxidants(pressure,temperature,solventflowrate, solvent-to-feed ratio, modifier type, and concentration) should be optimizedbeforeoperation.ThosemostinfluentialandspecificforSFEincomparisontoCSEarefurthercommented.

9.6.2.1 pressure and temperature

ThesolvatingpowerofSFEwithCO2dependsonpressureandtemperature.Densitygives an estimate of the joint effects of both variables on the solvating power.Besidestheoperationalconditions,theequilibriumsolubilityofpurecompoundsisinfluencedbymolecularweight,polarity,andpresenceoffunctionalgroups.Whenconsideringextractionfromasolidsubstrate,kineticsandyieldsalsodependontheinteractionwiththesolidmatrix.

Effect on the solubility of antioxidant compounds. Equilibriumsolubilitiesarebasicinformationforaddressingthedesignofextractionandseparationprocesses.Solubilitydataforsyntheticantioxidants[140],fat-solublevitamins[141],andmanyphenolic compounds have been obtained by different groups and were recentlycompiled[14,142].Solubilitydatahavebeenreportedforpurecompoundsandtheirmixtures[143,144]andforterpenoidsfromcitrusoils[145],aswellasforessentialoils[9]andtheircomponents[8].Mostsolubilitydatarefertoauniquesolute,andscarceinformationexistsfornaturalextracts,whicharemulticomponentmixtures.Since pioneer data on the solubility of tocopherols were published by Chrastil[146], severalother studies [147–149]havebeen reported.Data are alsoavailableformixtureswithmethyloleatetosimulatetheesterifiedby-productfromsoybeanoildeodorizerdistillate [150,151], formixturesof thisby-product [152,153]andforcrudepalmoil[132].Additionalliteratureconcerningpurecarotenoids,suchascapsaicin[148],β-carotene[148,154–156],andtheirmixtures[157],andfornaturalβ-carotene from carrots [156] has been published. Solubility data forβ-caroteneand tocopherols were compiled by Guglü-Üstündag and Temelli [137]. Table9.2summarizesdatafromreviewpapersonthesolubilityofdifferentcompoundshavingantioxidantactivity.

Yield and selectivity of antioxidant extraction. Increased pressure results inincreased solvent density, allowing higher extraction yields. Increasing pressurebeyond a threshold point results in higher fluid viscosity and reduced diffusioncoefficients.Pressuresover50MPa[160]havebeenreportedfortheextractionofantioxidants.Operatingathighpressure,increasedtemperaturesmaydecreasetheextractionyieldduetothereductionindensityandthesolventpowerofthefluid.Operatingatpressuresclose to thecriticalpoint,where thedensity showshigherinfluenceonthesolventpowerthanthevaporpressure,increasedtemperaturesmaydecreasetheextractionyieldduetothereductionindensityandthesolventpowerofthefluid.Athigherpressures,theincreasedinfluenceofthesolutevaporpressuregenerallyleadstoincreasedsolubility.Temperatureandpressureshowacrossover

7089_C009.indd 289 10/8/07 12:13:09 PM


effectwherebyhighertemperaturesimproveextractionathighpressuresandlowertemperaturesfavorextractionatlowpressures.Thecrossoverregionsinsupercriticalfluids,orthepointwheretheslopeofsolubilityvs. temperaturechanges,arealsofavorabletodesignseparationprocesses.

Theeffectsofpressureandtemperatureontheextractionyieldofsomeanti-oxidantsareshowninFigure9.2toFigure9.4.Effectsoftemperatureontextureandcoloroftheextractshavebeenreportedformoso-bambooextracts[128].Inothercases,slightchangesinappearance[176]andsignificantonesincompositionhavebeenreported.Thislattereffectisduethesolventpower(whichcontrolstheabilitytodissolvedifferentmolecules)andtothethermalstabilityofthesolutes.Highlythermal-sensitive compounds require mild extraction conditions (temperaturesbelow50ºC)toavoidalteration.Undertheseconditions,SFEoffershigheryieldsofactivecompounds—forexample,carnosicacidfromrosemary[122],anacardicacid from cashew nut shell [177], hyperforin from Hypericum perforatum [178],carnosol frommarjoram[179],antioxidants fromaloe [165],andmatricine fromchamomile[180].

9.6.2.2 Modifier

PureCO2undersupercriticalconditionsisagoodsolventforlipophiliccompoundsbutispoorforphenolics.Extractioncanbeenhancedusingamodifierabletointeractwith

tAble 9.2solubility of selected Antioxidant Compounds in supercritical Co2

Compound p (Mpa) t (K) solubility ref.

tocopherols(alpha,delta) 8–35

8–35.219.5–351–2.52

292–353298–353303–353298–313

(y2·104)———

2.59–7.31

[141][137][143][12]

Carotenoids(astaxanthin,canthaxanthin,capsanthin,β-carotene,lycopene,lutein,zeaxanthin)

0.15–502–3.55–1805–80

288–343313–353288–353288–353

(y2·106)—

0.09–3.24—

0.019–0.989

[141][12][137][58]

terpenoids(monoterpenehydrocarbons,sesquiterpenehydrocarbons,oxygenatedderivatives,aldehydes,ketones)

3–118–10

0.8–13

295–335313–333310–333

(mg/g)—

1.6–CMa

—

[59][8]

[143]

phenolic Compounds(benzoicacid,cinnamicacids,flavonoids) 2–50

0.91–2.532–40.479–5000.26–50

308–473308–318308–473308–373308–373

(y2·104)—

0.0788–5.61——

0.08·10–4–1730

[59][12][143][142][14]

a CM=Completemiscibility

7089_C009.indd 290 10/8/07 12:13:10 PM


thetargetcompounds,possiblyimprovingyieldandselectivity.However,highmodi-fierconcentrationsmaydecreaseselectivitydependingonthesizeofphenolics[181].Alcoholsarewidelyusedasmodifiers,ethanolbeingthemostrecommendedoneonthebasisoftoxicologicalandenvironmentalconsiderations.Ethanolhasbeenemployedtoincreasethesolubilityofginsenoids[182],phenols[183],flavonoids[163],terpenoids[184],andcarotenoids[166,170,185–187].Methanolhasbeenusedforextractingphe-nolics[188],flavonoids[106,131,135,180,187],and isoflavones[189](Figure9.2).Othermodifiersandtheirtargetcompoundsareisopropanolforterpenoids,phenolicketones,andcurcuminoids[190,191];propyleneglycolforpolyphenols[180];waterforphenolicditerpenesandphenolicacids[129];andacetone;2,2-dimethoxypropane,chloroformandn-hexane[138,168,192]forcarotenoids.Vegetableoilshavealsobeenproposedforextractinglycopene[139,193]andcaprylicacid[194].Mixturesoftwomodifiershavebeensuccessfullyassayed[107,190].Oppositely,ethylacetate,chloro-form[187],andaceticacid[129]werenotsuitableasmodifiers.

Phenolics and Terpenoids

0 10 15 20 Ethanol (%)

Yiel

d (m

g/g)

0

2

4

6

8

10

12

14

16

P (MPa)

0

1

2

3

4

5

6

7

5 10 15 20 25 30 35

Yiel

d (%

)

0

10

20

30

40

50

60

70

80

90

Yiel

d (%

)

Rosemary ( 30°C), ( 40°C) [122]

[161]

From:

Coriander ( 38°C), ( 58°C) [162]

Black Pepper ( 45°C), ( 50°C),

G. biloba ( ) [163]

Savory ( ) [126] Sweet Tamarind ( ) [164]

Naringin from citrus peel [165] ( Fresh citrus peel), ( Dry citrus peel)

Products from grape seed Conc.[131] Syringic acid ( ) Protocatechualdehyde ( )

Epicatechin ( )

5

( 55°C), ( 60°C), ( 65°C)

Catechin ( )

fIgure 9.2 Effectsofpressure,temperature,andethanolconcentrationontheextractionyieldofphenolicsandterpenoidsfromvarioussources.

7089_C009.indd 291 10/8/07 12:13:12 PM


9.6.3 sc-co2 exTracTs Versus conVenTional solVenT exTracTs

Dataconcerning theantioxidantactivityofSC-CO2extracts fromsolidbotanicalsamples,commercialandcrudeextractsproducedwithconventionalsolvents,andliquidstreamsaresummarizedinTable9.3.Optimizationoftheoperationalvariablesisrequiredforeachprocess,owingtothewidevarietyofstartingmaterials,targetcompounds,andconditionsemployedforextractionandseparation.Tocomparetheextracts,boththeproductionconditionsandtheassayusedtoquantifytheantioxidantactivitymustbeconsidered.AgeneralcomparisonbetweenSFEandCSEcannotbe established beforehand. Even though conventional, less-selective solvents mayallowhigherextractionyields[121,188],theisolatedfractionscouldhaveunpleasantaromas.FurtherfractionationbySFEcanbeusedtopurifytheextract,preservingtheantioxidantactivity[195].Similarcompositionoftheextractsobtainedusingthese

Carotenoids A

bsor

banc

e

0 5 10 15 20 Ethanol (%)

0

0.1

0.2

0.3

20

40

60

80

Yiel

d(%)

P (MPa)

Abs

orba

nce

Yiel

d (%

)

20

40

60

80

10 20 30 40 50 60 0

0.2

0.4

0.6

Astaxanthin ( ) [166]; ( ) [167]

β-Carotene ( 35°C), ( 45°C), ( 55°C), ( 65°C) [170]

Lycopene ( 35°C), ( 45°C), ( 55°C), ( 65°C) [170]

Astaxanthin ( ) [167]

Astaxanthin (+ 40ºC), ( 50°C), ( 60°C) [166]

β-Carotene ( ) [168]

Carotenoids ( 40°C), ( 50°C),

Lycopene ( 45°C), ( 60°C) [139] Lycopene ( ), β-Carotene ( ) [170]

( 60°C) [169]

fIgure 9.3 Effects of pressure, temperature, and ethanol concentration on absorbanceandextractionyieldofselectedcarotenoids.

7089_C009.indd 292 10/8/07 12:13:15 PM


technologieshasbeen reported forchamomile [180]andmarigold [160],whereasdifferent compositionwas found inextracts fromoregano [125] and fennel seeds[176].Reports indicate thatSFE results in higher extraction yields and enhancedselectivityofactivecompoundsthanCSE[124,183,196].SuperiorityofSFEwithrespecttoconventionalmethodshasbeenreportedforeucalyptusleaves[196],blackpepperoleoresin[161]andLippia albastemsandleaves[197],owingtothehigherconcentrationsofactivecompounds.SFEmayresultinextractswithhigheractivitywhen processing substrates with high contents of thermally unstable active com-pounds and in better odor and color of the isolates [122]. Short processing timeand lowsolventconsumptionareadditionaladvantagesofSFE [182].Oppositely,thelowerselectivityofconventionalsolventscouldfavortheantioxidantactivityofextractedfractionsshowingsynergismamongcomponents,asobservedforturmeric[191],tamarindseedcoat[164],marjoram[108],andblacksesame[198].

referenCes

1. Halliwell,B.andGutteridge,J.M.C.,Free Radicals in Biology and Medicine,3rded.,Oxford,OxfordUniversityPress,1989.

2. Halliwell,B.,Antioxidantcharacterization,Biochem. Pharmacol.,49,1341,1995. 3. Huang,D.,Ou,B.andPrior,R.L.,Thechemistrybehindantioxidantcapacityassays,

J. Agric. Food Chem.,53,1841,2005. 4. Willcox,J.K.,Ash,S.L.andCatignani,G.L.,Antioxidantsandpreventionofchronic

disease,Crit. Rev. Food Sci. Nutr.,44,275,2004. 5. Azzi,A.,Davies,K.J.A.andKelly,F.,Freeradicalbiology:Terminologyandcritical

thinking,FEBS Letters, 558,3,2004. 6. Halliwell,B.,Rafter,J.andJenner,A.,Healthpromotionbyflavonoids,tocopherols,

tocotrienols, andotherphenols:Director indirect effects?Antioxidantornot?,Am. J. Clin. Nutr.,81,268S,2005.

Tocopherols

0

5

10

15

20

25

0 10 20 30 40 50 60

Yiel

d(%)

0

0.5

1

1.5

2

2.5

3

Yiel

d (%

)P (MPa)

Tomato Seeds and Skins ( 32ºC), ( 50°C), ( 68°C), ( 86°C) [173]

Silybum Marianum Seeds ( 25°C),( 40°C), ( 60°C), ( 80°C) [171]

From:

Soy Deodorizer Distillate ( 40°C),( 50°C), ( 60°C) [172]

Wheat Germ ( 40°C), ( 45°C)( 50°C) [174]

fIgure 9.4 Effectsofpressure,temperature,andethanolconcentrationontheextractionyieldsoftocopherolsfromvarioussources.

7089_C009.indd 293 10/8/07 12:13:17 PM


tAb

le 9

.3A

ntio

xida

nt A

ctiv

ity

of e

xtra

cts

obt

aine

d by

sfe

of b

otan

ical

s, C

omm

erci

al a

nd C

onve

ntio

nal s

olve

nts

extr

acts

vege

tal M

ater

ial o

r ex

trac

tA

ntio

xida

nt A

ctiv

ity

vege

tal M

ater

ial o

r ex

trac

tA

ntio

xida

nt A

ctiv

ity

Alo

e ba

rbad

ensi

s le

afs

kin

DPP

Hr

adic

als

cave

ngin

gPr

opol

ise

than

ole

xtra

ctL

ipid

oxi

datio

n(%

)C

hela

ting

effe

cto

nfe

rrou

sio

nsR

educ

ing

pow

erFr

eer

adic

als

cave

ngin

g(D

PPH

,SO

· ,· O

H)

Bup

leur

um k

aoi r

oote

than

olic

ext

ract

sL

ipid

oxi

datio

n(%

)Fr

eer

adic

als

cave

ngin

g(D

PPH

,SO

· ,· O

H)

Cam

elia

sin

ensi

sL

ipid

oxi

datio

n

Cor

iand

rum

sat

ivum

see

dsD

PPH

rad

ical

sca

veng

ing

Ros

mar

inus

offi

cina

lis

Pero

xide

val

ueD

PPH

rad

ical

sca

veng

ing

β-ca

rote

ne-l

inol

eic

acid

Cur

cum

a lo

nga

Lin

olei

cac

ido

xida

tion

β-ca

rote

ne-l

inol

eic

acid

Euc

alyp

tus

cam

aldu

lens

is le

aves

L

inol

eic

acid

oxi

datio

nR

osm

arin

us o

ffici

nalis

sol

vent

ext

ract

DPP

Hra

dica

lsca

veng

ing

Gin

kgo

bilo

ba le

aves

met

hano

lice

xtra

ctD

PPH

rad

ical

sca

veng

ing

Ros

mar

inus

offi

cina

lis

com

mer

cial

ext

ract

Pero

xide

val

ue

Hel

ichr

ysum

ital

icum

flow

ers

Free

rad

ical

sca

veng

ing

(DPP

H,S

O· )

β-ca

rote

ne-l

inol

eic

acid

Salv

ia o

ffici

nali

sβ-

caro

tene

-lin

olei

cac

idV

eget

able

oil

oxid

atio

n

Satu

reja

hor

tens

isV

eget

able

oil

oxid

atio

n

Hum

ulus

lupu

lus

Lin

olei

cac

ido

xida

tion

Red

ucin

gpo

wer

Sesa

mum

indi

cum

DPP

Hr

adic

als

cave

ngin

gL

inol

eic

acid

oxi

datio

n

Mel

issa

offi

cina

lis

Lin

olei

cac

ido

xida

tion

Scut

ella

ria

baic

alen

sis

root

DPP

Hr

adic

als

cave

ngin

g

Nig

ella

sat

iva

seed

sβ−

caro

tene

-lin

olei

cac

idm

odel

Tam

arin

dus

indi

ca s

eed

coat

Pe

roxi

dev

alue

Ori

ganu

m v

ulga

rePe

roxi

dev

alue

Te

rmin

alia

cat

appa

leav

esa

nds

eeds

DPP

Hr

adic

als

cave

ngin

g

Ori

ganu

m m

ajor

ana

Veg

etab

leo

ilox

idat

ion

Thy

mus

vul

gari

sPe

roxi

dev

alue

Ort

hosi

phon

spi

catu

s m

etha

nolic

ext

ract

DPP

Hr

adic

als

cave

ngin

gT

hym

us v

ulga

ris

β-ca

rote

ne-l

inol

eic

acid

Peum

us b

oldu

s A

BT

Sra

dica

lsca

veng

ing

Viti

s vi

nife

rain

dust

rial

by-

prod

ucts

DPP

Hr

adic

als

cave

ngin

g

Zin

gibe

r of

ficin

ale

β-ca

rote

ne-l

inol

eic

acid

7089_C009.indd 294 10/8/07 12:13:18 PM


7. Pokorný,J.andKorczak,J.,Preparationofnaturalantioxidant,inAntioxidants in Food: Practical Applications, 1st ed., Pokorný, J., Yanishlieva, N. and Gordon, M., Eds.,WoodheadPublishingLimited,Abington,Cambridge,England,2001,pp.311–330.

8. Reverchon, E., Supercritical fluid extraction and fractionation of essential oils andrelatedproducts,J. Supercrit. Fluids,10,1,1997.

9. delValle,J.M.,delaFuente,J.C.andCardarelli,D.A., ContributionstosupercriticalextractionofvegetablesubstratesinLatinAmerica,J. Food Eng., 67,35,2005.

10. Brunner, G., Supercritical fluids: Technology and application to food processing,J. Food Eng.,67,21,2005.

11. Reverchon, E. and De Marco, I., Supercritical fluid extraction and fractionation ofnaturalmatter,J. Supercrit. Fluids,number38,p.146,2006,availableonline27April2006.

12. Mukhopadhyay,M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.

13. Herrero,M.,Cifuentes,A.and Ibáñez,E.,Sub-andsupercriticalfluidextractionoffunctionalingredientsfromdifferentnaturalsources:Plants,food-by-products,algaeandmicroalgae:Areview,Food Chem., 98,136,2006.

14. Diaz-Reinoso,B. et al.,SupercriticalCO2extractionandpurificationof compoundswithantioxidantactivity,J. Agric. Food Chem.,54,2441,2006.

15. Gordon, M.H., The development of oxidative rancidity in foods, in Antioxidants in Food: Practical Applications, 1st ed.,Pokorný, J.,Yanishlieva,N. andGordon,M.,Eds.,WoodheadPublishingLimited,Abington,Cambridge,England,2001.

16. Frankel,E.N. andMeyer,A.S.,Theproblemsofusingone-dimensionalmethods toevaluatemultifunctionalfoodandbiologicalantioxidants,J. Sci. Food Agric.,80,1925,2000.

17. Yanishlieva, N.V., Inhibition of oxidation, in Antioxidants in Food: Practical Applications,1sted.,Pokorný, J.,Yanishlieva,N.andGordon,M.,Eds.,WoodheadPublishingLimited,Abington,Cambridge,England,2001.

18. Antolovich, M. et al., Methods for testing antioxidant activity, Analyst, 127, 183,2002.

19. Roginsky,V.andLissi,E.A.,Reviewofmethods todeterminechain-breakinganti-oxidantactivityinfood,Food Chem.,92,235,2005.

20. Prior,R.L.,Wu,X.andSchaich,K.,Standardizedmethodsfor thedeterminationofantioxidantcapacityandphenolicsinfoodsanddietarysupplements,J. Agric. Food Chem.,53,4290,2005.

21. European Parliament and Council Directive No. 95/2/EC, Official Journal of the European Communities,1995.

22. Míková,K.,Theregulationofantioxidantsinfood,inFood Chemical Safety, Vol. 2: Additives, 1sted.,Watson,D.H.,Ed.,WoodheadPublishingLimited,BocaRaton,FL,2002.

23. Ross,J.A.andKasum,C.M.,Dietaryflavonoids:Bioavailability,metaboliceffects,andsafety,Annual Rev. Nutr.,22,19,2002.

24. Miller, N.J. and Ruiz-Larrea, M.B., Flavonoids and other plant phenols in the diet:Theirsignificanceasantioxidants,J. Nutr. Env. Med.,12,39,2002.

25. Soobrattee,M.A.etal.,Phenolicsaspotentialantioxidanttherapeuticagents:Mecha-nismandactions,Mutat. Res.,579,200,2005.

26. Meyer, A.S. and Frankel, E.N., Antioxidant activity of hydroxycinnamic acids onhumanlow-densitylipoproteinoxidation,Methods Enzymol.,335,256,2001.

27. Collins,A.R.andHarrington,V.,Antioxidants:Not theonlyreason toeat fruitandvegetables,Phytochem. Rev.,1,167,2003.

7089_C009.indd 295 10/8/07 12:13:18 PM


28. Mansouri, A., Makris, D.P. and Kefalas, P., Determination of hydrogen peroxidescavenging activityof cinnamic andbenzoic acids employing ahighly sensitiveper-oxyoxalatechemiluminescence-basedassay:Structure–activityrelationships,J. Pharm. Biomed. Anal., 39,22,2005.

29. Nenadis, N., Zhang, H.Y. and Tsimidou, M.Z., Structure-antioxidant activity rela-tionshipofferulicacidderivatives:Effectofcarbonsidechaincharacteristicgroups,J. Agric. Food Chem.,51,1874,2003.

30. vanAcker,S.A.etal.,Structuralaspectsofantioxidantactivityofflavonoids,Free Rad. Biol. Med.,20,331,1996.

31. Rice-Evans,C.A.,Miller,N.J.andPaganga,G.,Structure-antioxidantactivityrelation-shipsofflavonoidsandphenolicacids,Free Rad. Biol. Med., 20,933,1996.

32. Firuzi,O.etal.,Evaluationoftheantioxidantactivityofflavonoidsby“ferricreducingantioxidantpower”assayandcyclicvoltammetry,Biochim. Biophys. Acta, 1721,174,2005.

33. Robards,K.etal.,Phenoliccompoundsandtheirroleinoxidativeprocessesinfruits,Food Chem.,66,401,1999.

34. Macheix,J.J.,Fleuriet,A.M.andBillot,J.,Fruit Phenolics,CRCPress,BocaRaton,FL,1990.

35. Heinonen,I.M.andMeyer,A.S.,Antioxidantsinfruits,berries,andvegetables,Fruit Vegetable Proc.,23,2002.

36. Proteggente,A.R.etal.,Therelationshipbetween thephenoliccompositionand theantioxidant activity of fruits and vegetables, in Flavonoids in Health and Disease, 2ndEd., Rice-Evans,C.A.andPacker,L.,Eds.,MarcelDekker, London,2003,71.

37. Decker,E.etal.,Wholegrainsasasourceofantioxidants,Cereal Foods World,47,370,2002.

38. Higdon,J.V.andFrei,B.,Teacatechinsandpolyphenols:Healtheffects,metabolism,andantioxidantfunctions,Crit. Rev. Food Sci. Nutr.,43,89,2003.

39. Gramza,A.andKorczak,J.,Teaconstituents(Camellia sinensisL.)asantioxidantsinlipidsystems,Trends Food Sci. Technol.,16,351,2005.

40. Grassmann,J.,Terpenoidsasplantantioxidants,Vitam. Horm.,72,505,2005. 41. Escuder,B.etal.,AntioxidantcapacityofabietanesfromSphacele salviae,Nat. Prod.

Letters,16,277,2002. 42. Grassmann,J.,Hippeli,S.andElstner,E.F.,Plantdefenseanditsbenefitsforanimals

and medicine: Role of phenolics and terpenoids in avoiding oxygen stress, Plant Physiol. Biochem.,40,471,2002.

43. Milde, J., Elstner, E.F. and Grassmann, J., Synergistic inhibition of low-density lipo-proteinoxidationbyrutin,γ-terpinene,andascorbicacid,Phytomedicine,11,105,2004.

44. Schieber,A.andCarle,R.,Occurrenceofcarotenoidcis-isomersinfood:Technologi-cal,analytical,andnutritionalimplications,Trends Food Sci. Technol.,16,416,2005.

45. Di Mascio, P., Kaiser, S. and Sies, H., Lycopene as the most efficient biologicalcarotenoidsingletoxygenquencher,Arch. Biochem. Biophys.,274,532,1989.

46. Bohm, V. et al., Trolox Equivalent Antioxidant Capacity of different geometricalisomersofα-carotene,β-carotene,lycopene,andzeaxanthin.,J. Agric. Food Chem.,50,221,2002.

47. Stahl,W.andSies,H.,Antioxidanteffectsofcarotenoids:Implicationinphotoprotec-tion inhumans, inHandbook of Antioxidants, 2nded.,Cadenas,E. andPacker,L.,Eds.,MarcelDekker,NewYork,2002,chap.11.

48. Stahl, W. and Sies, H., Antioxidant activity of carotenoids, Molecular Aspects of Medicine,24(6),345,2003.

49. Kiokias,S.andGordon,M.H.,Antioxidantpropertiesofcarotenoids in vitroand in vivo, Food Rev. Int.,20,99,2004.

7089_C009.indd 296 10/8/07 12:13:19 PM


50. Weber, S.U. and Rimbach, G., Biological activity of tocotrienols, in Handbook of Antioxidants, 2nded.,Cadenas,E.andPacker,L.,Eds.,MarcelDekker,NewYork,2002,chap.6.

51. Kitts,D.D.,Antioxidantpropertiesofcasein-phosphopeptides,Trends Food Sci. Technol.,16,549,2005.

52. Chen,L.,Remondetto,G.E.andSubirade,M.,Foodprotein-basedmaterialsasnutra-ceuticaldeliverysystems,Trends Food Sci. Technol., 17,272,2006.

53. Billaud,C.etal.,Maillardreactionproductsderivedfromthiolcompoundsasinhibitorsof enzymatic browning of fruits and vegetables: The structure-activity relationship,Ann. NY Acad. Sci.,1043,876,2005.

54. Somoza,V.,Fiveyearsof researchonhealthrisksandbenefitsofMaillardreactionproducts:Anupdate,Molec. Nutr. Food Res.,49,663,2005.

55. Kim,S.K.andRajapakse,N.,Enzymaticproductionandbiologicalactivitiesofchitosanoligosaccharides(COS):Areview,Carbohydr. Polymers,62,357,2005.

56. Hall, C., Sources of natural antioxidants: Oilseeds, nuts, cereals, legumes, animalproducts and microbial sources, in Antioxidants in Food, 1st ed., Pokorny, J.,Yanishlieva, N. and Gordon, M., Eds., Woodhead Publishing Limited, Abington,Cambridge,England,2001.

57. Nakagami, T., Tamura, N. and Nakamura, T., Plant phenol compounds as anti-complement agents and therapeutics for complement-associated diseases and healthfoodscontainingthem,Jpn. Kokai Tokkyo Koho,7,1995,JP07223941.

58. Fernández,M.A.,Sáenz,M.T.andGarcía,M.D.,Anti-inflammatoryactivityinratsandmiceofphenolicacidsisolatedfromScrophularia frutescens,J. Pharm. Pharmacol.,50,1183,1998.

59. Zang,L.Y.etal.,Effectofantioxidantprotectionbyp-coumaricacidonlow-densitylipoproteincholesteroloxidation,Am. J. Physiol. Cell Physiol.,279,C954,2000.

60. Cartron,E.etal.,Specificantioxidantactivityofcaffeoylderivativesandothernaturalphenoliccompounds:LDLprotectionagainstoxidationanddecreaseintheproinflam-matorylysophosphatidylcholineproduction,J. Nat. Prod.,64,480,2001.

61. Kweon,M.H.,Hwang,H.J.andSung,H.C.,Identificationandantioxidantactivityofnovelchlorogenicacidderivativesfrombamboo(Phyllostachys edulis),J. Agric. Food Chem.,49,4646,2001.

62. Krishnaswamy,K.,Nonnutrientsandcancerprevention,ICMRBull,31,2001,availableathttp://www.icmr.nic.in/bujan01.pdf

63. Taniguchi,H.etal.,FerulicacidestersasantioxidantsandUVabsorbents,European Pat. Appl.,EP681825,1995.

64. Lee,H.S.,InhibitoryactivityofCinnamomum cassiabark-derivedcomponentagainstratlensaldosereductase,J. Pharm. Pharmaceut. Sci.,5,226,2002.

65. Aviram, M., Vaya, J. and Fuhrman, B., Licorice root flavonoid antioxidants reduceLDLoxidationandattenuatecardiovasculardiseases,Oxid. Stress Dis., 14(HerbalandTraditionalMedicine), MarcelDekker,Inc.,2004,595.

66. Aviram,M.,Kaplan,M.,Rosenblat,M. andFuhrman,B.,Dietary antioxidants andparaoxonases against LDL oxidation and atherosclerosis development, Handb. Exp. Pharmacol.,170(Atherosclerosis),2005,263.

67. Kris-Etherton, P.M. et al., Bioactive compounds in nutrition and health-researchmethodologiesforestablishingbiologicalfunction:Theantioxidantandanti-inflammatoryeffectsofflavonoidsonatherosclerosis,Annu. Rev. Nutr.,24,511,2004.

68. Fuhrman,B.andAviram,M.,PolyphenolsandflavonoidsprotectLDLagainstathero-genicmodifications,Oxididative Stress Disease,8(HandbookofAntioxidants),2002,303–336.

69. Nijveldt,R.J.etal.,Flavonoids:Areviewofprobablemechanismsofactionandpoten-tialapplications,Am. J. Clin. Nutr.,74,418,2001.

7089_C009.indd 297 10/8/07 12:13:19 PM


70. Marilyn, E., Dietary flavonoids: Effects on xenobiotic and carcinogen metabolism,Toxicol. in Vitro,20,187,2006.

71. Choi,M.S.etal.,Cholesterol-loweringpropertiesofcitrusflavonoidsandpolyphenoliccompoundsandtheirrelevancetoantioxidativeactivity,Nutr. Sci.,6,31,2003.

72. Horvathova,K.,Vachalkova,A.andNovotny,L.,Flavonoidsaschemoprotectiveagentsincivilizationdiseases,Neoplasma,48,435,2001.

73. Spencer, J.P.E., Interactions of flavonoids and their metabolites with cell signalingcascades,Oxididative Stress Disease,17(Nutrigenomics),2005,353.

74. Ghosh, D., Anthocyanins and anthocyanin-rich extracts in biology and medicine:Biochemical,cellular,andmedicinalproperties,Curr. Top. Nutraceutical Res.,3,113,2005.

75. Depeint,F. et al.,Evidence for consistentpatternsbetweenflavonoid structuresandcellularactivities,Proc. Nutr. Soc.,61,97,2002.

76. Erlund, I., Review of the flavonoids quercetin, hesperetin, and naringenin. Dietarysources,bioactivities,bioavailability,andepidemiology,Nutr. Res.,24,851,2004.

77. VanHoorn,D.E.C.etal.,Biologicalactivitiesofflavonoids, Sci. Med.,9,152,2003. 78. Das,S.andRosazza,J.P.N.,Microbialandenzymatic transformationsofflavonoids,

J. Nat. Prod.,69(3),499,2006. 79. Hodek,P.,Trefil,P.andStiborova,M.,Flavonoids:Potentandversatilebiologically

activecompounds interactingwithcytochromesP450,Chem.-Biol Interact.,139,1,2005.

80. Kim,H.P.etal.,Anti-inflammatoryflavonoids:Modulatorsofproinflammatorygeneexpression,Nat. Prod. Sci.,10,1,2004.

81. Kim,H.P.etal.,Anti-inflammatoryplantflavonoidsandcellularactionmechanisms,J. Pharmacol. Sci.,96,229,2004.

82. Strickland,F.M.,Boostingtheimmunesystem,Comprehensive Series in Photosciences,3(SunProtectioninMan),2001,613,615–636.

83. Hoensch,H.P.andKirch,W.Potentialroleofflavonoidsinthepreventionofintestinalneoplasia: A review of their mode of action and their clinical perspectives, Int.J. Gastroint. Cancer,35,187,2005.

84. Dajas,F. et al.,Flavonoidsand thebrain:Evidencesandputativemechanisms foraprotectivecapacity,Curr. Neuropharmacol.,3,193,2005.

85. Schroeter,H.andSpencer,J.P.E.,Flavonoids:Neuroprotectiveagents?Modulationofoxidativestress-inducedMAPkinasesignaltransduction,Oxididative Stress Disease,9(FlavonoidsinHealthandDisease(SecondEd.)),2003,233–272.

86. Choi,H.S.etal.,Radical-scavengingactivitiesofcitrusessentialoilsandtheircompo-nents:Detectionusing1,1-diphenyl-2-picrylhydrazyl,J. Agric. Food Chem., 48,4156,2000.

87. Singh,R.P.andAgarwal,R.,Flavonoidantioxidantsilymarinandskincancer,Antioxid. Redox Signaling,4,655,2002.

88. Walle,T.,Absorptionandmetabolismofflavonoids,Free Rad. Biol. Med.,36,829,2004.

89. Kroon,P.A.etal.,Howshouldweassesstheeffectsofexposuretodietarypolyphenolsinvitro?,Am. J. Clin. Nutr.,80,15,2004.

90. Schroeter,H.etal.,MAPKsignaling inneurodegeneration: Influencesofflavonoidsandofnitricoxide,Neurobiol. Aging,23,861,2002.

91. Youdim,K.A.etal.,Dietaryflavonoidsaspotentialneuroprotectants,Biol. Chem.,383,503,2002.

92. Wagner,K.H.andElmadfa,I.,Biologicalrelevanceofterpenoids.Overviewfocusingonmono-,di-andtetraterpenes,Ann. Nutr. Metab.,47,95,2003.

93. Pietri,S.etal.,Cardioprotectiveandanti-oxidanteffectsoftheterpenoidconstituentsofGinkgobilobaextract(EGb761), J. Mol. Cell. Cardiol., 29,733,1997.

7089_C009.indd 298 10/8/07 12:13:20 PM


94. Ulubelen, A., Cardioactive and antibacterial terpenoids from some Salvia species,Phytochem., 64,39,2003.

95. De las Heras, B. et al., Terpenoids: Sources, structure elucidation and therapeuticpotentialininflammation,Curr. Top. Med. Chem.,3,171,2003.

96. Perrucci, S. et al., Structure/activity relationship of some natural monoterpenes asacaricidesagainstPsoroptes cuniculi,J. Nat. Prod.,58,1261,1995.

97. Wang,B.J.etal.,AntioxidantactivityofBupleurum kaoiLiu(ChaoetChuang)frac-tionsfractionatedbysupercriticalCO2,Lebensm. Wiss. Technol.,38,281,2005.

98. Deming,D.M.etal.,Carotenoids:Linkingchemistry,absorption,andmetabolismtopotential roles inhumanhealth anddisease, inHandbook of Antioxidants, 2nded.,Cadenas,E.andPacker,L.,Eds.,MarcelDekker,NewYork,2002,chap.10.

99. Granado,F.,Olmedilla,B.andBlanco,I.,Nutritionalandclinicalrelevanceofluteininhumanhealth,British J. Nutr.,90,487,2003.

100. Hix, L.M., Lockwood, S.F. and Bertram, J.S., Bioactive carotenoids: Potent anti-oxidantsandregulatorsofgeneexpression,Redox Report,9,181,2004.

101. Lowe,G.M.,Vlismas,K.andYoung,A.J.,Carotenoidsasprooxidants?,Mol. Aspects Med.,24,363,2003.

102. Sharoni,Y. et al.,Modulationof transcriptional activitybyantioxidant carotenoids,Mol. Aspects Med.,24,371,2003.

103. Landvik,S.V.,Diplock,A.T.andPacker,L.,EfficacyofvitaminE inhumanhealthanddisease,inHandbook of Antioxidants,2nded.,Cadenas,E.andPacker,L.,Eds.,MarcelDekker,NewYork,2002,chap.4.

104. Violi,F.,Cangemi,R.andLoffredo,L.,VitaminsEandCforpreventionofcardio-vasculardisease,Curr. Dev. Atheroscler. Res.,117,2006.

105. Traber, M.G., Vitamin E bioavailability, biokinetics and metabolism, Handbook of Antioxidants,2nded.,Cadenas,E.andPacker,L.,Eds.,MarcelDekker,NewYork,2002,chap.5.

106. Palma,M.etal.,Fractionalextractionofcompoundsfromgrapeseedsbysupercriticalfluidextractionandanalysis forantimicrobial andagrochemical activities,J. Agric. Food Chem.,47,5044,1999.

107. Leal,P.F.etal.,Functionalpropertiesofspiceextractsobtainedviasupercriticalfluidextraction,J. Agric. Food Chem., 51,2520,2003.

108. Vági, E. et al., Essential oil composition and antimicrobial activity of Origanum majorana L. extracts obtained with ethyl alcohol and supercritical carbon dioxide,Food Res. Int.,38,51,2005.

109. Arlorio, M. et al., Antioxidant and biological activity of phenolic pigments fromTheobroma cacao hulls extracted with supercritical CO2, Food Res. Int., 38, 1009,2005.

110. McHugh, M.A. and Krukonis, V.J., Supercritical Fluid Extraction: Principles and Practice,Butterworth,Stoneham,MA,1986.

111. Ko,T.F.etal.,AntimutagenicityofsupercriticalCO2extractsofTerminalia catappaleavesandcytotoxicityoftheextractstohumanhepatomacells,J. Agric. Food Chem.,51,3564,2003.

112. Miquel,E.M.,Nissen,L.R.andSkibsted,L.H.,Antioxidantevaluationprotocols:Foodqualityorhealtheffects.Eur. Food Res. Technol.,219,561,2004.

113. Gordon, M.H., Measuring antioxidant activity, in Antioxidants in Food: Practical Applications,1sted.,Pokorný, J.,Yanishlieva,N.andGordon,M.,Eds.,WoodheadPublishingLimited,Abington,Cambridge,England,2001.

114. Decker,E.A.etal.,Measuringantioxidantreffectivenessinfood,J. Agric. Food Chem.,53,4303,2005.

7089_C009.indd 299 10/8/07 12:13:20 PM


115. Halliwell,B.,Food-derivedantioxidants:Howtoevaluatetheirimportanceinfoodandin vivo,Handbook of Antioxidants,2nded.,Cadenas,E.andPacker,L.,Eds.,MarcelDekker,NewYork,2002,chap.1.

116. Sánchez-Moreno,C.,Methodsusedtoevaluatethefreeradicalscavengingactivityinfoodsandbiologicalsystems,Food Sci. Technol. Int.,8,121,2002.

117. Liu,R.H.andFinley,J.,Potentialcellculturemodelsforantioxidantresearch,J. Agric. Food Chem., 53,4311,2005.

118. Halliwell,B.,Oxidativestressincellculture:Anunder-appreciatedproblem?,FEBS Letters,540,1,2003.

119. Halliwell, B. and Whiteman, M., Measuring reactive species and oxidative damageinvivoandcellculture:Howshouldyoudoitandwhatdotheresultsmean?,Br. J. Pharmacol.,142,231,2004.

120. El-Ghorab,A.H.etal.,AntioxidantactivityofEgyptianEucalyptus camaldulensis var. brevirostrisleafextracts,Nahrung,47,41,2003.

121. Dapkevicius,A.etal.,AntioxidantactivityofextractsobtainedbydifferentisolationproceduresfromsomearomaticherbsgrowninLithuania,J. Sci. Food Agric.,77,140,1998.

122. Carvalho,R.N.,Supercriticalfluidextractionfromrosemary(Rosmarinus officinalis):Kineticdata,extract’sglobalyield,composition,andantioxidantactivity,J. Supercrit. Fluids,35,197,2005.

123. Ashraf-Khorassani,M.andTaylor,L.T.,Sequentialfractionationofgrapeseedsintooils,polyphenols,andprocyanidinsviaasinglesystememployingCO2-basedfluids,J. Agric. Food Chem.,2440,2004.

124. Nguyen,U.etal.,Processforextractingantioxidantsfromlabiataeherbs,U.S. Patent,US5017397,1991.

125. Simándi,B.etal.,Supercriticalcarbondioxideextractionandfractionationoforeganooleoresin,Food Res. Int.,31,723,1998.

126. Esquível, M.M., Ribeiro, M.A. and Bernardo-Gil, M.G., Supercritical extraction ofsavory oil: Study of antioxidant activity and extract characterization, J. Supercrit. Fluids,14,129,1999.

127. del Valle, J.M. et al., Recovery of antioxidants from boldo (Peumus boldus M.) byconventionalandsupercriticalCO2extraction.Food Res. Int.,37,695,2004.

128. Quitain,A.T.,Katoh,S.andMoriyoshi,T.,Isolationofantimicrobialsandantioxidantsfrommoso-bamboo (Phyllostachys heterocycla)by supercriticalCO2extractionandsubsequenthydrothermal treatmentof the residues, Ind. Eng. Chem. Res.,43,1056,2004.

129. López-Sebastian,S.etal.,Dearomatizationofantioxidantrosemaryextractsbytreat-mentwithsupercriticalcarbondioxide,J. Agric. Food Chem.,46,13,1998.

130. Hadolin, M. et al., Isolation and concentration of natural antioxidants with high-pressureextraction,Innov. Food Sci. Emerging Technol.,5,245l,2004.

131. Murga,R.etal.,Extractionofnaturalcomplexphenolsandtanninsfromgrapeseedsbyusingsupercriticalmixturesofcarbondioxideandalcohol,J. Agric. Food Chem.,48,3408,2000.

132. Gast,K.,Machado,N.andBrunner,G.,Countercurrentextractionofvitaminsfromcrudepalmoil,inState of the Art Book on Supercritical Fluids, Ainia,Spain,2004,chap.20.

133. Señoráns,F.J.etal., Isolationofantioxidantcompoundsfromorange juicebyusingcountercurrentsupercriticalfluidextraction(CC-SFE),J. Agric. Food Chem.,49,6039,2001.

134. Ribeiro,M.A.,Bernardo-Gil,M.G.andEsquível,M.M.,Melissa officinalisL.:Studyofantioxidantactivityinsupercriticalresidues,J. Supercrit. Fluids,21,51,2001.

7089_C009.indd 300 10/8/07 12:13:21 PM


135. Louli,V.,Ragoussis,N. andMagoulas,K.,Recoveryofphenolic antioxidants fromwineindustryby-products,Biores. Technol., 92, 201,2004.

136. Ibáñez, E. et al., Supercritical fluid extraction and fractionation of different pre-processedRosemaryplants,J. Agric. Food Chem.,47,1400,1999.

137. Guglü-Üstündag,O.andTemelli,F.,Correlatingthesolubilitybehaviorofminorlipidcomponentsinsupercriticalcarbondioxide,J. Supercrit. Fluids,31,235,2004.

138. Sanal,I.S.etal.,RecyclingofapricotpomacebysupercriticalCO2extraction,J. Super-crit. Fluids,32,221,2004.

139. Vasapollo,G.etal.,InnovativesupercriticalCO2extractionoflycopenefromtomatointhepresenceofvegetableoilasco-solvent,J. Supercrit. Fluids,29,87,2004.

140. Cortesi,A.etal.,Effectofchemicalstructureonthesolubilityofantioxidantsinsuper-critical carbon dioxide: Experimental data and correlation, J. Supercrit. Fluids, 14,139,1999.

141. Johannsen,M.andBrunner,G.,Solubilitiesofthefat-solublevitaminsA,D,E,andKinsupercriticalcarbondioxide,J. Chem. Eng. Data,42,106,1997.

142. Fornari,T.etal.,Anewdevelopmentintheapplicationofthegroupcontributionasso-ciatingequationofstatetomodelsolidsolubilitiesofphenoliccompoundsinSC-CO2,Ind. Eng. Chem. Res.,44,8147,2005.

143. Christov, M. and Dohrn, R., High-pressure fluid phase equilibria: Experimentalmethods and systems investigated (1994–1999), Fluid Phase Equilibria, 202, 153,2002.

144. Lucien, F. and Foster, N.R., Solubilities of solid mixtures in supercritical carbondioxide:Areview,J. Supercrit. Fluids,17,111,2000.

145. Díaz,S.,Espinosa,S.andBrignole,E.A.,Citruspeeloildeterpenationwithsupercriticalfluids,J. Supercrit. Fluids,35,49,2005.

146. Chrastil,J.,SolubilityofsolidsandliquidsinsupercriticalCO2,J. Phys. Chem.,86,3016,1982.

147. Chen, C.-C. et al., Vapor-liquid equilibria of carbon dioxide with linoleic acid,α-tocopherol,andtrioleinatelevatedpressures,Fluid Phase Equilibria,175,107,2000.

148. Skerget,M.andKnez,Z.,Solubilityofbinarysolidmixtureβ-carotene-capsaicinindenseCO2,J. Agric. Food Chem., 45,2066,1997.

149. Pereira, P.J. et al., High pressure phase equilibrium forα-tocopherol + CO2, Fluid Phase Equilibria,216,53,2004.

150. Fang,T.etal.,Phaseequilibriaforbinarysystemsofmethyloleate-supercriticalCO2andα-tocopherol-supercriticalCO2,J. Supercrit. Fluids,30,1,2004.

151. Fang,T.etal.,Phaseequilibria for the ternarysystemmethyloleate+ tocopherol+supercriticalCO2,J. Chem. Eng. Data,50,390,2005.

152. Stoldt,J.,Saure,C.andBrunner,G.,Phaseequilibriaoffatcompoundswithsupercriticalcarbondioxide,Fluid Phase Equilibria,116,399,1996.

153. Stoldt,J.andBrunner,G.,Phaseequilibriummeasurementsincomplexsystemsoffats,fat compounds, and supercritical carbon dioxide, Fluid Phase Equilibria, 146, 269,1998.

154. Cygnarowicz, M.L., Maxwell, R.J. and Seider, W.D., Equilibrium solubilities ofβ-caroteneinsupercriticalcarbondioxide,Fluid Phase Equilibria,59,57,1990.

155. Tuma,D.andSchneider,G.M.,Determinationofthesolubilitiesofdyestuffsinnear-and supercriticalfluidsbya staticmethodup to180MPa.,Fluid Phase Equilibria,158–160,743,1999.

156. Saldaña,M.D.A.etal.,Comparisonofthesolubilityofβ-caroteneinsupercriticalCO2basedonabinaryandamulticomponentcomplexsystem,J. Supercrit. Fluids,37,342,2006.

7089_C009.indd 301 10/8/07 12:13:21 PM


157. Skerget,M.,Knez,Z.andHabulin,M.,Solubilityofβ-caroteneandoleicacidindenseCO2anddatacorrelationbyadensitybasedmodel,Fluid Phase Equilibria,109,131,1995.

158. de la Fuente, J.C., Solubility of carotenoid pigments (lycopene and astaxanthin) insupercriticalcarbondioxide,Fluid Phase Equilibria,247,90,2006.

159. Dohrn,R.andBrunner,G.,High-pressurefluid-phaseequilibria:Experimentalmethodsandsystemsinvestigated(1988–1993),Fluid Phase Equilibr., 106,213,1995.

160. Baumann, D. et al., Supercritical carbon dioxide extraction of marigold at highpressures:Comparisonofanalyticalandpilot-scaleextraction,Phytochem. Anal.,15,226,2004.

161. Tipsrisukond,N.,Fernando,L.N.andClarke,A.D.,Antioxidanteffectsofessentialoilandoleoresinofblackpepperfromsupercriticalcarbondioxideextractionsingroundpork,J. Agric. Food Chem.,46,4329,1998.

162. Yépez,B.etal.,Producingantioxidantfractionsfromhernaceousmatricesbysuper-criticalfluidextraction,Fluid Phase Equilibria,194–197,879,2002.

163. Yang, C., Xu, Y.R. and Yao, W.X., Extraction of pharmaceutical components fromGinkgo biloba leaves using supercritical carbon dioxide, J. Agric. Food Chem., 50,846,2002.

164. Luengthanaphol,S.etal.,ExtractionofantioxidantsfromsweetThaitamarindseedcoat-preliminaryexperiments,J. Food Eng.,63,247,2004.

165. Giannuzzo,A.N.etal.,SupercriticalfluidextractionofnaringinfromthepeelofCitrus paradisi,Phytochem. Anal.,14,221,2003.

166. López,M.etal.,Selectiveextractionofastaxanthinfromcrustaceansbyuseofsuper-criticalcarbondioxide,Talanta,64,726,2004.

167. Machmudah,S.etal.,ExtractionofastaxanthinfromHaematococcus pluvialisusingsupercriticalCO2andethanolasentrainer,Ind. Eng. Chem. Res.,45,3652,2006.

168. Jarén-Galán, M., Nienaber, U. and Schwartz, S.J., Paprika (Capsicum annuum)oleoresinextractionwithsupercriticalcarbondioxide, J. Agric. Food Chem.,47,3558,1999.

169. Montero,O.etal.,SupercriticalCO2extractionofβ-carotenefromamarinestrainoftheCyanobacterium synechococcusspecies, J. Agric. Food Chem.,53,9701,2005.

170. Baysal, T. et al., Supercritical CO2 extraction of beta-carotene and lycopene fromtomatopastewaste, J. Agric. Food Chem.,48,5507,2000.

171. Hadolin, M. et al. High pressure extraction of vitamin E–rich oil from Silybum marianum, Food Chem.,74,355,2001.

172. Nagesha, G. K., Manohar, B. and Udaya Sankar, K., Enrichment of tocopherols inmodifiedsoydeodorizerdistillateusingsupercriticalcarbondioxideextraction,Eur. Food Res. Technol.,217,427,2003.

173. Rozzi,N.L.etal.,Supercriticalfluidextractionof lycopene fromtomatoprocessingby-products,J. Agric. Food Chem.,50,2638,2002.

174. Ge,Y.etal.,ExtractionofnaturalvitaminEfromwheatgermbysupercriticalcarbondioxide,J. Agric. Food Chem.,50,685,2002.

175. Ge,Y.etal.,OptimizationofthesupercriticalfluidextractionofnaturalvitaminEfromwheatgermusingresponsesurfacemethodology,J. Food Sci.,67,239,2002.

176. Damjanovic, B. et al., Extraction of fennel (Foeniculum vulgare Mill.) seeds withsupercriticalCO2:Comparisonwithhydrodistillation,Food Chem.,92,143,2005.

177. Smith, R.L., Jr. et al., Separation of cashew (Anacardium occidentale L.) nut shellliquidwithsupercriticalcarbondioxide,Biores. Technol.,88,1,2003.

178. Seger, C. et al., Characterization of supercritical fluid extracts of St. John’s Wort(Hypericum perforatumL.)byHPLC-MSandGC-MS,Eur. J. Pharm. Sci.,21,453,2004.

7089_C009.indd 302 10/8/07 12:13:22 PM


179. Vági,E.etal.,PhenolicandtriterpenoidantioxidantsfromOriganum majoranaL.herbandextractsobtainedwithdifferentsolvents,J. Agric. Food Chem.,53,17,2005.

180. Hu,Q.,Hu,Y.andXu,J.,Freeradical-scavengingactivityofAloe vera(Aloe barbadensisMiller)extractsbysupercriticalcarbondioxideextraction,Food Chem.,91,85,2005.

181. Scalia, S., Giuffreda, L. and Pallado, P., Analytical and preparative supercriticalfluidextractionofchamomileflowersanditscomparisonwithconventionalmethods,J. Pharm. Biomed. Anal.,21,549,1999.

182. Wang,H.C.,Chen,C.R.andChang,C.J.,Carbondioxideextractionofginsengroothairoilandginsenosides,Food Chem., 72,505,2001.

183. Vaher,M.andKoel,M.,Separationofpolyphenoliccompoundsextractedfromplantmatricesusingcapillaryelectrophoresis,J. Chromatogr. A,990,225,2003.

184. Daukšas,E.etal.,Rapidscreeningofantioxidantactivityofsage(Salvia officinalisL.)extractsobtainedbysupercriticalcarbondioxideatdifferentextractionconditions,Nahrung,45,338,2001.

185. Lim,G.B.etal.,SeparationofastaxanthinfromredyeastPhaffia rhodozymabysuper-criticalcarbondioxideextraction,Biochem. Eng. J.,11,181,2002.

186. Suto, K. et al.,Determination ofmagnolol andhonokiol inMagnoliae cortexusingsupercriticalfluidchromatographyon-linecoupledwithsupercriticalfluidextractionbyon-columntrapping,J. Chromatogr. A, 786,366,1997.

187. Moraes, M.L.L., Vilegas, J.H.Y. and Lanças, F.M., Supercritical fluid extraction ofglycosilatedflavonoidsfromPasiflora leaves,Phytochem. Anal., 8,257,1997.

188. Goli, A.H. et al., Antioxidant activity and total phenolic compounds of pistachio(Pistachia vera)hullextracts,Food Chem.,92,521,2005.

189. Rostagno,M.A.,Araujo, J.M.A. andSandi,D.,Supercriticalfluid extractionof iso-flavonesfromsoybeanflour,Food Chem.,78,111,2002.

190. Zancan,K.C.etal.,Extractionofginger(Zingiber officinaleRoscoe)oleoresinwithCO2andco-solvents:Astudyof theantioxidantactionof theextracts,J. Supercrit. Fluids,24,57,2002.

191. Braga,M.E.M.,Comparisonofyield,composition,andantioxidantactivityofTurmeric(Curcuma longaL.)extractsobtainedusingvarioustechniques,J. Agric. Food Chem.,51,6604,2003.

192. Cadoni,E.etal.,SupercriticalCO2extractionof lycopeneandβ-carotenefromripetomatoes,Dyes Pigments,44,27,1999.

193. Sun,M.andTemelli,F.,Supercriticalcarbondioxideextractionofcarotenoidsfromcarrotusingcanolaoilasacontinuousco-solvent,J. Supercrit. Fluids,37,397,2006.

194. Grigonis,D.etal.,Comparisonofdifferentextractiontechniquesforisolationofanti-oxidantsfromsweetgrass(Hierochloë odorata),J. Supercrit. Fluids,33,223,2005.

195. Simándi,B.etal.,Antioxidantactivityofpilot-plantalcoholicandsupercriticalcarbondioxideextractsofthyme,Eur. J. Lipid Sci. Technol.,103,355,2001.

196. Fadel,H.etal.,Effectofextractiontechniquesonthechemicalcompositionandanti-oxidantactivityofEucalyptus camaldulensisvar.brevirostrisleafoils,Z. Lebensm. Unters. Forsch.,208,212,1999.

197. Stashenko,E.E.,Jaramillo,B.E.andMartínez,J.R.,Comparisonofdifferentextrac-tionmethodsfortheanalysisofvolatilesecondarymetabolitesofLippia alba(Mill.)N.E. brown, grown in Colombia, and evaluation of its in vitro antioxidant activity,J. Chromatogr. A,1025,93,2004.

198. Xu,J.,Chen,S.andHu,Q.,Antioxidantactivityofbrownpigmentandextractsfromblacksesameseed(Sesamum indicumL.), Food Chem.,91,79,2005.

7089_C009.indd 303 10/8/07 12:13:23 PM

7089_C009.indd 304 10/8/07 12:13:23 PM

305

10 Essential Oils Extraction and Fractionation Using Supercritical Fluids

Ernesto Reverchon and Iolanda De Marco

Contents

10.1 Introduction.................................................................................................30510.2 SolidsProcessing........................................................................................307

10.2.1 SelectionoftheOperatingParameters..........................................30810.2.2 Examples........................................................................................ 312

10.2.2.1 Leaves............................................................................ 31210.2.2.2 Flowers.......................................................................... 31410.2.2.3 Seeds.............................................................................. 31410.2.2.4 OtherMatrices............................................................... 31410.2.2.5 FlowerConcretesFractionation..................................... 316

10.3 LiquidFeedProcessing............................................................................... 31810.3.1 SelectionoftheOperatingParameters.......................................... 31910.3.2 Examples........................................................................................ 320

10.4 AntisolventExtraction................................................................................ 32210.4.1 SelectionoftheOperatingParameters.......................................... 32210.4.2 Examples........................................................................................ 323

10.4.2.1 ProteinsandAromaExtractionfromTobacco.............. 32310.5 MathematicalModelling.............................................................................324References.............................................................................................................. 328

10.1 IntroduCtIon

The extraction from natural sources is the most widely studied application ofsupercriticalfluids(SCFs),andseveralhundredscientificpapersonthetopichavebeenpublishedandreviewed[1–9].Indeed,supercriticalfluidextraction(SFE)hasimmediateadvantagesovertraditionalextractiontechniques;itisaflexibleprocessduetothepossibilityofcontinuousmodulationofthesolventpower/selectivityoftheSCF,anditallowstheeliminationofpollutingorganicsolventsandoftheexpensivepostprocessingoftheextractsforsolventelimination.

Several compounds have been examined as SFE solvents, including hydrocarbonssuchashexane,pentaneandbutane,nitrousoxide,sulfurhexafluoride,andfluorinatedhydrocarbons[10].However,carbondioxide(CO2)isthemostpopular

7089_C010.indd 305 10/8/07 12:15:39 PM


SFEsolventbecauseitissafe,isreadilyavailable,andhasalowcost.Itallowssupercriticaloperationsatrelativelylowpressuresandatnearroomtemperatures.

TheonlyseriousdrawbackofSFEisthatinvestmentcostsarehigherthanthosefortraditionalatmosphericpressureextractiontechniques.However,thebaseprocessscheme(extractionplusseparation)isrelativelycheapandverysimpletobescaleduptoindustrialscale.

Inthischapter,wefocusouranalysisontheextraction,isolation,andfractionationof essentialoils.EarlyworksonSFEof essentialoils frequentlyusedhighpressures (>350bar),evenwhensupercriticalCO2 (SCCO2)–solublecompoundshadtobeextracted(forexample,terpenes,sesquiterpenes,fattyacids).Operatinginthismanner,thesolventpoweroftheSCFwasenhanced,butitsselectivitywasverylow.Sincethen,theconceptoftheoptimizationbetweensolventpowerandselectivityhasbeenappliedandSFEoperatingconditionshavebeenchosentoobtaintheselectiveextractionofthecompoundsofinterest,reducingtoaminimumthecoextractionofundesiredcompounds[1].Moreover,forsuccessfulextraction,notonlymustthesolubilityofthecompoundstobeextractedbetakenintoaccountbutalsothesolubilitiesoftheundesiredcompounds.Masstransferresistancesduetothestructureof therawmaterialand to thespecific locationof thecompounds tobeextractedcanalsoplayarelevantrole.Microscopicanalysisofthenaturalstructurecanhelpinunderstandingwheremasstransferresistancesarelocated.Specificexperimentsperformedvaryingparticlesizeandsupercriticalsolventresidencetimecanalsobehelpful in thissense.Thecomplex interplaybetween thermodynamics(solubility)andkinetics(masstransfer)hastobeunderstoodtoproperlyperformSFE.

Fractionalseparationoftheextractsisanotherwellknownconceptthatcanbeuseful in improvingSFEselectivity. Indeed, in severalcases, it isnotpossible toavoidthecoextractionofsomecompoundfamiliesthatshowdifferentsolubilities,buttherearealsodifferentmasstransferresistancesintherawmatter.Inthesecases,onecanperformanextractioninsuccessivestepsatincreasingpressurestoobtainthefractionalextractionofthesolublecompoundscontainedintheorganicmatrix.FractionalseparationallowsfractionationoftheSCFextracts,equippingtheplantwithsomeseparationvesselsoperatinginseriesatdifferentpressuresandtemperatures.ThescopeofthisoperationistoinducetheselectiveprecipitationofdifferentcompoundfamiliesbasedondifferentsaturationconditionsintheSCF.

Inseveralothercases,thefeedisaliquidmixture.Therefore,theprocesstobeappliedisthecontinuousliquidextractioninapackedtower.Notethat,althoughtheextractionfromsolidsisadiscontinuousoperation,thepackedtoweriscapableofcontinuoussteadystateoperation thatallows theprocessingof largequantitiesofliquidmixturesinarelativelysmallapparatusandinashorttime.

Insomeothercases,thematerialtobetreatedisaliquidmixturethatcontainssolidcompoundsdissolvedinit.Theextractionofthesecompoundsfromtheliquidsolutioncannotbeperformedinapackedtowersincethesolidmatterprecipitatesonthepackingsofthebed.Inthiscase,supercriticalantisolventextraction(SAE)canbeadopted.ThepreconditionstoapplySAEaresimilartotheonescharacteristicof supercritical antisolventmicronization (SAS): the liquid solventhas tobeverysoluble inSCCO2,whereas the solidshave tobe insoluble in theSCF.Thescope of SAE is not the micronization but the purification of the liquid solution

7089_C010.indd 306 10/8/07 12:15:39 PM

Essential Oils Extraction and Fractionation Using Supercritical Fluids 307

fromundesiredsubstances.Theseconditionscanbefrequentlyobtainedsincemanyorganic solvents are readily soluble inSCCO2evenatmildoperatingconditionsand many highmolecularweight solids show negligible solubilities in SCCO2,especiallyatlowCO2densities.

Duetothestructuralcomplexityandvariability(withseason,kind,crop,etc.)of the materials to be treated and to the large variety of compounds that can beextracted (different molecular weights, polarities, links with the structure, etc.),theseprocessesarefar frombeingexhaustivelystudied,althoughsomeindustrialapplicationshavealreadybeendeveloped.Moreover,anincreasinginteresthasbeenregisteredintheextractionofhighaddedvalueessentialoils,suchasoilsthatshowantioxidantandpharmaceuticalproperties.

Therefore,inthischapter,weanalyzeSFE,SAE,andliquidfractionationstudiesperformedonessentialoils and relatedmaterials andconsider the evolutionof theextractionprocesses,products,andmaterialstreated.Acriticalanalysisisperformed.

10.2 solIds ProCessIng

SolidsprocessingisthemoststudiedSCFapplicationbecausethemostfrequentlyrequiredseparationprocessistheextractionofoneormorecompoundfamiliesfromasolidnaturalmatrix.Thebasicextractionschemeconsistsofanextractionvesselchargedwiththerawmattertobeextracted.SCFattheexitoftheextractorflowsthroughadepressurizationvalvetoaseparatorinwhich,duetothelowerpressure,the extracts are released from the gaseous medium and collected. As a rule, thestartingmaterialisdriedandgrindedtofavortheextractionprocessandisloadedin a basket located inside the extractor to allow fast charge anddischargeof theextractionvessel.

Moresophisticatedextractionschemes,suchastheonereportedinFigure10.1,containtwoormoreseparators.Inthiscase,itispossibletofractionatetheextractintwoormorefractionsofdifferentcompositionsbysettingopportunetemperaturesandpressuresintheseparators[11–28].Solidspreprocessingisalsoaparameterthatcanlargelyinfluenceseparationperformance.Forexample,soliddryingandparticlesizeoptimization,asarule,havetobetakenintoaccount.

Dryingof thesolidmaterials is frequentlyrequiredbeforeextractionbecauserawvegetablemattercancontainupto90%water.Waterisonlyslightlysolublein

2

1

3 4 OutSCF IN

FIgure 10.1 1)CO2pump;2)extractor;3)firstseparator;4)secondseparator.

7089_C010.indd 307 10/8/07 12:15:41 PM


SCCO2atthecommonextractionconditions;but,duringthepressurizationoftheextractorcanbepartlyexpressedfromthevegetablematerialandtravelsalongtheplanttogetherwiththesupercriticalsolvent.Moreover,waterinthesolidstructurecan obstacle SCCO2 penetration (diffusion), lowering extraction efficiency. As arule,watercontentsbetween5%and10%w/warerequiredtoperformSFEproperly.Fortunately,thesewaterpercentagesarealsotheonesusuallypresentindriedmaterials.Attention shouldbepaid to the selecteddryingprocessbecause it canlargelyinfluencethefinalcontentofvolatilecompoundsinthetreatedmaterial.Insomeparticularcases,thepresenceofwaterisnotdetrimentalforSFE,forexample,inthecaseofcaffeineextractionfromcoffeebeans,becauseinthatcasewaterunhooksthecaffeinesodiumsaltfromthevegetablematrix.

Other possible variations of the SFE solid processing scheme are multistageextractionandcosolventsaddition.Multistepoperationinvolvesvaryingpressureortemperatureineachprocessstep[29,30].ThisstrategycanbeusedwhentheextractionofseveralcompoundfamiliesfromthesamematrixthatshowdifferentsolubilitiesinSCCO2isrequired.IttakesadvantageofthefactthatSCCO2solventpowercanbecontinuouslyvariedwithpressureandtemperature.Forexample,itispossibletoperformafirstextractionoperatingatlowCO2density(e.g.,0.29g/cm3,90bar,50°C) followedbya secondextraction stepathighCO2density (e.g.,0.87g/cm3,300bar,50°C).Themostsolublecompounds(suchastheessentialoils)areextractedduringthefirststep,whereasthelesssolublecompounds(forexample,antioxidantsandcoloringmatter)areextractedinthesecondone[31–34].

AliquidcosolventcanbeaddedtoSCCO2toincreaseitssolventpowertowardpolarmolecules. Indeed,SCCO2 isagoodsolvent for lipophilic(nonpolar)compounds,whereasithasalowaffinityforpolarcompounds.Variousauthorsaddedsmall quantities of liquid solvents (for example, ethyl alcohol) that are readilysolubilizedbySCCO2andmodifyitssolventpower[22,26,34–69].Thisstrategyhasthedrawbackthatalargersolventpoweralsoimpliesalowerprocessselectivityand because, as a rule, the cosolvent is liquid at atmospheric pressure, itwill becollectedintheseparatortogetherwiththeextractedcompounds.Subsequentprocessingforsolventeliminationisrequired;therefore,oneoftheadvantagesofSFE(i.e.,solventlessoperation)islost.

Anotherpossibleprocessarrangementisthecontinuousfeedinganddischargingofthesolidtoobtaincontinuousprocessingofthesolidmatter[70].Thisoperationismadepossiblebyaddingtwosolidextrudersatthetopandbottomoftheextractorandcanavoidtheuseoftwoormoreextractorstosimulatecontinuoussolidprocessing.Designandoperationof the twoextruders isnotcheapandsimple.Apatentexistsonthisoperationmode,butithasnotyetbeenindustriallyapplied.

10.2.1 Selection of the operating parameterS

Selection of the operating conditions depends on the specific compound or compoundfamilytobeextracted;molecularweightandpolarityhavetobetakenintoaccount case by case. However, some general rules can be applied. First of all,processingtemperatureforthermolabilecompoundshastobefixedbetween35°Cand60°C (specifically, in thevicinityof thecriticalpoint andas lowaspossible

7089_C010.indd 308 10/8/07 12:15:41 PM


toavoiddegradation).TheincreaseoftemperaturereducesthedensityofSCCO2(forafixedpressure), thusreducingthesolventpowerofthesupercriticalsolvent.However,italsoincreasesthevaporpressureofthecompoundstobeextracted;thus,thetendencyofthesecompoundstopassinthefluidphaseisincreased.Themostrelevantprocessparameter is theextractionpressure thatcanbeused to tune theselectivityoftheSCF.Thegeneralruleisthis:thehigherthepressure,thelargerthesolventpowerandthesmallertheextractionselectivity.Frequently,solventpowerisdescribedintermsoftheSCCO2densityatthegivenoperatingconditions.CO2densitycanvaryfromabout0.15to1.0g/cm3andisconnectedtobothpressureandtemperature.Itsvariationisstronglynonlinear;therefore,properselectionrequirestheuseoftablesofCO2properties[71,72].

The other crucial parameters in SFE are CO2 flow rate, particle size of thematrix,anddurationoftheprocess(extractiontime).Theproperselectionoftheseparametershasthescopeofproducingthecompleteextractionofthedesiredcompoundsintheshortesttime.Theyareconnectedtothethermodynamics(solubility)andkineticsoftheextractionprocessinthespecificrawmatter(masstransferresistances).Theproperselectiondependsonthemechanismthatcontrolstheprocess;theslowestonedeterminestheoverallprocessvelocity.CO2flowrateisarelevantparameteriftheprocessiscontrolledbyexternalmasstransferresistanceorbyequilibrium;theamountofsupercriticalsolventfeedtotheextractionvessel,inthiscase,influencestheextractionrate.Particlesizeplaysadeterminingroleinextractionprocessescontrolledbyinternalmasstransferresistances;asmallermeanparticlesizereducesthelengthofdiffusionofthesolvent.However,ifparticlesaretoosmall,theycancausechannelingproblems inside theextractionbed.Partof thesolventflowsthroughchannelsformedinsidetheextractionbedanddoesnotcontact thematerialtobeextracted,thuscausingalossofefficiencyandyieldoftheprocess.Asarule,particleswithmeandiametersrangingbetweenapproximately0.25and2.0mmareused.Theoptimumdimensioncanbechosencasebycaseconsideringthewatercontent in thematrixand thequantityofextractable liquidcompoundsthatcanproducecoalescenceamongtheparticles,thusfavoringirregularextractionalongtheextractionbed.Moreover,theproductionofverysmallparticlesbygrindingcouldproducethelossofvolatilecompounds.ProcessdurationisinterconnectedwithCO2flowrateandparticlesizeandhastobeproperlyselectedtomaximizetheyieldoftheextractionprocess.

Essentialoilsaremainlyformedbyhydrocarbonandoxygenatedterpenesandbyhydrocarbonandoxygenatedsesquiterpenes.Theycanbeextractedfromseeds,roots, flowers, herbs, and leaves using the process of hydrodistillation (HD). HDis a very simple process but suffers from many drawbacks: thermal degradation(forexample,ofcissabinenehydrateandcissabinenehydrateacetateinmarjoramessentialoil),hydrolysis(forexample,oflinalylacetateinlavenderessentialoil),andsolubilizationinwaterofsomecompoundsthataltertheflavorandfragranceprofileofmanyessentialoilsextractedbythesetechniques.Insomecases,acomparisonbetweenthecompositionsoftheessentialoilsobtainedbySFEandthoseobtainedbyHDhasbeenmade.Forexample,inthecaseofrosemaryoil[1],theSFEessentialoilcontainedhigherpercentagesoflinalool,verbenone,andisobornylacetate;theircontentwasalmostdouble that in thehydrodistilledoil.Thedifferencewas

7089_C010.indd 309 10/8/07 12:15:41 PM


moreevidentintermsofthetotalpercentageofoxygenatedmonoterpenes(whichstronglycontribute to thefragrance):73.7%for theSFEoilagainst59.4%for thehydrodistilledoil.Organoleptictestsconfirmedthatthehydrodistilledoilpossessedalessintenserosemaryaroma.

Insomeothercases,liquidsolventextractionisalsoperformed;theresultsarethesocalledoleoresinsorconcretesthataresolidorsemisolidandcontainessentialoilcompoundstogetherwithwaxes,fattyacids,coloringmatter,antioxidants,andothercompounds.Volatileoilorabsoluteisalsosometimesthetargetoftheprocessthatisroughlyspeakingthevolatilefractionoftheoleoresin.

Essential oil isolation is an example of extraction plus fractional separation.Indeed,thisprocesscanbeoptimallyperformedoperatingatmildpressures(from90to100bar)andtemperatures(from40°Cto50°C)because,attheseprocessconditions,alltheessentialoilcomponentsarelargelysolubleinSCCO2[73–76].Forexample,at40°C,linalooliscompletelymisciblewithSCCO2atpressuresgreaterthanabout85bar[73],andlimonene[74–76],αpinene[76],andfenchone[76]arecompletelysolubleatabout80bar.

However,essentialoilcompoundsareatleastpartlylocatedinsidethevegetablestructure;therefore,masstransferresistanceshavetobeconsidered,too.Atthepreviously discussed operating conditions, essential oil components are extractedtogetherwithcuticularwaxes(i.e.,paraffiniccompoundslocatedonthesurfaceofvegetablematterwiththescopeofcontrollingitsperspiration).Paraffinsexhibitarelativelylowsolubilityattheseoperatingconditions[77].Whenextractionpressureisincreased,theircontributionintheextractismoreprevalentandothercompounds(suchasfattyacids)canalsobeincreasinglyextracted.Sincewaxesareonthestructuresurface,theirextractioniscontrolledbytheirsolubility,whereasessentialoilextractioniscontrolled,atleastinpart,byinternalmasstransferresistancesinthevegetable structure. As a result of these interactions, essential oil and waxes arecoextractedatalloperatingconditions.Toisolate theessentialoil, it isnecessarytotakeadvantageofthefactthat,atlowtemperatures(from–5°Cto+5°C),waxesarepracticallyinsolubleinCO2,whereasterpeniccompoundsmaintainverylargesolubilities (they are completely miscible in liquid CO2). Therefore, it is possibletoobtainafractionationoperating,forexample,theextractionat90barand40°Cand,then,performingafirstseparation,forexampleat0°C,90bar,andasecondseparationat15°C,20bar. In thismanner, theselectiveprecipitationofwaxes isobtainedinthefirstseparatorandnoprecipitationoftheotherextractedcompoundsoccurs,whereas, in the secondseparator, essentialoil is recovered.An industrialplant(V=1200dm3)thatusesthisprocessarrangementhasbeenconstructedandsuccessfully operated since 1996 (Essences, Italy). One must take into account,however,thatitisnotpossibletoperformSFEdirectlyat0°Cand90barbecausethevegetablemattercontainsmanyothercompoundfamilies(antioxidants,colors,etc.)thataresolubleattheseprocessconditionsand,therefore,acomplexmixtureofessentialoilplustheseothercompoundsisobtained.

Dataonessentialoils,volatileoils,andoleoresinsobtainedbySFEareshowninTable10.1,alphabeticallyorganizedbythecommonname(rawmaterial),thebotanicalname,and the targetcomponents (theextract). InTable10.1, laboratory,pilotplant, and analytical studies performed using very small extractors are included.

7089_C010.indd 310 10/8/07 12:15:42 PM


table 10.1sFe of oleoresins (or), essential oils (eo), and Volatile oils (Vo)

raw Material botanical name extract references

leaves

Basil Ocimum basilicum EO [73,78]

Eucalyptus Eucalyptus globulus L. EO [11]

Laurel Laurus nobilis EO [14]

Lemonbalm Melissa officinalis EO [79]

Lemonbergamot Monarda citriodora EO [79]

Lemoneucalyptus Eucalyptus citriodora EO [79]

Lemongrass Cymbopogon citratus EO [79,80]

Lovage Levisticum officinale Koch. EO [22,63,81]

Marjoram Origanum majorana L. EO [82,83]

Mint Mentha spicata insularis EO [15,78]

Oregano Origanum vulgare L. EO [84]

Sage Salvia desoleana EO [15,23,63,85]

Spikedthyme Thymbra spicata EO [86]

Thyme Thyme zygis sylvestris EO [87]

Flowers

Chamomile Chamomilla recutita L.R. EOandOR [88,89]

Lavender Lavandula angustifolia EO [90]

seeds

Aniseed Pimpinella anisum L. EO [91]

Fennel Foeniculum vulgare Mill. EO [17]


roots

Celery Apium graveolens L. EO [22]


other Matrices

Bacurifruitshells Platonia insignis Mart. EO [93]

Blackpepperfruits Piper nigrum L. EO [94,95]

Cashewnutshell Anacardium occidentale VO [30,96]

Clovebud Eugenia caryophyllata EO [13]

Lemonbalmherb Melissa officinalis EO [16,21]

Oreganoherb Origanum vulgare L. EO [61,63,78,97]

Pennyroyalplant Mentha pulegium L. EO [50]

continued

7089_C010.indd 311 10/8/07 12:15:43 PM


Inonlysomecasestheoperatingconditionshaveoptimizedtomaximizeboththeyieldandtheselectivityoftheprocess.Therefore,theyieldandtheoperatingconditionsindicatedbytheauthorsarelargelyinfluencedbythefinalscopeofthepaper:toisolatetheessentialoilortoextrapolateitscompositionfromtheunfractionatedextract(“concretelike”).Ananalysisontheinfluenceofsomeprocessparameters,suchaspressure,temperature,extractiontime,percentageofcosolvents,andsolventflowrates,isavailableinsomeofthepapersconsideredinTable10.1.

Inseveralcases, thesamematrixcontainsessentialoils(orvolatileoils)withknownbiologicalactivityandhighmolecularweightcompoundsthatexhibitnutraceutical or pharmaceutical activity (see Table10.2 for several examples). A largespectrumofcompoundscanbeinsertedinthesecategoriesbecausefoodadditiveswithnutritionalandpharmaceuticalproperties(nutraceuticals)rangefromtocopherolstocarotenoidstoalkaloidstounsaturatedfattyacids.Pharmaceuticalcompoundslike Artemisinin (an antimalaria drug), Hyperforin (an antidepressant drug), andsterolscanbeextractedfromvariousmatters.Inthesepapers,adifferentemphasisisgiventothesecharacteristicsandtheextractischaracterizedmoreforitsfunctionalitythanwithrespecttoessentialoilcomposition.

10.2.2 exampleS

Aspreviouslystated,essentialoilscanbeextractedfromdifferentmatrices,includingleaves,flowers,andseeds.Inthissection,weillustratesomeexamplesofextraction.

10.2.2.1 leaves

Essentialoilisolationhasbeenperformedasaruleinplantsoperatedwithatleasttwoseparatorsinseries.AnexampleofSFEfromleavesissage(Salvia officinalis)essentialoilextraction[85].Thebestisolationconditionshavebeenfoundat90barand50°C.Sagewaxeseliminationhasbeendemandedtothefirstseparator,fixingthe conditions at 85bar and–12°C. In the second separator, operating at 17bar,

table 10.1 (continued)sFe of oleoresins (or), essential oils (eo), and Volatile oils (Vo)


other Matrices (continued)

Redpepperfruits Capsicum frutescens L. OR [98]

Staranise Illicium anisatum EO [13]

Juniperfruits Juniperus communis L. VO [99]

Concretes

Jasmine Jasminum grandiflorum L. VO [100]

Rose Rosa damascena Mill. VO [101]

Tuberose Nepeta tuberosa L. EO [12]

7089_C010.indd 312 10/8/07 12:15:43 PM


–6°C,onlyessentialoilisfound.Theessentialoilobtainediswaxfreeandcontainsonly traces of highmolecularweight compounds. These highmolecularweightcompounds were identified as two flavones. The main compounds extracted are1,8cineole,camphor,andcaryophyllene.Theasymptoticvalueofthesageessentialoilyield,expressedasweightofextractdividedbytheweightofthestartingmaterial,is1.35wt%ofthechargedmaterial.

table 10.2essential oil–related and biologically active Compounds


leaves

Aloevera Aloe barbadensis Miller αtocopherol [45]

Eucalyptus Eucalyptus camaldulensis var. brevirostris

Gallicandellagicacids [39]

Hawthorn Crataegus sp. Flavonoidsandterpenoids [48]

Marjoram Origanum majorana L. Carotenoidsandchlorophylls [19]

Marjoram Origanum majorana L. Phenolicandtriterpenoidantioxidants

[102,103]

Rosemary Rosmarinus officinalis L. Rosmanol,carnosicacid,andcarnosol

[31–33,36,41,104–107]

Sage Salvia officinalis L. Carnosolicacid [23]

Savory Satureja hortensis L. Oil [20]

Flowers

Chamomile Matricaria recutita Flavonoidsandterpenoids [48]

Hawthorn Crataegus sp. Flavonoidsandterpenoids [48]

Marigold Calendula officinalis Flavonoidsandterpenoids [48]

seeds

Coriander Coriandrum sativum Tocopherols,flavonoids,andterpenoids

[108]

other Matrices

Aniseverbena Lippia alba Limoneneandcarvone [109,110]

Coffeepowder Coffea arabica Aroma [111]

Gingerrhyzomes Zingiber officinale Roscoe Gingerolsandshogaols [40,41]

Horsetailplant Equisetum giganteum L. Oleoresin [112]

Mosobambooplant Phyllostachys heterocycla EthoxyquinA,sesquiterpeneA,andcyclohexanoneA

[46]

Paprikaflake Capsicum annuum L. Carotenoids,tocopherols,andcapsaicinoids

[113,114]

Sawpalmettoberries Serenoa repens Fattyacidsandβsitosterol [56]

Spearmintplant Mentha spicata Tocopherol [115]

7089_C010.indd 313 10/8/07 12:15:43 PM


10.2.2.2 Flowers

As examples of SFE of essential oils from flowers, we consider lavender [90]andchamomile[89]essentialoilextractionandisolation.Inthecaseoflavenderflowers[90],theoperatingconditions,intermsofpressureandtemperature,havebeenfixedasfollows:intheextractor,90barand48°C;inthefirstseparator,80barand–10°C;inthesecondseparator,25barand0°C.Waxesprecipitateinthefirstseparator,andthelavenderessentialoilprecipitatesinthesecondseparatorandmainlyconsistsof1,8cineole,linalool,camphor,4terpineol,αterpineol,andlinalylacetate.Theasymptoticvalueofthelavenderessentialoilyieldis4.9wt%ofthechargedmaterial.

In the case of chamomile flower [89], the operating conditions are similar.Themajor constituentsof this essential oil areoxygenated sesquiterpenes,whichrepresent 78.48% of the oil composition. In Table10.3, the compositions of thechamomileessentialoilandthechamomilecuticularwaxeshavebeenreportedtogiveanexampleofdetailedidentificationofessentialoilcomponents.Oxygenatedsesquiterpenescontainthemostcharacteristicchamomileessentialoilcompounds,namelybisabololoxideB(16.88%),αbisabolol(0.35%),bisaboloneoxide(7.76%),andbisabololoxideA(50.42%).Matricine(evaluatedaschamazulene)represents,inthiscase,3.52%anddicycloetherscontributemorethan12.97%tothetotalextract.Theyieldsobtainedare1.18%foressentialoiland0.8%forcuticularwaxes.

10.2.2.3 seeds

Inthecaseofseedoils,atleasttwoSCCO2extractablecompoundfamiliesarecontainedinthevegetablematrix:essentialoilandseedoil;therefore,extractionconditionshavetobesettoavoidtheircoextraction.Anexampleisgivenbyfennelessentialoilisolation[116].Thefirststepoftheextractionprocessisperformedat90barand50°C,withtheaimofselectivelyextractingfennelessentialoil.Waxeseliminationisdemandedtothefirstseparator.Theextractionandsimultaneousisolationoffennelessentialoilhasbeensuccessful.Inthefirstseparator,paraffinicwaxesarecollectedwithcarbonatomnumbersbetween25and37; thiswaxcompositionagreeswellwiththatofvariousothervegetablematterextractedbySCCO2[1].Inthesecondseparator,fennelessentialoil iscollected.It ismainlyformedbyestragole(about80%), anethole, fenchone, and limoneneand isnot contaminatedbywaxesorbyhighermolecularweightcompounds.Anessentialoilasymptoticyieldof1.8wt%oftheloadedmaterialhasbeenobtained.

Thesecondstepoftheextractionprocess,performedat40°Cand200bar,producestheextractionoffennelvegetableoil.Also,inthiscase,thefirstseparatorisusedtoprecipitatecoextractedwaxes.Thewhitemasscollectedinthefirstseparatorisagainformedbyparaffins,althoughtheirmolecularweightisslightlylarger(carbonatomsfrom25to41).Fenneloiliscollectedinthesecondseparator.However,higherpressuresarecommonlyusedinthisstepoftheprocesstoacceleratetheextractionofthevegetableoil.

10.2.2.4 other Matrices

Asexamplesofextractionfromdifferentmatrices,weconsiderclovebudandstaraniseessentialoils.Inbothcases,thebestessentialoilprocessconditionsare90bar

7089_C010.indd 314 10/8/07 12:15:44 PM


table 10.3Composition of the Chamomile essential oil and of Cuticular Waxes (adapted from [89])

Compoundretention time

(min)area (%)

essential oil

6methyl5hepten2one 20.02 0.07

Ocimene 25.33 0.11

Linalool 28.26 0.57

Isoborneol 33.07 0.10

Menthol 33.43 <0.05

4terpineol 34.02 0.07

αterpineol 35.00 0.09

nid.C10H16O 38.25 0.11

Nerol 39.58 0.65

Geraniol 41.07 0.24

Menthylacetate 45.13 0.17

n.id.C12H22O2 48.10 0.17

βelemene 49.17 <0.05

βcaryophyllene 51.01 0.13

βfarnesene 53.33 1.53

trans-nerolidol 60.07 0.42

Spathulenol 60.53 0.65

Caryophylleneoxide 63.18 0.17

n.id.C15H26O 64.03 0.39

Tcadinol 64.39 0.36

BisabololoxideB 65.38 16.88

αbisabolol 66.14 0.35

Bisaboloneoxide 67.09 7.76

Matricine(chamazulene) 69.39 3.52

BisabololoxideA 70.53 50.42

n.id.C15H26O 71.43 0.34

n.id. 72.09 0.56

n.id.C15H26O 74.07 0.18

cis-dicycloetherMW200 77.46 9.64

trans-dicycloetherMW200 78.23 3.33

trans-farnesol 79.02 0.32

cis,trans-farnesol 79.55 0.42

cis-dicycloetherMW214 79.92 <0.05

trans-dicycloetherMW214 81.18 <0.05

continued

7089_C010.indd 315 10/8/07 12:15:44 PM


and50°Cforextraction,90barand–10°Cinthefirstseparator,and15barand10°Cinthesecondseparator.

Theobtainedclovebudessentialoilcomprisesmainlyeugenol(65.9%),caryophyllene(11.1%),andeugenylacetate(19.0%).Theyieldis20.7%byweightofthematerialchargedintheextractor.

Theobtainedstaraniseessentialoilcontains94.2%anethole,1.4%estragole,1.7%limonene,and0.3%linalool.Theasymptoticyield is7.3%byweightof thechargedmaterial.

10.2.2.5 Flower Concretes Fractionation

Whenvegetablematerialscharacterizedbyaveryshortlifehavetobeprocessed,asinthecaseofmanyflowers,thefragranceproductionisperformedintwosteps.Thefirststepconsistsofsolventextraction,usuallybyhexane,whichyieldsanintermediateproductcalled“concrete.”Itismainlycomposedoffragrancerelatedcompoundsbutalsocontainslargequantitiesofparaffins,fattyacids,fattyacidsmethylesters, diterpenic and triterpenic compounds, pigments, and other substances. In

table 10.3 (continued)Composition of the Chamomile essential oil and of Cuticular Waxes (adapted from [89])

Compoundretention time

(min)area (%)

Waxes

Hexadecane 13.12 0.44

Octadecene 17.43 2.17

Docosene 26.82 0.83

Tricosane 29.59 1.64

Tetracosane 32.09 0.31

Pentacosane 34.65 10.50

Hexacosane 36.98 1.52

Methylhexacosane 38.73 0.26

Heptacosane 39.74 17.56

Octacosane 41.78 2.74

Methylheptacosane 43.07 0.85

Nonacosane 43.94 24.12

Triacontane 45.54 2.86

Entriacontane 47.73 19.71

Methyltriacontane 49.24 1.26

Dotriacontane 49.80 1.54

Methylentriacontane 51.39 1.16

Tritriacontane 52.57 9.46

Methyldotriacontane 54.70 1.13

7089_C010.indd 316 10/8/07 12:15:45 PM


the second step, theconcrete ispostprocessedby steamdistillationor solubilization ina largeexcessofalcohol toobtainavolatileoil containing the fragrance.Theseconventionalpostprocessing techniquesare subject to somedisadvantages,suchasthermaldegradation,incompleteeliminationofnonvolatilecompounds,andfractionationofthecompoundsthatformthefragrance.Becausefloweressentialoilshavealargecommercialvalue(thousandsofdollarsperliter),exploringtheuseofSCCO2extractionasanewwaytofractionatetheconcretecouldbebeneficial.

In thecaseof roseconcrete [101],apreliminarystudyshowed that themajorvolatilecompoundscontainedinthestartingmaterialwere2phenylethanol(25.1%),citronellol(4.7%),and2phenylethylacetate(2.7%).However,italsocontainedmanyothercompoundsthatdonotcontributetorosefragranceformation,likeparaffins.Amongthese,twolongchainparaffinicalcoholshavebeendetected.Theyarecommonly called “steroptens” and are characteristic of rose concrete and adverselycontributetorosefragrance.

Roseconcretehasbeenwarmedupto35°C,mixedwith2mmdiameterglassbeads to perform SCCO2 processing, and then charged into the extractor. Themixinghasbeenperformedtoobtainathinlayerofconcretearoundtheglassbeads.Thisprocedurehasbeenusedtomaximizethecontactsurfacebetweentheconcreteandthesupercriticalsolventandtoavoidchanneling.Thesolutionattheexitoftheextractor,asforsolidextraction,flowedintothetwoseparatorsoperatedinseries,inordertofractionatetheextract.Thefirstseparatorwassetat80barand–16°C,whereasthesecondseparatorwassetat15barand0°CtominimizethelossofvolatilecompoundsinthegaseousCO2streamattheexitoftheapparatus.Attheendoftheextractionprocess,glassbeadswererecoveredbywashingwithwarmethanol.

TheoptimizedSFEisperformedat80barand40°C,andamaximumyieldofvolatilecompoundsof49%byweightofthechargedmaterialhasbeenrecoveredinthesecondseparatorusingfractionalseparation.Theprocesswasextremelyselective:nounwantedcompoundsweredetected.Thewaxesyieldinthefirstseparatorwasabout2%byweightoperatingat80barand40°Cbutcanreachupto10.4%byweightiftheextractionisperformedat120barand40°C.

At the endof theSFEprocess at80bar and40°C,on the exhaustedcharge,a further extraction step has been performed operating at 120 bar and 40°C for100min.Thehigherpressurestepyieldsafurther14.9%byweightofthecharge.The extract is still liquid but contains only 1.0% 2phenylethanol, whereas thepercentage of steroptens is 21.3%. The second step was performed to evaluatewhetherothervaluablecompoundswerestillcontainedinthestartingmaterialandwerenotpreviouslyextractedat80barand40°C.

Inthecaseoftuberoseflowers,concretefractionationisalsorequiredbecauseSCCO2 extraction performeddirectly on the tuberose flower is not applicable atanindustrialscalesincetheyieldinessentialoilfromflowersislessthan0.1%byweight.TuberoseconcretefractionationusingSCCO2hasbeenperformed[12]withtheaimofseparatingvolatileoilfromthehighermolecularweightcompounds.Theextractionprocesshasbeencoupledagainwiththefractionalseparationtechniquethatusestwoseparationstagesoperatinginseries.

SystematicSFEtestsontuberoseconcretehavebeenperformedintherangeof80to100bar,operatingatatemperatureof40°Candanalyzingtheproductcollected

7089_C010.indd 317 10/8/07 12:15:45 PM


in thetwoseparatorsbygaschromatography–massspectrometry.Byoperatingat80bar,theresearchersobtainedthemaximumcontentoffragrancecompoundsintheextractcollectedinthesecondseparator.

Since the different compound families that constitute the tuberose volatile oil(hydrocarbonterpenes,oxygenatedterpenes,andbenzenederivatives)showdifferentextraction rates, thecompositionsof the tuberoseoilchangesduring theextractionprocess.Theextractrecoveredafter20minutesofextractioncontainsahighpercentage(>83%)ofoxygenatedcompounds(monoterpenesandbenzenederivatives).Thevolatilefractionrecoveredintheextractiontimeintervalbetween360and480minutesconsistsofalowerpercentage(<79%)ofoxygenatedcompoundswithanincrementofthepercentageofthetransmethylisoeugenolandoftheeugenylacetate,whereasthemostvolatilecompounds,suchasmethylbenzoateandmethylsalicylate,arereduced.

The fraction recovered in the extraction time interval between 690 and 750minutescontainsverylowquantitiesofaromacompoundsbutstillcontainslargequantitiesoftransmethylisoeugenol,eugenylacetate,andlactones.

Bydividingthetuberoseoilcompoundsintothreefamilies,hydrocarboncompounds(monoterpenesandsesquiterpenes),oxygenatedcompoundswith10orlesscarbonatoms(monoterpenesandbenzenederivatives),andoxygenatedcompoundswith 15 or more carbon atoms (sesquiterpenes and lactones), the contribution ofeach compound family can be calculated as the sum of the area contribution ofall compounds belonging to that family. Tuberose oil contains a low percentageofhydrocarboncompounds(monoterpenesandsesquiterpenes)andtheirpercentagedecreasebyincreasingtheextractiontime.Thepercentageofoxygenatedcompoundswith 10 or less carbon atoms also decreases during the extraction, whereas thepercentage of oxygenated compounds with 15 or more carbon atoms increases,especially at extraction times longer than450minutes.Thismeans thatdifferentsolubilitiesandperhapsmasstransferresistancescharacterizethevariouscompoundfamiliesduringtheextractionprocessandtheextractiontimeplaysarelevantroleinthefinalcompositionoftuberoseoil.Moreover,byinterruptingtheextractionoftheoilatdifferenttimes,itispossibletofractionatethefragranceandtoobtainanextractinwhichtopnotesorbottomnotesprevail.

It is also possible to divide the tuberose oil compounds in two groups: thefragrance compounds (oxygenated compounds) and the nonfragrance compounds(hydrocarboncompounds).Theyieldcurvesshowanexponentialtrendagainsttheextractiontime;thehydrocarboncompoundscurvegetsflatafterthefirst300minutesofextraction,whereas theyieldcurveoffragrancecompoundsasymptotizesonlywhencompleteextractionisperformed(after750minutes).

Asconfirmedbythetwoexamples,SFEisgenerallyapplicabletoflowerconcretesandcanbeveryselective.Thus,itcanbeusedtorecoverinarelativelycheap,singlestepSFEoperationessentialoilswidelyusedintheperfumeindustry.AbetterproductisalsoobtainedwithSFEthanwithtraditionaltechniques.

10.3 lIquId Feed ProCessIng

Thefractionationofliquidmixturesintotwoormorefractionsisanotherrelevantprocess.Inatypicalapparatus,twopumpsdelivertheliquidsolutionandSCCO2to

7089_C010.indd 318 10/8/07 12:15:45 PM


thepackedcolumn.ThepackingisaninertmaterialcharacterizedbyalargespecificsurfacewhosescopeistofavorthecontactbetweentheliquidandtheSCF.SCCO2generallyflowsalong the column from thebottom to the top,whereas the liquidsolutionisusuallyaddedtothetop.However,itisalsopossibletofeedtheliquidatanintermediatepositionalongthecolumnandtoaddarecycleofpartofthefluidphaseexitingatthetop.AschemeoftheapparatusisshowninFigure10.2.


Selectionof theoperatingparameters is basedon thedifferent solubilitiesof theliquidstobeseparatedinSCCO2.TheidealcaseisobtainedwhenonlythecompoundstobeextractedaresolubleinSCCO2andalltheotherliquidcomponentsarecompletelyinsoluble.However,thiscaseisrareandalimitedsolubilityoftheotherliquidcompoundsformingthemixturehastobetakenintoaccount.Forthisreason, pressure and temperature of the process have to be accurately chosen toselect theconditionsatwhichthemaximumdifferenceinsolubilityexistsamongthecompounds tobeextractedandall theothercompounds in themixture.Alsointhiscase,CO2densityisfrequentlyusedasacriteriontofindtheconditionsofmaximumselectivity.ThedifferenceindensitybetweentheliquidandSCCO2isanotherparametertobetakenintoaccount;toallowthecountercurrentoperation,SCFdensityhastobelowerthanthedensityoftheliquidmixture.

The traditionaloperationofpackedcolumns requires that liquidflow ratebelargerthantheminimumamountthatassuresthecompletewettingofthepacking.Thefeedratioisalsoselectedtoavoidthemassiveentrainmentoftheliquidinthefluidphase(flooding).TheseconditionshavetoberespectedalsowhenaSCFisusedasthefluidprocessingmedium.Theclassicalcalculationintermsofthenumberoftheoreticalequilibriumstagesrequiredforseparationcanalsobeapplied.Apossiblevariation of this processing scheme can consist of the adoption of a temperature

Liquid IN

2

3 4 5

Out

SCF IN

1

FIgure 10.2 1)CO2pump;2)liquidpump;3)packedtower;4)firstseparator;5)secondseparator.

7089_C010.indd 319 10/8/07 12:15:47 PM


profilealongthecolumn,withtheaimofoptimizingtheseparationtemperaturewithrespecttothecompositionofthemixturesatdifferentlevelsinsidethecolumn.

The extraction of liquid mixtures is controlled by the relative solubilities inSCCO2ofthevariouscompoundsformingthemixture—thatis,thethermodynamiclimitationoftheprocess.Masstransferbetweenthetwophasesrepresentsthekineticlimitation.Thedistancefromtheequilibriumconditionisthedrivingforcefortheseparationalongthecolumn.

10.3.2 exampleS

ThefractionationofliquidmixturesbySFEhasbeenproposedforvariousapplications, including some essential oils, with the aim of improving their fragranceandeliminatinghydrocarbonterpeniccompoundsthatcanrapidlydecomposeandtherefore can shorten the shelf life of the product. The problem does not have asimple solution. Selective elimination of hydrocarbon terpenes (deterpenation) isrequiredincitruspeeloilsbecausethesecompoundscontributetoasmallextenttothecitruspeelfragranceandarerapidlyoxidizedbyairandcanundergostructuralrearrangements. These essential oils are mainly formed by hydrocarbon andoxygenatedterpenes,butalsocontainsmallquantitiesofsesquiterpenesandhighmolecularweightcompounds likecoumarins,psoralens,andwaxes.Hydrocarbonterpenepercentagecanrangefrom60%to99%.InTable10.4,someexamplesoffractionationsofliquidmixturesarereported.

The fractionation of a peel oil with SCCO2 was studied using a mixture offour key compounds [117]. The composition of this mixture was determined bythefollowingconsiderations.Limoneneisahydrocarbonterpeneanditisthepredominant compound inallpeeloils,withconcentrationsbetween30%and80%.Therefore, it canbechosenas themost abundantkeycompound,withaconcentration of 60% by weight. Linalool is one of the most representative among theoxygenatedcompoundsand,therefore,itsconcentrationwassetat20%byweight,representingthesecondmostabundantcomponentinthekeymixture.γTerpinenehasamolecularweightsimilartolimonenebutisahydrocarbonterpenethathasavolatilityveryneartooxygenatedcompounds.Therefore,itspresenceinthemixture(10%)allowsevaluationoftheeffectivenessofseparationforthosecompoundsthat

table 10.4Fractionation of liquid Mixtures by sFe in Continuous (C) and semicontinuous (sC) Plants

Initial mixture objective Process references

Citruspeeloilkeymixture Separationoflimonenefromlinalool C [117]

Citrusoil Deterpenation C [118]

Citrusoil Deterpenation SC [119]

Orangepeeloil Deterpenation C [120]

Oreganumoil Deterpenation SC [121]

7089_C010.indd 320 10/8/07 12:15:47 PM


aremoredifficulttoseparatethanlimonene.Linalylacetate(10%)isrepresentativeofoxygenatedcompoundswithamolecularweighthigherthanlinalool.

Systematicexperiments[117]havebeenperformedona2mcolumnoperatedincountercurrentandequivalenttoabout2.5theoreticalequilibriumstages.Theoperatingpressureandtemperaturerangedbetween75and90barandbetween40°Cand80°C,respectively.Solventtofeedratiosof60,80,and120wereused.Theeffectsofdifferentfeedinsertionpointsandcolumnpackingswerealsotested.Experimentalresultsindicatedthatfractionationcouldbesuccessfullyobtainedbetween75and80barandbetween50°Cand80°C.Ingeneral,anincreaseinsolubilitycorrespondsto a decrease in selectivity and, thus, optimization of the separation is required.Experiments also indicated that temperature helps separation and, furthermore,increasestherecoveryofoxygenatedcompounds.Theupperlimittotheoperatingtemperature is given, however, by the thermal stability of the product. The totalandpartialrefluxesoftheextractatthecolumntopshowadefinitelypositiveeffecton theseparation.However,onlywhen theoperation ina twocolumnserieswasperformed(i.e.,usingalengthofcolumnequivalenttoabout5equivalentstages),researchersobserved theelimination in the topproductof linalylacetateand thereductionoflinaloolcontenttoabout0.8%.

Liquid feed can be alternatively fractionated by adsorption/desorption of theliquidmixtureonanadsorbent.Forexample,SCCO2hasbeenusedtoselectivelydesorbbergamotpeeloilcomponentsfromsilicagel[122].Themaximumdesorptionselectivityhasbeenobtainedoperatingat40°C, in twosuccessivepressuresteps.Thefirststep,performedat75bar,producestheselectivedesorptionofhydrocarbonterpenes;thesecondone,performedat200bar,assuresthefastdesorptionofalltheoxygenatedcompounds.

Inanotherwork[123],thirteenterpenesformingamixturecharacteristicofpeelessentialoilswereselectivelyadsorbedonsilicagelusingSCCO2.Thesecompoundsareαpinene,βpinene,myrcene,limonene,γterpinene,βcaryophyllen,citronellylacetate,geranylacetate,linalylacetate+geraniol,linalool,citronellal,andcitral.Theycanbegroupedinfourfamiliesofpseudocomponents:hydrocarbonterpenes(likelimonene),asesquiterpene(βcaryophyllene),terpeneacetates(likegeraniol),andoxygenatedcompounds(likelinalool).Theexperimentswereperformedatdifferentpressures(130to210bar),temperatures(37°Cto57°C),andconcentrations(0.9 to 7.6 g/kgsolvent). The different pseudocomponents show a different adsorptionbehaviorthatcanbejustifiedlookingattheinteractionswiththeactivesitesof the adsorbent. Hydrocarbon terpenes and sesquiterpenes can be adsorbed onCH3groupsofsilicagelandarerapidlyshiftedbythemorepolarcompounds(displacement).OxygenatedcompoundscanalsobondwithOHgroups(silanol).Compoundswithhighermolecularweightsorhigherpolaritiescanmove(displace)theotherspeciesfromtheadsorptionsiteinwhichtheywerelocated.Thedisplacementof the lessstronglyadsorbedcompoundsismoreevidentat lowerconcentrationsandallowstheselectiverecoveryofthevariousfractionsattheexitoftheadsorption/desorptioncolumn.

7089_C010.indd 321 10/8/07 12:15:47 PM


10.4 antIsolVent extraCtIon

Therecoveryofsolidcompoundsfromaliquidmixturerequiresdifferentprocessapproachessincethefixedbedextractordoesnotadapttoprocessliquidmixtures.Inaddition,thepackedtowercannotbeusedinthesecasesbecausethesolidcompoundsprecipitateontheinternalpackings.

The Supercritical antisolvent extraction (SAE) process is conceptually verysimilartoSupercriticalantisolventmicronization(SAS),butthescopeoftheprocessistherecoveryofoneormoresolidcompoundsfromaliquidmixture.ItconsistsofthecontinuousflowofSCCO2andoftheliquidmixtureinapressurizedprecipitationvessel.Iftheprocessconditionshavebeenproperlyselected,theliquidisrapidlydissolvedintheSCF,whereasthesolidprecipitatesatthebottomoftheprecipitationvessel.Therefore,inapossiblerepresentationoftheprocess,twopumpsdelivertheliquidsolutionandtheSCF,respectively.Theprecipitationvesselisusedtocollectthe solid and a vessel located downstream the precipitator and operated at lowerpressure(forexample,30barand25°C)isusedtorecovertheliquid.AschematicoftheapparatusisshowninFigure10.3.


Thefirststepofthisprocessistheformationofasprayoftheliquidsolution.Theintentofthisoperationistoproduceaverylargeliquidsurfaceduetotheformationofsmallliquiddropletstostronglyenhancetherateofsolubilizationoftheliquid

Liquid IN

2

3

41

Liquid Recovery

Out

SCF IN

FIgure 10.3 1)CO2pump;2)liquidpump;3)precipitator;4)separator.

7089_C010.indd 322 10/8/07 12:15:49 PM


phaseinthesupercriticalmedium.Forthesamereason,theprocessisperformedatoperatingconditionsatwhichtheliquidsolventiscompletelysolubleinSCCO2.TheknowledgeofsolubilitydataontheliquidsolventsandofthesolidsinSCCO2ismandatoryfortheproperselectionofprocesstemperatureandpressure.

InthecaseofSAE,theinteractionsbetweenthermodynamicsconstraintsandmass transfer mechanisms also control the process performance. The enhancedmasstransferthatcharacterizesSCFsisagainadistinctiveadvantageoftheiruseasextractionmedia,togetherwiththefastandcompleteseparationbysimpledepressurizationbetweenthesupercriticalsolventandtheliquid.

Alimitationofthisprocessisthepossibleformationofaternarymixtureliquid/solid/SCCO2.Indeed,thepresenceoftheliquidcaninduceanincreaseofthesolubilityofthesolidcompoundsinSCCO2.Inthiscase,theliquidcanactasacosolventfromthepointofviewofsolidsolubilization.Whenthisphenomenonoccurs,thepart of the solid retained in thefluidphaseobviouslydoesnot precipitate and islostintheliquidrecoveredintheseparationvessel.Thelimitcaseisthecompletesolubilizationofthesolidinthefluidphasethatproducestheprocessfailure.

10.4.2 exampleS

Untilnow,SAEhasbeenusedinalimitednumberofprocesses(forexample,therecoveryofessentialoilfromamixtureessentialoil+triglycerideoil[124,125])butithasalargepotentialforfutureapplications,someofwhicharediscussedhere.

10.4.2.1 Proteins and aroma extraction from tobacco

Aprocesshasbeendeveloped for the recoveryof tobaccoproteins, aminoacids,andaromausingethylalcohol[126].Afractionationprocessisrequiredtoseparateproteinrelated(solid)compoundsfromtobaccoaroma,obtainingthesimultaneouseliminationoftheliquidsolvent.Theethanolicextractispreparedusinganethanolsolution 1% potassium hydroxide using a solution containing a tobacco blend ofapproximately10to1(v/w),for3hoursat40°C.Potassiumhydroxideisaddedtothesolutionforitsstabilization.

EthanolisreadilysolubleinSCCO2;therefore,thecouplesolventantisolventshouldnotdeserveproblemsinSAEprocessing.Proteinsandtheirderivativeaminoacidsshouldbevirtually insoluble inSCCO2dueto theircompositionandtheirmolecularweights.Flavoringcompoundscanalsobereadilyextracted.Therefore,basedontheseconsiderations,afractionationof theethanolicextract is, inprinciple, possible if processing conditions are selected to induce proteic compoundprecipitationandthetransferinthefluidphaseoftheliquidsolventtogetherwithflavoringcomponents.

Experimentshavebeenperformedatdifferentpressuresandsolidconcentrations.Goodresultshavebeenobtainedat150bar,40°C,and100mg/mL.Theethanolicextracthasbeenefficientlyfractionated,andtheyieldofprecipitatedmaterialwasabout40%(w/wofextract).

7089_C010.indd 323 10/8/07 12:15:49 PM


10.5 MatheMatICal ModellIng

Mathematical modelling gives the possibility to generalize experimental resultsand,ifsuccessful,toobtainindicationsaboutsystemsdifferentfromthosestudied(simulation).Moreover,itisusefulinthedevelopmentofscalingupproceduresfromlaboratorytopilotandindustrialscales.Forthesereasons,severalattemptsatmathematicalmodellingofSFEhavebeenpresentedin the literature[1,127,128]andsomeofthesemodelsarerelatedtoSFEofessentialoils.

Amodelshouldnotbeameremathematicalinstrument,butitshouldreflectthephysicalknowledgeofthesolidstructureandtheexperimentalobservations.Therefore,mathematicalmodels that havenophysical correspondence to thematerialsandtheprocessstudiedareoflimitedvalidity,althoughtheycanbeusedtofitsomeexperimentaldata.

ThreedifferentapproacheshavebeenproposedforthemathematicalmodellingofSFE:(1)empirical[129,130],(2)basedonheatandmasstransferanalogy[131,132],and(3)differentialmassbalancesintegration[127,133,134].Themostproperanalysis is obtained from the integration of the differential mass balances: timedependentconcentrationprofilesareobtainedforfluidandsolidphases.

In facingmathematicalmodellingofSFE, severalgeneral aspectshave tobetakenintoaccount:

1.Solid material structure:Knowledgeofthebotanicalaspectsorscanningelectronmicroscope(SEM)analysisofthematerialisnecessarytovisualizethestructure.Forexample,seedsareessentiallyformedbyspecializedstructuresthatoperateassmallrecipientscontainingtheoil.Theirshapeandstructurechangeseedbyseed,butthegeneralorganizationisalwaysthesame.

2.Location of the compounds to be extracted: Thedistributionofthesolutewithinthesolidsubstratemaybeverydifferent.Theextractablesubstancesmaybefreeonthesurfaceofthesolidmaterialorinsidethestructureofthematerialitself.Essentialoilcanbelocatedneartheleafsurfaceinglandulartrichomes or in vacuoles (intracellular structures located well inside theleaf)[29,135].

3. Interactions of solutes with the solid matrix: Dependingontheinteractionsbetween thecompoundsand the solid structure,different equilibriamaybe involved. Indeed, if the material has no interactions with the matrix,equilibriumsolubilityhas tobe taken intoaccount.Thematerial canbeadsorbedontheoutersurfaceorinsidethesolidstructure;inthiscase,apartitioningequilibriumbetweensolidandfluidphaseoccurs.

4.Broken-intact cell structures: Partofthecompoundstobeextractedmaybenearthesurfaceofthestructureduetocellbreakingduringgrinding.Moreover,membranesmodificationsmayoccurduetodrying,freeingpartofthesolublematerial.

5.Shape of particles:Particlesmaybespherical,platelike,orothershapesasaresultoftheoriginalshapeofthematerial(forexample,leaves)andofthegrindingprocess.Theirshapecaninfluencethediffusionpathof thesupercriticalsolvent[29,127].

7089_C010.indd 324 10/8/07 12:15:49 PM


Fromthepointofviewoftheextractionmechanisms,otherconsiderationsarenecessary.Theequilibriummayexistif:

1. thematerialislargelyavailable 2. itisdistributedonornearthesurface 3. thekindofequilibriumdependsontheinteractions(ifany)withthesolid

structure

Masstransferresistances,ingeneral,maybeoftwotypes:externalorinternal(and,inthiscase,variouspossibilitieshavetobeconsidered).Totakeintoaccountmasstransferresistances,differentialmassbalancesareapplied.

Essential oils forwhichmathematicalmodellingofSFEhasbeenattemptedarereportedinTable10.5.Wehavealsoindicatedifthemodelisbasedonempiricalkineticequations,on theanalogybetweenheatandmass transfer (HMT),orondifferentialmassbalancesequations(DMBE)alongtheextractionbedoronasingleparticle.

A brokenintact cell model of the SFE of essential oils can be based on thefollowinghypotheses:

1.Thebehaviorofallcompoundsextractedissimilarandcanbedescribedbyasinglepseudocomponentwithrespecttothemasstransferphenomena.

2.Concentrationgradientsinthefluidphasedevelopatlargerscalesthantheparticlesize(i.e.,concentrationvariationsinthefluidphasehaveacharacteristiclengthscalelargerthanthediameterofparticles).

3.Thesolventflowrate,withsuperficialvelocityu,isuniformlydistributedinallthesectionsoftheextractor.

table 10.5Mathematical Modelling of sFe of essential oils

raw Material extract type of Model references

Basilleaves Essentialoil DMBE [136]

Carawayseeds Essentialoil DMBE [136]

Clovebud Essentialoil DMBE [137]

Fennelseeds Essentialoil DMBE [116,138]

Gingerrhizomes Oleoresin DMBE [139]

Jalapenopepperflakes Oleoresin DMBE [140]

Lavenderflower Essentialoil DMBE(shrinkingcore) [141]

Marigold Oleoresin Variousmodelsproposed [142]

Marjoram Essentialoil DMBE [136]

Orangeflowerconcrete Volatileoil DMBE [143]

Oreganobracts Essentialoil HMT(singleplatemodel) [29]

Pennyroyal Essentialoil DMBE [135]

Pepper,black Essentialoil DMBE [144]

Rosemaryleaves Essentialoil DMBE [136]

7089_C010.indd 325 10/8/07 12:15:50 PM


4.Thevolumefractionofthefluid,ε,isnotaffectedbythereductionofthesolidmassduringextraction.

5.Thesoluteinthesolidispresentintwoseparatephases.Onephaseincludesthesolutecontainedinsidetheinternalstructureoftheparticles.Itfillsafractionφtoftheoverallvolumeoccupiedbytheseedparticles.Theotherphase ismade of the solute freely available on the particle surface.Theconcentrationhereisalwaysthesameand,accordingtothehypotheses,itisequaltothepuresolutedensityρ0(thesoluteisfreelyavailableonthesurfaceand,therefore,itsconcentrationisconstant).

6.The fraction of the volume filled by the free solute before extraction isφf=1–φt.

7.Thefractionofthevolumeoccupiedbythefreesoluteduringtheextractionisψφf,whereisψ≤1.

8.Alinearequilibriumrelationshipappliesbetweenphases.

According to the above hypotheses, the mass balance on the solute in theextractoris:

ε ρ ε ρ ρ ε φ⋅ ⋅ ⋅ ∂∂

+ ⋅ ⋅ ∂∂

+ ⋅ ⋅ ∂∂

+ −( ) ⋅f L f f fDC

z

Ct

uCz

2

21 ρρ

ε φ ρ ψ

s

f

Pt

t

⋅ ∂∂

+ −( ) ⋅ ⋅ ∂∂

=1 00 (10.1)

whereDListheaxialdispersion;uthesuperficialvelocity;ρfthefluiddensity,whichissupposedlynotaffectedbythepresenceofthesolute;ρs thebulkdensityofthenonsolublesolidthatisthemassofnonsolublesolidsinthevegetablematerialsperunitoffilledparticlevolume,thatisthetotalvolumeoftheparticleminusthevolumeofbrokencells.

Thegeneralmassbalanceonthephaseofthefreesolutealoneis:

ρψ ρ

ε φψ

00

1∂∂=−

−( )−( )t

k a K Cf

funtil ψ > 0 , (10.2)

otherwise,

∂∂

=ψt

0 (10.3)

whereaisthespecificsurfaceoftheparticlesandkfistheexternalmasstransferresistance.

Themassbalanceontiedsoluteis:

∂∂

= −⋅ −( )

−( )Pt

k a P K Ci p

t1 ε φ (10.4)

7089_C010.indd 326 10/8/07 12:15:54 PM


wherekiistheinternalmasstransferresistance.Thissystemofequationshasauniquesolutionwhentheinitialconditions(i.c.)

onC,P,andψandtheboundarycondition(b.c.)onCaregiven:

(i.c.) Att=0: C=C0; P=P0; ψ=ψ0; foreachz (10.5)

(b.c.) Atz=0:u

C DCzLε

⋅ − ⋅ ∂∂

= 0 foreacht (10.6)

(b.c.) Atz=L:∂∂

=Cz

0 foreacht. (10.7)

Thesetofdifferentialequationscanbenumericallyintegratedusingafinitedifferencemethod.

Reverchonetal.[145–148]usedSEManalysistoconfirmthepresenceofbrokencellsonparticlesurfaces.Theconceptofbrokenandintactcellswascombinedwithequilibriumrelationshipsforeitherfreesolute[149,150]orsoluteinteractingwithmatrix[133,135].Bothtypesofequilibriumwerealsoassumedtooccursimultaneouslybyvariousauthors,thefreesoluteinbrokencellsandtheinteractingsoluteinintactcells[135,137,146,147,151].

Sovovà[128]proposedthatageneralmodelapproachcanbeappliedtoseedoilandessentialoilextraction.Themodelisbasedonthedivisionoftheprocessintotwoextractionperiods:thefirstonegovernedbyphaseequilibriumandthesecondonebyinternaldiffusioninparticles,takingintoaccounttheconceptofbrokenandintactcellstoexplainthesuddenreductionoftheextractionrateafterthefirstextractionstep.Thiseffectisparticularlyevidentinthecaseofseedoilextraction.Thenewfeatureofthemodelisthedescriptionofthefirstextractionperiodconsideringdifferenttypesofphaseequilibria:independentonmatrix(solubilityequilibrium),adsorbedonmatrix(partitionbetweenthetwophases),anddifferentflowpatterns,mainlydispersion.Themodelhasbeenverifiedondatasetsfromliteraturerelatedtoseeds(almond)andessentialoils(orangepeels,pennyroyal).Thismodelpresentsalimitinthecaseofessentialoilextractionwhentheextractablematerialislocatedonlyinsidethematrix;theconceptofbroken(inthesurface)andintactcells(insidethe particle) is no longer applicable and the first part of extraction controlled byequilibriumdoesnotapply.

Gaspar et al. [29]modeled theextractionoforeganoessentialoil.Themodelisbasedontheprevalentgeometryofparticles:thoseobtainedfromleavestendtomaintainaplatelikegeometry.Massbalancesontheparticlehavebeenproposed.

Adsorptiondesorption processes can also be treated as extraction processes:adsorptiondesorptionisothermsbeingtheequilibriumcurvesduetointeractionsofthesolutesbetweenthesolidmatrixandthefluidphase.Differentialmassbalancesinthiscasecanalsodescribetheextractionprocess.Thisapproachhasbeenusedby Reverchon [152] to model the selective desorption from silica gel of two keycompoundsofessentialoils:limonene(representativeofthehydrocarbonterpenesfraction) and linalool (representative of the oxygenated terpenes fraction). The

7089_C010.indd 327 10/8/07 12:15:55 PM


modelhasalsobeenextendedtothefractionaldesorptionofbergamotpeeloil[122],describingatwostepdesorptionprocess,withthefirststepperformedat40°C,75bartodesorbhydrocarbonterpenesandthesecondstepat40°C,200bartodesorbtheoxygenatedcompounds.Later,Reverchonetal.[123]modeledtheselectiveadsorption on silica gel of a complex terpenic mixture formed by 13 components. Themixturewasdividedintofourfamiliesconsideredasfourpseudo–keycomponents.Theintegrationofdifferentialmassbalancesgaveaccountofthecompetitionamongthedifferentcompoundsfortheoccupationoftheadsorptionsitesandofdisplacementeffectsobservedattheexitoftheadsorptionbed.

Mathematicalmodelling has alsobeenperformed in this case [143, 153]; thevolatileoilhasbeenconsideredasamixtureoffourcompoundfamilies(pseudocomponents)extractedfromanactivelayerofconcreteputonasphericalinertcore.Successful modelling of terpenes oxygenated terpenes and oxygenated sesquiterpenesextractionwasobtained.

Mathematicalmodellingofcountercurrentpackedcolumnhasbeenstudiedbyonlyafewauthors[154–158]andonlyinsomecaseswithreferencetonaturalmatterfractionation[154,157].ThemostinterestingworkistheoneproposedbyRuivoetal.[154],whichperformedthedynamicmodellingandsimulationofapackedcolumn.Theyusedexperimentaldatafromamodelbinarymixtureformedbysqualeneandmethyloleate,fractionatedusingSCCO2.Themodelwasformedbyasetofpartialdifferentialequationsthatcorrespondtothedifferentialmassbalancesonthepackedcolumnandalgebraicequationsthatdescribethemasstransfer,thehydrodynamicsofthetwophaseflowthroughthepackingsandtheternarythermodynamicequilibriumforthestudiedsystem.Thecolumnwasconsideredatconstanttemperature.Afairlygoodagreementwasobtainedbetweenmeasuredandpredictedcompositionprofilesoftheoutletstreamsovertime.

reFerenCes

1. Reverchon, E., Supercritical fluid extraction and fractionation of essential oils andrelatedproducts,J. Supercrit. Fluids,10,1,1997.

2. Meireles,M.A.A.,Supercriticalextractionfromsolid:Processdesigndata(2001–2003),Current Opin. Solid State Mat. Sci.,7,321,2001.

3. Lang,Q.andWai,C.M.,Supercriticalfluidextraction inherbalandnaturalproductstudies—Apracticalreview,Talanta,53,771,2001.

4. Turner,C.,Eskilsson,C.S.andBjorklund,E.,Review:Collectioninanalyticalscalesupercriticalfluidextraction,J. Chromat. A,947,1,2002.

5. Brunner, G., Supercritical fluids: Technology and application to food processing,J. Food Eng.,67,21,2005.

6. Stahl,E.,Quirin,K.W.andGerard,D.,Verdichtete Gase zur Extraction und Raffination,Springer,Berlin,1997.

7. Moyler,D.A.,ExtractionofflavoursandfragranceswithcompressedCO2,inExtrac-tion of Natural Products Using Near-Critical Solvents, King, M.B. and Bott, T.R.,Eds.,Blackie,Glasgow,1993.

8. Kerrola,K.,Literature review: Isolationof essential oils andflavour compoundsbydensecarbondioxide,Food Rev. Int.,11,547,1995.

9. Rosa,P.T.V.andMeireles,M.A.A.,SupercriticaltechnologyinBrazil:Systemsinvestigated(1994–2003),J. Supercrit. Fluids,34,109,2005.

7089_C010.indd 328 10/8/07 12:15:56 PM


10. Smith,R.M.,Supercriticalfluidsinseparationscience—Thedreams,thereality,andthefuture,J. Chromatogr. A,856,83,1999.

11. Della Porta, G. et al., Isolation of eucalyptus oil by supercritical fluid extraction,Flavour Fragr. J.,14,214,1999.

12. Reverchon, E. and Della Porta, G., Tuberose concrete fractionation by supercriticalcarbondioxide,J. Agric. Food Chem.,45,1356,1997.

13. DellaPorta,G.etal.,IsolationofclovebudandstaraniseessentialoilbysupercriticalCO2extraction,Lebensm. –Wiss. U. –Technol.,31,454,1998.

14. Caredda, A. et al., Supercritical carbon dioxide extraction and characterization ofLaurus nobilisessentialoil,J. Agric. Food Chem.,50,1492,2002.

15. Marongiu,B.etal.,ExtractionandisolationofSalvia desoleanaandMentha spicatasubsp.insularisessentialoilsbysupercriticalCO2,Flavour Fragr. J.,16,384,2001.

16. Marongiu,B.etal.,AntioxidantactivityofsupercriticalextractofMelissa officinalis subsp.officinalisandMelissa officinalissubsp.inodora,Phytother. Res.,18,789,2004.

17. Simandi,B.etal.,Supercriticalcarbondioxideextractionandfractionationoffenneloil,J. Agric. Food Chem.,47,1635,1999.

18. Simandi,B.etal.,Pilotscaleextractionandfractionalseparationofonionoleoresinusingsupercriticalcarbondioxide,J. Food Eng.,46,183,2000.

19. Vagi,E.etal.,RecoveryofpigmentsfromOriganum majoranaL.byextractionwithsupercriticalcarbondioxide,J. Agric. Food Chem.,50,2297,2002.

20. Esquivel, M.M., Ribeiro, M.A. and BernardoGil, M.G., Supercritical extraction ofsavory oil: Study of antioxidant activity and extract characterization, J. Supercrit. Fluids,14,129,1999.

21. Ribeiro,M.A.,BernardoGil,M.G.andEsquivel,M.M.,Melissa officinalisL.:Studyofantioxidantactivityinsupercriticalresidues,J. Supercrit. Fluids,21,51,2001.

22. Dauksas,E.etal.,EffectoffastCO2pressurechangesontheyieldoflovage(Levisticum officinaleKoch.)andcelery(Apium graveolens L.)extracts,J. Supercrit. Fluids,22,20,2002.

23. Dauksas,E.etal.,Rapidscreeningofantioxidantactivityofsage(Salvia officinalisL.)extractsobtainedbysupercriticalcarbondioxideatdifferentextractionconditions,Nahrung/Food,45,338,2001.

24. Wang,B.J.,Lien,Y.H.andYu,Z.R.,Supercriticalfluidextractive fractionation—Studyoftheantioxidantactivitiesofpropolis,Food Chem.,86,237,2004.

25. Wang, B.J. et al., Hepatoprotective and antioxidant effects of Bupleurum kaoi Liu(Chao et Chuang) extract and its fractions fractionated using supercritical CO2 onCCl4–inducedliverdamage,Food Chem. Toxicol.,42,609,2004.

26. Vasapollo,G.etal.,InnovativesupercriticalCO2extractionoflycopenefromtomatointhepresenceofvegetableoilascosolvent,J. Supercrit. Fluids,29,87–96,2004.

27. Kiriamiti,H.K.etal.,Supercriticalcarbondioxideprocessingofpyrethrumoleoresinandpale,J. Agric. Food Chem.,51,880,2003.

28. Kiriamiti, H.K. et al., Pyrethrin extraction from pyrethrum flowers using carbondioxide,J. Supercrit. Fluids,26,193,2003.

29. Gaspar,F. et al.,Modelling the extractionof essential oilswith compressed carbondioxide,J. Supercrit. Fluids,25,247,2003.

30. Smith, R.L., Jr., et al., Separation of cashew (Anacardium occidentale L.) nut shellliquidwithsupercriticalcarbondioxide,Biores. Technol.,88,1,2003.

31. Ibanez,E. et al.,Supercriticalfluidextractionand fractionationofdifferentpreprocessedrosemaryplants,J. Agric. Food Chem.,47,1400,1999.

32. Senorans, F.J. et al., Liquid chromatographicmass spectrometric analysis of supercriticalfluidextractsofrosemaryplants,J. Chromatogr. A,870,491,2000.

7089_C010.indd 329 10/8/07 12:15:56 PM


33. Ramirez,P.etal.,Separationofrosemaryantioxidantcompoundsbysupercriticalfluidchromatography on coated packed capillary columns, J. Chromatogr. A, 1057, 241,2004.

34. Grigonis,D.etal.,Comparisonofdifferentextractiontechniquesforisolationofantioxidantsfromsweetgrass(Hierochloe odorata),J. Supercrit. Fluids,33,223,2005.

35. Dauksas,E.,Venskutonis,P.R.andSivik,B.,Supercriticalfluidextractionofborage(Borago officinalisL.)seedswithpureCO2anditsmixturewithcaprylicacidmethylester,J. Supercrit. Fluids,22,211,2002.

36. Bicchi, C., Binello, A. and Rubiolo, P., Determination of phenolic diterpene antioxidantsinrosemary(Rosmarinus officinalisL.)withdifferentmethodsofextractionandanalysis,Phytochem. Anal.,11,236,2000.

37. LeFloch,F.etal.,Supercriticalfluidextractionofphenolcompoundsfromoliveleaves,Talanta, 46,1123,1998.

38. delValle,J.M.etal.,Extractionofboldo(Peumus boldusM.)leaveswithsupercriticalCO2andhotpressurizedwater,Food Res. Intern.,38,203,2005.

39. ElGhorab,A.H.etal.,AntioxidantactivityofEgyptianEucaliptus camaldulensisvar.brevirostrisleafextracts,Nahrung/Food,47,41,2003.

40. Zancan,K.C.etal.,Extractionofginger(Zingiber officinaleRoscoe)oleoresinwithCO2andcosolvents:Astudyof theantioxidantactionof theextracts,J. Supercrit. Fluids,24,57,2002.

41. Leal,P.F.etal.,Functionalpropertiesofspiceextractsobtainedviasupercriticalfluidextraction,J. Agric. Food Chem.,51,2520,2003.

42. Braga,M.E.M.etal.,Comparisonofyield,composition,andantioxidantactivityofturmeric(Curcuma longaL.)extractsusingvarioustechniques,J. Agric. Food Chem.,51,6604,2003.

43. Yoda,S.K.etal.,SupercriticalfluidextractionfromStevia rebaudianaBertoniusingCO2andCO2+water:Extractionkineticsandidentificationofextractedcomponents,J. Food Eng.,57,125,2003.

44. Rostagno,M.A.,Araujo, J.M.A. andSandi,D.,Supercriticalfluid extractionof isoflavonesfromsoybeanflour,Food Chem.,78,111,2002.

45. Hu, Q., Hu, Y. and Xu, J., Free radicalscavenging activity of Aloe vera (Aloe barbadensisMiller)extractsbysupercriticalcarbondioxideextraction,Food Chem.,91,85,2005.

46. Quitain,A.T.,Katoh,S.andMoriyoshi,T.,Isolationofantimicrobialsandantioxidantsfrommosobamboo (Phyllostachys heterocycla)by supercriticalCO2extractionandsubsequenthydrothermal treatmentof the residues, Ind. Eng. Chem. Res.,43,1056,2004.

47. Saldana,M.D.A.etal.,Extractionofmethylxanthinesfromguaranàseeds,matèleaves,andcocoabeansusingsupercriticalcarbondioxideandethanol,J. Agric. Food Chem.,50,4820,2002.

48. Hamburger,M.,Baumann,D.andAdler,S.,Supercriticalcarbondioxideextractionofselectedmedicinalplants—Effectsofhighpressureandaddedethanolonyieldofextractedsubstances,Phytochem. Anal., 15,46,2004.

49. Klimes,J.,Sochor,J.andKriz,J.,Astudyoftheconditionsofthesupercriticalfluidextractionintheanalysisofselectedantiinflammatorydrugsinplasma,Il Farmaco,57,117,2002.

50. Aghel,N.etal.,SupercriticalcarbondioxideextractionofMentha pulegiumL.essentialoil,Talanta,62,407,2004.

51. Cui,Y. andAng,C.Y.W.,Supercriticalfluidextractionandhighperformance liquidchromatographic determination of phloroglucinols in St. John’s wort (Hypericum perforatumL.),J. Agric. Food Chem.,50,2755,2002.

7089_C010.indd 330 10/8/07 12:15:57 PM


52. Cao,X.L.etal.,SupercriticalfluidextractionofcatechinsfromCratoxylum prumifoliumDyer and subsequent purification by highspeed countercurrent chromatography,J. Chromatogr. A,898,75,2000.

53. Cao,X.andIto,Y.,Supercriticalfluidextractionofgrapeseedoilandsubsequentseparationoffreefattyacidsbyhighspeedcountercurrentchromatography,J. Chromatogr. A,1021,117,2003.

54. Yang,C.,Xu,Y.R.andYao,W.X.,ExtractionofpharmaceuticalcomponentsfromGinkgo biloba leaves using supercritical carbon dioxide, J. Agric. Food Chem., 50,846,2002.

55. Shu,X.S.,Gao,Z.H. andYang,X.L.,Supercriticalfluid extractionof sapogeninsfromtubersofSmilax china,Fitoterapia,75,656,2004.

56. Catchpole,O.J.etal.,SupercriticalextractionofherbsI:Sawpalmetto,St.John’swort,kavaroot,andechinacea,J. Supercrit. Fluids,22,129,2002.

57. Sovovà,H.etal.,Nearcriticalextractionofpigmentsandoleoresinfromstingingnettleleaves,J. Supercrit. Fluids,30,213,2004.

58. Lim, G.B. et al., Separation of astaxanthin from red yeast Phaffia rhodozyma bysupercriticalcarbondioxideextraction,Biochem. Eng. J.,11,181,2002.

59. Quan,C.etal.,Supercriticalfluidextractionandcleanupoforganochlorinepesticidesinginseng,J. Supercrit. Fluids,31,149,2004.

60. Ling,Y.C.,Teng,H.C.andCartwright,C.,SupercriticalfluidextractionandcleanupoforganochlorinepesticidesinChineseherbalmedicine,J. Chromatogr. A,835,145,1999.

61. Leeke,G.,Gaspar,F.andSantos,R.,Influenceofwaterontheextractionofessentialoilsfromamodelherbusingsupercriticalcarbondioxide,Ind. Eng. Chem. Res.,41,2033,2002.

62. Illés,V.etal.,ExtractionofhiprosefruitbysupercriticalCO2andpropane,J. Super-crit. Fluids,10,209,1997.

63. Menaker, A. et al., Identification and characterization of supercritical fluid extractsfromherbs,C. R. Chimie,7,629,2004.

64. Chiu,K.L.etal.,SupercriticalfluidsextractionofGinkgoginkgolidesandflavonoids,J. Supercrit. Fluids,24,77,2002.

65. King,J.W.,SupercriticalfluidextractionofVernonia galamensisseeds,Ind. Crops and Products,14,241,2001.

66. Sanal,I.S.etal.,DeterminationofoptimumconditionsforSC(CO2+ethanol)extractionofβcarotenefromapricotpomaceusingresponsesurfacemethodology,J. Super-crit. Fluids,34,331,2005.

67. daCruzFrancisco,J.etal.,Applicationofsupercriticalcarbondioxidefortheextractionofalkylresorcinolsfromryebran,J. Supercrit. Fluids,35,220,2005.

68. Teberikler,L.,Koseoglu,S.S.andAkgerman,A.,Deolingofcrudelecithinusingsupercriticalcarbondioxideinthepresenceofcosolvents,J. Food Sci.,66,850,2001.

69. Teberikler,L.,Koseoglu,S.S.andAkgerman,A.,Selectiveextractionofphosphatilylcholinefromlecithinbysupercriticalcarbondioxide/ethanolmixture,JAOCS,78,115,2001.

70. Eggers,R.,Voges,S.K. and Jaeger,Ph.T.,Solidbedproperties in supercritical processing,inI.Kikic,M.Perrut,Eds.,Proceedings of the 9th Meeting on Supercritical Fluids, Trieste(Italy)2004,E11(pdf).

71. http://webbook.nist.gov/chemistry/fluid. 72. Span,R.andWagner,W.,Anewequationofstateforcarbondioxidecoveringthefluid

regionfromthetriplepointtemperatureto1100Katpressuresupto800MPa,J. Phys. Chem. Ref. Data,25,1509,1996.

73. Raeissi,S.andPeters,C.J.,Bubblepointpressuresofthebinarysystemcarbondioxide+linalool,J. Supercrit. Fluids,20,221,2001.

7089_C010.indd 331 10/8/07 12:15:58 PM


74. Iwai,Y.etal.,Highpressurevaporliquidequilibria forcarbondioxide+ limonene,J. Chem. Eng. Data,41,951,1996.

75. Marteau,P.,Obriot, J.andTufeu,R.,Experimentaldeterminationofvaporliquidequilibria of CO2 + limonene and CO2 + citral mixtures, J. Supercrit. Fluids, 8,1995,20.

76. Akgün,M.,Akgün,N.A.andDinçer,S.,Phasebehaviourofessentialoilcomponentsinsupercriticalcarbondioxide,J. Supercrit. Fluids,15,117,1999.

77. Reverchon,E.,Russo,P.andStassi,A.,Solubilitiesofsolidoctacosaneandtriacontaneinsupercriticalcarbondioxide,J. Chem. Eng. Data,38,458,1993.

78. DýazMaroto, M.C., PerezCoello, M.S. and Cabezudo, M.D., Supercritical carbondioxideextractionofvolatilesfromspices:Comparisonwithsimultaneousdistillationextraction,J. Chromatogr. A,947,23,2002.

79. Rozzi,N.L.etal.,Supercriticalfluidextractionofessentialoilcomponentsfromlemonscentedbotanicals,Lebensm. –Wiss. U. –Technol.,35,319,2002.

80. Carlson,L.H.C.etal.,Extractionoflemongrassessentialoilwithdensecarbondioxide,J. Supercrit. Fluids,21,33,2001.

81. Dauksas,E.,Venskutonis,P.R.andSivik,B.,SupercriticalCO2extractionofthemainconstituentsoflovage(Levisticum officinaleKoch.)essentialoilinmodelsystemsandovergroundbotanicalpartsoftheplant,J. Supercrit. Fluids,15,51,1999.

82. Reverchon, E., Fractional separation of SCF extracts from marjoram leaves: Masstransferandoptimization,J. Supercrit. Fluids,5,256,1992.

83. Rodrigues,M.R.A.etal.,TheeffectsoftemperatureandpressureonthecharacteristicsoftheextractsfromhighpressureCO2extractionofMajorana hortensisMoench,J. Agric. Food Chem.,51,453,2003.

84. Simandi,B.etal.,Supercriticalcarbondioxideextractionandfractionationoforeganooleoresin,Food Res. Intern.,31,723,1998.

85. Reverchon,E.,Taddeo,R.andDellaPorta,G.,ExtractionofsageessentialoilbysupercriticalCO2:Influenceofsomeprocessparameters,J. Supercrit. Fluids,8,302,1995.

86. Sonsuzer,S.,Sahin,S.andYilmaz,L.,OptimizationofsupercriticalCO2extractionofThymbra spicataoil,J. Supercrit. Fluids,30,189,2004.

87. MoldaoMartins, M. et al., Supercritical CO2 extraction of Thymus zygis L. subsp.sylvestris aroma,J. Supercrit. Fluids,18,25,2000.

88. Povh,N.P.,Marques,M.O.M.andMeireles,M.A.A.,SupercriticalCO2extractionofessential oil and oleoresin from chamomile (Chamomilla recutita [L.] Rauschert),J. Supercrit. Fluids,21,245,2001.

89. Reverchon,E.andSenatore,F.,Supercriticalcarbondioxideextractionofchamomileessentialoilanditsanalysisbygaschromatographymassspectrometry,J. Agric. Food Chem.,42,154,1994.

90. Reverchon,E.,DellaPorta,G.andSenatore,F.,SupercriticalCO2extractionandfractionationoflavenderessentialoilandwaxes,J. Agric. Food Chem.,43,1654,1995.

91. Rodrigues,V.M.etal.,Supercriticalextractionofessentialoilfromaniseed(Pimpi-nella anisumL.)usingCO2:Solubility,kinetics,andcompositiondata,J. Agric. Food Chem.,51,1518,2003.

92. Dauksas,E.andVenskutonis,P.R.,Extractionoflovage(Levisticum officinaleKoch.)rootsbycarbondioxide.1.EffectofCO2parametersontheyieldoftheextract,J. Agric. Food Chem.,46,4347,1998.

93. Monteiro,A.R.etal.,Extractionofthesolublematerialfromtheshellsofthebacurifruit (Platonia insignisMart)withpressurizedCO2andothersolvents,J. Supercrit. Fluids,11,91,1997.

94. Ferreira,S.R.S.etal.,Supercriticalfluidextractionofblackpepper(Piper nigrunL.)essentialoil,J. Supercrit. Fluids,14,235,1999.

7089_C010.indd 332 10/8/07 12:15:58 PM


95. Tipsrisukond,N.,Fernando,L.N.andClarke,A.D.,Antioxidanteffectsofessentialoilandoleoresinofblackpepperfromsupercriticalcarbondioxideextractionsingroundpork,J. Agric. Food Chem.,46,4329,1998.

96. Patel,R.N.,Bandyopadhyay,S. andGanesh,A.,Extractionof cashew (Anacardium occidentale)nutshellliquidusingsupercriticalcarbondioxide,Biores. Techn.,97,847,2006.

97. Rodrigues,M.R.A.etal.,ChemicalcompositionandextractionyieldoftheextractofOriganum vulgareobtainedfromsubandsupercriticalCO2,J. Agric. Food Chem.,52,3042,2004.

98. Duarte,C.etal.,Supercriticalfluidextractionofredpepper(Capsicum frutescens L.),J. Supercrit. Fluids,30,155,2004.

99. Barjaktarovic,B.,Sovilj,M.andKnez,Z.,ChemicalcompositionofJuniperus com-munisL.fruitssupercriticalCO2extracts:Dependenceonpressureandextractiontime,J. Agric. Food Chem.,53,2630,2005.

100. Reverchon,E.,DellaPorta,G.andGorgoglione,D.,SupercriticalCO2fractionationofjasmineconcrete,J. Supercrit. Fluids,8,60,1995.

101. Reverchon,E.andDellaPorta,G.,RoseconcretefractionationbysupercriticalCO2,J. Supercrit. Fluids,9,199,1996.

102. Vagi,E.etal.,PhenolicandtriterpenoidantioxidantsfromOriganum majoranaL.herbandextractsobtainedwithdifferentsolvents,J. Agric. Food Chem.,53,17,2005.

103. ElGhoran,A.H.,Mansour,A.F.andElmassry,K.F.,EffectofextractionmethodsonthechemicalcompositionandantioxidantactivityofEgyptianmarjoram(Majorana hortensisMoench),Flavour Fragr. J.,19,54,2004.

104. LopezSebastian,S.etal.,Dearomatizationofantioxidantrosemaryextractsbytreatmentwithsupercriticalcarbondioxide,J. Agric. Food Chem.,46,13,1998.

105. Ibanez,E.etal.,Combineduseofsupercriticalfluidextraction,micellarelectrokineticchromatography,andreversephasehighperformanceliquidchromatographyfor theanalysis of antioxidants from rosemary (Rosmarinus officinalis L.), J. Agric. Food Chem.,48,4060,2000.

106. Tena,M.T.etal.,Supercriticalfluidextractionofnaturalantioxidantsfromrosemary:Comparisonwithliquidsolventsonication,Anal. Chem.,69,521,1997.

107. Hadolin, M. et al., Isolation and concentration of natural antioxidants with highpressureextraction,Inn. Food Science Emerg. Technol.,5,245,2004.

108. Yepez,B.etal.,Producingantioxidantfractionsfromherbaceousmatricesbysupercriticalfluidextraction,Fluid Phase Equil.,194–197,879,2002.

109. Braga,M.E.M.etal.,Supercriticalfluidextraction fromLippia alba:Globalyields,kineticdata,andextractchemicalcomposition,J. Supercrit. Fluids,34,149,2005.

110. Stashenko,E.E.,Jaramillo,B.E.andMartinez,J.R.,ComparisonofdifferentextractionmethodsfortheanalysisofvolatilesecondarymetabolitesofLippia alba(Mill.)N.E. Brown, grown in Colombia, and evaluation of its in vitro antioxidant activity,J. Chromatogr. A,1025,93,2004.

111. Ramos,E.etal.,ObtentionofabrewedcoffeearomaextractbyanoptimizedsupercriticalCO2basedprocess,J. Agric. Food Chem.,46,4011,1998.

112. Michielin,E.M.Z.etal.,Compositionprofileofhorsetail (Equisetum giganteumL.)oleoresin:ComparingSFEandorganic solvents extraction,J. Supercrit. Fluids, 33,131,2005.

113. Daood,H.G.etal.,Extractionofpungentspicepaprikabysupercriticalcarbondioxideandsubcriticalpropane,J. Supercrit. Fluids,23,143,2002.

114. Uquiche,E.,delValle,J.M.andOrtiz,J.,Supercriticalcarbondioxideextractionofredpepper(Capsicum annuumL.)oleoresin,J. Food Eng.,65,55,2004.

115. GomezCoronado, D.J.M. et al., Tocopherol measurement in edible products ofvegetableorigin,J. Chromatogr. A,1054,227,2004.

7089_C010.indd 333 10/8/07 12:15:59 PM


116. Reverchon,E.etal.,Supercriticalfractionalextractionoffennelseedoilandessentialoil:Experimentsandmathematicalmodelling,Ind. Eng. Chem. Res.,38,3069,1999.

117. Reverchon,E.,Marciano,A.andPoletto,M.,FractionationofapeeloilkeymixturebysupercriticalCO2inacontinuoustower,Ind. Eng. Chem. Res.,36,4940,1997.

118. Budich,M.etal.,CountercurrentdeterpenationofcitrusoilswithsupercriticalCO2,J. Supercrit. Fluids,14,105,1999.

119. Sato,M.,Goto,M.andHirose,T.,Fractionalextractionwithsupercriticalcarbondioxidefortheremovalofterpenesfromcitrusoil,Ind. Eng. Chem. Res.,34,3941,1995.

120. Stahl,E.,Quirin,K.W.andGerard,D.,Verdichtete Gase zur Extraction und Raffination, Springer,Berlin,1987.

121. Kubat,H.,Akman,U.andHortacsu,O.,Semibatchpackedcolumndeterpenationoforiganumoilbydensecarbondioxide,Chem. Eng. Proc.,40,19,2001.

122. Reverchon,E.andIacuzio,G.,Supercriticaldesorptionofbergamotpeeloilfromsilicagel—Experimentsandmathematicalmodelling,Chem. Eng. Sci.,52,3553,1997.

123. Reverchon, E., Lamberti, G. and Subra, P., Modelling and simulation of the supercriticaladsorptionofcomplexterpenemixtures,Chem. Eng. Sci.,53,3537,1998.

124. Catchpole,O.J.andBergmann,C.,Continuousgasantisolventfractionationofnaturalproducts, inFifth Meeting on Supercritical Fluids,Vol.1,Perrut,M.andSubra,P.,Eds.,Nice,France,1998,257.

125. Catchpole,O.J.,Hochmann,S.andAnderson,S.R.J.,Gasantisolventfractionationofnaturalproducts,inProcess Technology Proceedings 12 (High Pressure Engineering),R.vonRohr,Ch.Trepp,Eds.,Elsevier,Amsterdam,1996,309.

126. Scrugli,S.etal.,Apreliminarystudyon theapplicationofsupercriticalantisolventtechniquetothefractionationoftobaccoextracts,inEighth Meeting on Supercritical Fluids,Besnard,M.andCansell,F.,Eds.,Bordeaux,France,2002,901.

127. Reverchon,E.,Mathematicalmodellingofsupercriticalextractionofsageoil,AIChE J.,42,1765,1996.

128. Sovová,H.,Mathematicalmodelforsupercriticalfluidextractionofnaturalproductsandextractioncurveevaluation,J. Supercrit. Fluids,33,35,2005.

129. Kandiah,M.andSpiro,M.,Extractionofgingerrhizomes:Kineticstudieswithsupercriticalcarbondioxide,Int. J. Food Sci. Technol.,25,328,1990.

130. Nguyen, K., Barton, P. and Spencer, J., Supercritical carbon dioxide of vanilla,J. Supercrit. Fluids,4,40,1991.

131. Bartle,K.D.etal.,Amodelfordynamicextractionusingasupercriticalfluid,J. Super-crit. Fluids,3,143,1990.

132. Reverchon,E.,Donsì,G.andSestiOsséo,L.,Modelingofsupercriticalfluidextractionfromherbaceousmatrices,Ind. Eng. Chem. Res.,32,2721,1993.

133. Sovovà, H. et al., Supercritical carbon dioxide extraction of caraway essential oil,Chem. Eng. Sci.,49,2499,1994.

134. Roy,B.C.,Goto,M.andHirose,T.,Extractionofgingeroilwithsupercriticalcarbondioxide:Experimentsandmodeling,Ind. Eng. Chem. Res.,35,607,1996.

135. ReisVasco,E.M.C.etal.,Mathematicalmodellingandsimulationofpennyroyalessentialoilsupercriticalextraction,Chem. Eng. Sci.,55,2917,2000.

136. Goodarznia,I.andEikani,M.H.,Supercriticalcarbondioxideextractionofessentialoils:modellingandsimulation,Chem. Eng. Sci.,53,1387,1998.

137. Reverchon, E. and Marrone, C., Supercritical extraction of clove bud essential oil:Isolationandmathematicalmodelling,Chem. Eng. Sci.,52,3421,1997.


139. Martinez,J.etal.,Multicomponentmodel todescribeextractionofgingeroleoresinwithsupercriticalcarbondioxide,Ind. Eng. Chem. Res.,42,1057,2003.

7089_C010.indd 334 10/8/07 12:15:59 PM


140. delValle,J.M.,Jimenez,M.anddelaFuente,J.C.,ExtractionkineticsofprepelletizedjalapenopepperswithsupercriticalCO2,J. Supercrit. Fluids,25,33,2003.

141. Akgun,M.,Akgun,N.A.andDincer,S.,Extractionandmodellingoflavenderfloweressentialoilusingsupercriticalcarbondioxide,Ind. Eng. Chem. Res.,39,473,2000.

142. Campos, L.M.A.S. et al., Experimental data and modelling the supercritical fluidextractionofmarigold(Calendula officinalis)oleoresin,J. Supercrit. Fluids,34,163,2005.

143. Reverchon,E.,DellaPorta,G.andLamberti,G.,ModellingoforangeflowerconcretefractionationbysupercriticalCO2,J. Supercrit. Fluids,14,115,1999.

144. Ferreira,S.R.S.andMeireles,M.A.M.,Modelingthesupercriticalfluidextractionofblackpepper(Piper nigrumL.)essentialoil,J. Food Eng.,54,263,2002.

145. Reverchon, E. and Marrone, C., Modeling and simulation of the supercritical CO2extractionofvegetableoils,J. Supercrit. Fluids,19,161,2001.

146. Marrone, C. et al., Almond oil extraction by supercritical CO2: Experiments andmodelling,Chem. Eng. Sci.,53,3711,1998.

147. Reverchon,E.,Kaziunas,A.andMarrone,C.,SupercriticalCO2extractionofhiproseseedoil:Experimentsandmathematicalmodelling,Chem. Eng. Sci.,55,2195,2000.


149. Sovová,H.,RateofthevegetableoilextractionwithsupercriticalCO2.I.Modellingofextractioncurves,Chem. Eng. Sci.,49,409,1994.

150. Štástová,J.etal.,RateofthevegetableoilextractionwithsupercriticalCO2.III.Extractionfromseabuckthorn,Chem. Eng. Sci.,51,4347,1996.

151. ReisVasco,E.M.C.etal.,Mathematicalmodellingandsimulationofpennyroyalessentialoilsupercriticalextraction,Chem. Eng. Sci.,55,2917,2000.

152. Reverchon, E., Supercritical desorption of limonene and linalool from silica gel:Experimentsandmodelling,Chem. Eng. Sci.,52,1019,1997.

153. Reverchon,E.andPoletto,M.,MathematicalmodellingofsupercriticalCO2fractionationofflowerconcretes,Chem. Eng. Sci.,51,3741,1996.

154. Ruivo,R.etal.,Dynamicmodelofacountercurrentpackedcolumnoperatingathighpressureconditions,J. Supercrit. Fluids,32,183,2004.

155. Ramchandran,B.etal.,Dynamicsimulationofasupercriticalfluidextractionprocess,Ind. Eng. Chem. Res.,31,281,1992.

156. Camy,S.andCondoret,J.S.,Dynamicmodellingofafractionationprocessforaliquidmixtureusingsupercriticalcarbondioxide,Chem. Eng. Proc.,40,499,2001.

157. Benvenuti, F. and Gironi, F., Dynamic simulation of a semicontinuous extractionprocessusingsupercriticalsolvent,inProceedings of the Fifth Italian Conference on Supercritical Fluids and their Applications, A.Bertucco,Ed.,1999,281.

158. Cesari, G. et al., A computerprogram for the dynamic simulation of a semibatchsupercriticalfluidextractionprocess,Comput. Chem. Eng.,13,1175,1989.

7089_C010.indd 335 10/8/07 12:16:00 PM

7089_C010.indd 336 10/8/07 12:16:00 PM

337

11 Processing of Spices Using Supercritical Fluids

Mamata Mukhopadhyay

Contents

11.1 Introduction................................................................................................. 33711.2 ImportanceofSpices.................................................................................. 33811.3 BeneficialAttributesofSpices.................................................................... 33911.4 BioactiveIngredientsinSpices................................................................... 34111.5 SaleableSpiceProducts.............................................................................. 34311.6 ConventionalExtractionMethods..............................................................34611.7 SupercriticalCarbonDioxideastheExtractant......................................... 34711.8 CommercialSCFEProcess......................................................................... 34911.9 ComparisonofSpiceExtractsbyConventionalandSCFEProcesses....... 35111.10 ProcessAnalysisofSCFEfromSelectedSpices........................................ 354

11.10.1 CelerySeed(Apium graveolen).................................................... 35411.10.2 RedChili....................................................................................... 35611.10.3 Paprika.......................................................................................... 35711.10.4 Ginger........................................................................................... 35811.10.5 Nutmeg.......................................................................................... 35811.10.6 BlackPepper................................................................................. 35911.10.7 Vanilla........................................................................................... 35911.10.8 Cardamom.................................................................................... 35911.10.9 Fennel,Caraway,andCoriander...................................................36011.9.10 Garlic............................................................................................ 36111.10.11 Cinnamon...................................................................................... 362

11.11 CorrelationforSpiceOilSolubilityinSC-CO2.......................................... 36211.12 Conclusions.................................................................................................364References.............................................................................................................. 365

11.1 IntroduCtIon

In recent years, increasing demand for superior quality and safety of foods andmedicines, as well as concern for environmental pollution during their commer-cialproduction,havetriggeredstringentregulationsonthetoxinlevelsinfoodsandmedicinesaswellasonthedischargeofpollutantstotheenvironment.Inaddition,there has been increasing consumer preference for natural substances. All thesefactorshavegiven strong impetus todevelopmentofcost-effectivenew technolo-gies,suchastheoneforeco-friendlyextractionfromnaturalsubstancesemploying

7089_C011.indd 337 10/8/07 12:19:04 PM


greenandsafesolvents. In recentyears, supercriticalfluidextraction (SCFE)hasemergedasahighlypromisingenvironmentallybenigntechnologyforproductionofnaturalextracts,suchasflavors,fragrances,spiceoils,andoleoresins;naturalanti-oxidants;naturalcolors;nutraceuticals;andbiologicallyactiveprinciples.Thestateofasubstance iscalledsupercriticalwhenboth temperatureandpressureexceedtheircriticalpointvalues.Asupercriticalfluid(SCF)combinesthetwinbeneficialproperties,namelyhighdensity(whichimpartshighsolventpower)andhighcom-pressibility(whichpermitshighselectivityduetolargevariabilityofsolventpowerbysmallchangesintemperatureandpressure).Inaddition,itoffersveryattractiveextractioncharacteristics,owingtoitsfavorablediffusivity,viscosity,surfacetension,andotherthermo-physicalproperties.

Since the1980s,severalpotentialapplicationsofSCFEtechniqueshavebeenreported.Sofar,themostpopularSCFhasbeencarbondioxide(CO2),owingtoitseasyavailability,lowcost,nonflammability,nontoxicity,andaspectrumofsolventproperties in a single substance. Its critical temperature is 31.1°C and its criticalpressure is 73.8 bar. Dense or supercritical carbon dioxide (SC-CO2) could verywell be the most commonly used solvent in this century due to its wide-rangingapplications. Its near-ambient critical temperature makes it ideally suitable forprocessingof thermally labilenatural substances. It isgenerally regardedas safe(GRAS),andityieldsmicrobial-inactivated,contaminant-free,tailor-madeextractsof superior organoleptic profile and longer shelf life,withhighpotencyof activeingredients. The SCFE technique ensures high consistency and reliability in thequalityandsafetyofthebioactiveheat-sensitivebotanicalproducts,asitdoesnotalter thedelicatebalanceofbioactivityofnaturalmolecules.Allof theseadvan-tagesarealmostimpossibleinconventionalprocesses.Therefore,SCFEtechnologyusingSC-CO2asthesolventisanidealalternativetotheconventionaltechniquesforextractionofbioactive ingredients fromspices.Thischapterpresentssomeof theprinciplesandmethodsofSCFEtechnologyforprocessingofspicesforuseinfoodproducts,medicines,anddietarysupplements.

11.2 ImportanCe of spICes

Bydefinition,aspiceisan“aromatic,pungentvegetablesubstanceusedtoflowerfood,”asoriginatedfromtheLatinname“speciesaromatacea.”Anherbisdefinedasa“plantwithoutwoodytissuethatwithersanddiesafterflowering.”Spicesandherbsbothfunctionasflavoringagentsforfoodand,accordingly,theU.S.FoodandDrugAssociationincludesspicesandherbstogetherinaclassofaromaticvegetablesthatimpartflavorandseasoningtofoodratherthannutritionalvalue.Thus,spiceimplies a tropical herbal plant or some part of it that is used in cooking and incondiments aswellasincandies,cosmetics,fragrances,andmedicationsinordertoprovidearoma,flavor,andcolor,alongwithstimulatingpungencyandtaste[1].

In ancient ages, spices were employed for embalming, preserving foods, andmaskingbadodors.TheEbersPapyrus,writteninEgyptinabout1500B.C.,evenmentions some common spices, such as coriander, cumin, fenugreek, and mint,and describes how these spices were used in foods and medicines. In MedievalandRenaissance times,GreeksandRomansused tospendvast fortuneson trade

7089_C011.indd 338 10/8/07 12:19:04 PM

Processing of Spices Using Supercritical Fluids 339

with Arabia, which was then the center of the spice trade. Exotic spices used tobeexhibitedasa symbolofwealthandpower, somuchso that thereweremanylongexplorationsinsearchofsourcesofspices.TheextraordinaryvoyagesmadeforthesespicesresultedinthediscoveryoftheNewWorldand,inturn,demonstratedthat theglobecouldbecircumnavigatedbysea.Thestrongmotivation tocontrolspice sources lured the British to India, the Portuguese to Brazil, the Spanish toCentralandSouthAmericaandthePhilippines,theFrenchtoAfrica,andtheDutchtoIndonesia.However,countriesexploringthesenewregionshadtodealwiththenatives toestablishmonopolisticcontrolofpowerover thespice-growing regionsandmajorspicetraderoutes.Overthislonghistoricaljourney,manyoftheexoticspices of earlier attraction, such as nutmeg and saffron, lost their pride of place,whilelessvaluedspiceshavingsomemedicinalvalues,suchasgarlic,peppers,andothercommonherbs,havenowbecomeincreasinglypopular[2].

11.3 BenefICIal attrIButes of spICes

Inpresentdays,mostpeopleaspireforgoodhealthandthereisagrowingfascina-tion in theuseofnaturalhealth careproducts. It is interesting tonote thatmorethan80%oftheworld’spopulationbelievesthat“preventionisbetterthancure,”forwhichtheypreferbotanicalproductshavinghealthpromoting,diseasepreventing,medicinal properties. Hardly any spices have no medicinal effects [2]. The mostcommonlyusedspicesarewellproventobemedicinal;forexample,blackpepper,cayenne,cinnamon,garlic,ginger,licorice,onion,andchivesallcontainavarietyofbiologicallyactivecompounds[3]thatareGRAS.Table11.1listssomespicesandtheirsynergistictherapeuticbenefitsbasedonthenumberofbioactivecomponentspresentforaspecificbiologicalaction.Itisnowbelievedthatthenaturallyoccur-ringsynergisticeffectofthetotalextractrendersbettereffectivenessforaspecificbiologicalactionthantheisolatedactiveingredient[2].

Spicescanbeusedtopromotehealth,curedisorders,andpreventdiseases,fromcancer anddiabetes to liver andheartproblems toobesity.Diverseagro-climaticzonesprevalentinsomegeographicallocationsareresponsibleforproducingbio-diversefloraandfauna.Thesezonesledtothedevelopmentoftheancientmedicinalsciences,likeAyurveda,Sidda,andUnani,basedontheregionalnaturalresourcesavailableinIndiaandChina.Naturalflavorsandfragrancesobtainedfromspicesand herbs are often used to relieve stress by aromatherapy. Spices and herbs arealsoused for gastrointestinal therapies, aphrodisiacs, and nonspecific tonics. Themore pungent ones are counterirritants and can be used for pain relief and anti-inflammatoryeffects.Manyhaveantibacterialorantifungalproperties.Spicesareclaimedtopreventcancersduetotheirstrongantioxidantproperties,thoughmostofthemorepotentmedicalbenefitshavenotbeenvalidatedowingtothedifficultyofidentifyingtherelevantbioactivecompounds.

For years, researchers have recognized that garlic reduces hypertension,cholesterol, respiratory and urinary tract infections, and digestive and liverdisorders.Italsocuresdiphtheria,hepatitis,ringworm,typhoid,andbronchitisandinhibitspathogenicbacteria,amoebae,fungi,andyeast,evenatlevelsof10ppm.Itisbothanantioxidantandanantiseptic.Itisthuspopularlybelievedthat“agarlic

7089_C011.indd 339 10/8/07 12:19:05 PM


taBle 11.1number of Bioactive Components responsible for specific therapeutic Benefits of Common spicesspice (part) therapeutic Benefits (Correlated to number of Bioactive Compounds)*

Allspice P(8),AC(5),AI(3),AB(2),AU(2),AA(2),H(3),AD(2),AAM(2)

(Fruit) AG(2),AM(2)

Blackmustard P(8),AC(10),AS(3),FC(3),AB(6),AO(4),AV(4),AG(3),L(2)

Blackpepper P(31),AC(21),AS(16),AI(8),FC(10),AB(14),AO(4),AV(6),

(Fruit) ST(7),AA(4),AN(8),H(8),AG(5)

Cardamon P(2),AC(8),AI(2),HG(2),AM(2),HP(3)

Cassia P(10),AC(7),AS(5),ADB(3),AO(3),AU(4),AV(3),AA(3),

(Bark) AD(3),AM(3),I(4)

Cinnamon P(29),AC(14),AS(10),AI(7),FC(10),AB(11),AU(5),AV(6),

(Bark) ST(8),AA(4),H(8),AG(4),ADB(3)

Clove P(11),AC(10),AS(4),AI(6),FC(5),AO(3),AU(5),ST(3),

(Bud) AN(2),AA(3),AG(2)

Coriander P(40),AC(27),AS(9),AI(8),FC(11),AB(20),AO(7),AV(12),

(Fruit) ST(8),H(7),AG(6),AAM(5)

Cumin P(27),AC(11),AS(5),AI(7),FC(6),AB(11),AO(5),AU(5),

(Fruit) AV(7),ST(6),H(6),AG(3),AAM(3)

Garlic P(23),AC(21),AS(6),FC(8),AB(13),AO(9),AU(6),AV(5),

(Bulb) ST(5),AA(9),AD(5),HG(6),HP(5)

Ginger P(43),AC(25),AS(11),FC(18),AB(17),AO(6),AU(13),AV(6)

(Rhizome) ST(11),H(7),HP(8)

Licorice P(45),AC(26),AS(23),AI(12),FC(21),AB(20),AO(10),AU(6),

(Root) AV(8),ST(6),AN(9),AA(5),E(8)

Nutmeg P(32),AC(15),AS(11),FC(14),AB(15),AU(4),AV(4),ST(6),

(Seed) AN(5),AA(6),E(4),H(6),AG(3)

Poppy P(5),AO(3),AU(6),HPT(4),AD(5),AM(4)

Sesame ADB(4),P(7),AC(17),AB(5),AO(7),AD(7)

Turmeric P(15),AC(9),AS(4),AI(5),FC(7),AB(8),AO(3),AU(6),AV(3)

(Rhizome) AN(3),H(4),AG(3),I(4)

Vanilla P(20),AC(7),AS(9),FC(9),AB(7),AO(7),AU(3),AV(3),AN(5)

(Fruit) E(4)

* P:Pesticidal E:Expectorant

AC:Anticancerous H:Herbicidal

AS:Antiseptic AG:Analgesic

AI:Anti-inflammatory AD:Antidepressant

FC:Fungicidal HG:Hypoglycermic

AB:Antibacterial AM:Antimigraine

7089_C011.indd 340 10/8/07 12:19:05 PM


clove a day keeps the doctor away.” Ginger is often used to cure colds, cough,asthma, tuberculosis, joint pain, high cholesterol, low blood pressure, and evenmotion sickness. It is a heart stimulant, bactericide, and antidepressant. Onion isknown tobe abloodcleanser,weight regulator, andantidiabetic. It alsopreventscoldsandinfections. Cloveandcinnamonareknownpainkillers,antifungals,andantidiabeticsandcanbeusedforsparinginsulin.Theygenerallycureindigestion,nausea,andhyperacidity;inhibittuberculosis;relievefever,insomnia,andallergies;andevenlowerbloodpressure.Cumincurespiles,hoarsenessofvoice,dyspepsia,jaundice,insomnia,colds,andfever.Italsocureshook-worminfection. Turmericiswellknownforitsanti-inflammatory,antiseptic,andanticarcinogenicproperties.Itcuresarthritis,respiratorytractinfection,skinallergies,bronchialasthma,andviralhepatitis.Essenceofjojobaisconsideredauniqueproductofnatureasitisalmostidenticaltonaturalskinoilandisusedforherbalskincare.Moisturizersandbeautyoilsarealsomadefromnaturaloils(e.g.,olive,almond,wheatgrass,andaloevera).Teatreeoilisanuniqueherbaloilthatisanantibacterial,antifungal,anti-infective,and antiseptic. It is obtained fromneedle-like leavesof a small herb, a nativeofAustraliathatisnowextensivelygrownintheU.S.

11.4 BIoaCtIve IngredIents In spICes

Spicesmaybeclassifiedaccordingtotheirtherapeuticbenefitsorbioactiveingredi-ents,inadditiontotheiraroma,taste,color,andconsistencyimpartedtofoodsandmedicines.Table11.2listsgroupsofactiveingredientspresentinspicesandtheirtherapeuticvalues.Thevolatilefractionofaspiceisknownasitsessential oilandisresponsiblefortheessenceorflavorofthespice.Essentialoilsarefoundinvariouspartsofaplant(e.g.,sandalwood,clovebud,cinnamonbark,orangepeel,roseandjasmine flowers). The essential oil constituents may be classified into four majorgroups:monoterpenes,diterpenes,sesquiterpenes,andoxygenatedcompounds.Thecompoundsbelongingtothelastgroup,namelyesters,ketones,alcohols,andethers,areveryspecifictothespeciesorthegenusofthespiceplant.Eventhoughthesecompoundsarepresentinverysmallquantities,theyarethesubstancesresponsibleforthecharacteristicflavorofthespice,theabsenceofwhichsometimeschangesthearomacompletely.

taBle 11.1 (continued)number of Bioactive Components responsible for specific therapeutic Benefits of Common spices

AO:Antioxidant ADB:Antidiabetic

AU:Antiulcerous AAM:Antiasthmatic

AV:Antiviral L:Laxative

ST:Sedative I:Immunostimulant

AN:Anaesthetic HP:Hepatoprotective

AA:Antiaggregant

Source: Mukhopadhyay,M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.

7089_C011.indd 341 10/8/07 12:19:05 PM


The relatively nonvolatile fraction of a spice extract is viscous and resinous,andsoitiscalledoleoresin. Itisresponsibleforthetasteorpungencyofthespice.Thiscomprisesnonvolatileconstituentsorlarge-molecular-weightcompounds,suchas fatty acids, resins, paraffin waxes, and alkaloids. Most of the ingredients thatare responsible for themedicinalattributesofaspicearepresent in this fraction.Forexample,thecompoundresponsibleforthemedicinalvalueinblackpepperispiperine, an alkaloid having bitter taste that is present in the oleoresin. It is notpresent in theessentialoilanddoesnotcontribute to thearomaofblackpepper.Among the bioactive compounds present in oleoresin, another important groupdeters the formation and propagation of free radicals and is called antioxidants.Theypreventdiseasescausedbyoxidativedamage(e.g.,aging,cataracts,coronaryheartdisease,cancer,memoryloss,Alzheimer’sdisease,andkidney-failure).Thesenaturalbioactivechemicalcompoundsareoftencommerciallycallednutraceuticals,inlinewiththetermpharmaceuticals, andarealsotermedphytochemicals iftheyarederivedfromleaves,roots,stems,seeds,orfruits.Thephytochemicalspresentin functional foods include phenolics and polyphenolics, and some are commer-ciallyusedasnaturalantioxidants.VitaminsA,C,andEandflavonoidsaresomenaturalantioxidantsthatareaddedinsmallconcentrationstofoodsassupplementsforpreservation.TheprovitaminAactivityinspicesisduetothecarotenoidspresentinspicesandplaysanimportantroleasanantioxidant.Spicesandherbsthatpossessantioxidant properties include clove, turmeric, allspice, rosemary, mace, sage,oregano,thyme,nutmeg,ginger,cassia,cinnamon,savory,blackandwhitepepper,aniseed,andbasil.Inthefoodindustry, thesenaturalantioxidantsareusedalongwith syntheticantioxidants, suchasbutylatedhydroxyanisole,butylatedhydroxytoluene,tertiarybutylhydroquinone,andpropylgallate.

taBle 11.2Classification of Bioactive Constituents in spices

group examplespice: active Ingredient therapeutic value

Alkaloids Bitteramines Chili:Capsaicin Counter-irritantforpain

Bioflavonoids Phenolicpigments Rosemary:Luteolin Antioxidant

Essentialoils Mixturesofvolatiles Clove:Various Aphrodisiac,perfume

Glycosides Carbohydratederivatives Garlic:Alliin Expectorant

Phenylpropanoids Cinnamicacidderivatives Cinnamon:Eugenol Topicalanesthetic

Resins Terpeneoxidants Myrrh:Resinacids Antibacterial

Saponins Soapyhemolysants Licorice:Glycyrrhizin Anti-inflammatory

Sterols Steroidprecursors Sesame:Linoleicacid Antioxidant

Tannins Polyphenolics Tea:Catechin Antioxidant

Terpenes Isoprenederivatives Ginger:Zingiberene Antinauseant

Carotenoids Carotenes Paprika,redchili:β-carotene

Antioxidant,color

Anthocyanins Curcuminderivatives Turmeric:Kokum Naturalcolor

7089_C011.indd 342 10/8/07 12:19:06 PM


In addition to the antioxidants, some spices (e.g., turmeric, paprika, saffron,mustard,andblackpepper)havenatural colorasanimportantingredient,whichisusedforfoodcolor.Occurrenceofthecomponentcurcumininturmeric(Curcuma longa)hasmadeitbeneficialnotonlyasanaturalcolortofoodprocessingindustriesbutalsoasanantioxidant,anti-inflammatory,antimutagenic,andantivenomagentforhumanhealth.Alargenumberofbioactivecomponentsarepresentintheessentialoilobtainedfromturmeric,suchas,curcumin,ar-turmeric,andturmerone.SomeofthebioactivecompoundspresentinothercommonlyuseddomesticspicesarelistedinTable11.3.

11.5 saleaBle spICe produCts

Spicesarenotnecessarilysoldaspurespicesinwholeorgroundformbutarepre-ferredintheformofblendsandformulationsfortheeaseofusage.Inmanycases,additivesareaddedtoimprovethequalityorshelflife.Manypurespicessoldintheformofpowderundergocaking.Accordingly,anticakingadditivesareoftenaddedtomaintainthespicedryandfreeflowing.Forexample,asilicagel(sodiumsilicate)isoftenaddedasananticakingagent.Calciumstearate,magnesiumstearate,andpotassiumstearatearealsousedaseffectiveanticakingagents.Somespicesaresoldasblendsofspices,suchascurrypowder.Theingredientsandtheircompositionsinaspiceblendmaychangewiththefoodapplications,asinthecaseofchilipowder.

Alternatively, spice extracts can replace spice powder in food and flavorformulations.Spiceextractscanprovidethetrueessenceofspiceintheformofthevolatile essential oil, the taste components in the formof thenonvolatile resinousfraction, and the food colors in the form of the pigments. The formulations withflavors,spiceoils,andoleoresinareverymuchanart rather thanastandardtech-nology and vary depending on the buyer’s preferences for food habits and healthcareproducts.Liquidspiceflavorsareaddedtotheediblegum,powderedstarch,orcellulosesubstanceandcareistakensothattheblendcanretainitspowderynaturebytheadditionofanticakingagents.Furthermore,flavordehydrates,suchasdehydratedchicken,meat,andcheesepowders,areaddedtotheblend.Eachspiceformulationisdevisedsothatitiseffectiveduetoitsowncharacteristics,andeachformulahastobewithinthelimitsspecifiedbytheregulatoryboards.Spiceblendingequipment,suchasblendersandfilters,shouldbecleanedonaregularbasistoensurethatthereisnocontaminationwithresidualspiceblendfromthepreviousoperation.

Liquidspiceextracts,suchasessentialoilsandoleoresins,providefoodtech-nologists with many advantages, as food manufacturers can select the specificflavorprofileswithmuchgreaterprecisionthanif theyweretosimplyuseblendsofwholespices.Inaddition,hygienicconcernsaswellastransportationcostsaregreatlyreducediftheoilsandoleoresinsareextractedclosetotheareaswherethespicesaregrown.Spiceoilsandoleoresinsprimarilyfindusesinprocessedmeat,fishandvegetables;soups,sauces,chutneysanddressings;cheesesandotherdairyproducts;bakedfoods,confectionery,snacks,andbeverages.Thedemandforspiceoilsandoleoresinsisincreasinggloballydaybyday,duetotheincreasingdemandforspicyfast-foodsnackstobeintroducedintothemarketandwithaneyetowarddevelopingacharacteristictasteinthesnacksforthefuture.Spiceoilsandoleoresins

7089_C011.indd 343 10/8/07 12:19:07 PM


taB

le 1

1.3

Bio

logi

cally

act

ive

Con

stit

uent

s in

Com

mon

spi

ces

spic

epl

ant

part

Bio

-act

ive

Con

stit

uent

s (p

pm)

Gar

lic

(All

ium

sat

ivum

)B

ulb

Ajo

ene,

Alli

cin,

Alli

in,A

llist

atin

-I, A

llist

atin

-II,

Arg

inin

e(6

000–

1500

0),A

scor

bic

Aci

d(1

00–8

00),

Cho

line,

Citr

al,D

ially

ldi

sulfi

de,G

eran

iol,

Glu

tam

ica

cid

(805

0–19

320)

Lin

aloo

l,N

iaci

n(4

–17)

,Sco

rodi

n-A

,Try

ptop

han

(660

–158

4)

Gin

ger

(Zin

gibe

r of

ficin

ale)

Roo

tA

ceta

ldeh

yde,

Asc

orbi

cA

cid

(0–3

10),

Asp

arag

ine

Bor

neol

(55

–110

0),B

orny

lace

tate

(2–

50),

Cam

phen

e(2

5–55

0)C

havi

col,

1-8

Cin

eole

(30

–650

)C

itral

(0–

1350

0),D

ehyd

rogi

nger

dion

e,G

eran

iol(

2–5

0),G

inge

rdio

nes,

Gin

gero

ls(

1820

0),

Hex

ahyd

rocu

rcum

in,L

imon

ene,

Lin

aloo

l(30

–650

),M

ethi

onin

e(6

70–7

35),

Myr

cene

(2–

50),

Pin

ene

(5–2

00),

Se

linen

e(3

5–70

0),S

hoga

ols

(180

0),Z

inge

rone

,Zin

giba

in,T

rypt

opha

n(6

30–6

90).

Clo

ve

(Syz

ygiu

m a

rom

atic

um)

Bud

Ane

thol

e,B

enza

ldeh

yde,

Car

vone

,Car

yo-p

hyle

ne(

7400

–816

0),C

havi

col(

465–

510)

,Cin

nam

alde

hyde

,Ela

gic-

Aci

d,

Eug

enol

(10

8000

–120

000)

,Eug

enol

ace

tate

(36

000–

4000

0),F

urfu

ral,

Gal

lica

cid,

Kae

mfe

rol,

Lin

aloo

l(1)

,M

ethy

lEug

enol

(31

0–34

0)

Cas

sia

and

cinn

amon

(C

inna

mon

um, c

assi

a &

ve

rum

)

Bar

kB

enza

ldeh

yde

(25–

100)

,Cam

phen

e,C

amph

or,C

aryo

phyl

lene

(13

5–13

15),

1,8

Cin

eole

(16

5–18

00),

Cin

nam

alde

hyde

(6

000–

3000

0),C

umin

alde

hyde

(5–

100)

,p-c

ymen

e(5

5–44

5), E

ugen

ol(

220–

3520

),F

arne

sol(

3–10

),F

urfu

ral(

3–10

),

Lim

onen

e(4

5–18

0),L

inal

ool(

230–

950)

,Met

hylE

ugen

ol,M

yrce

ne(

5–20

),N

iaci

n(8

),P

inen

e(2

0–23

5),P

iper

itone

(7–

25),

Sa

frol

e,T

erpi

neol

(1–

260)

Cum

in

(Cum

inum

cym

inum

)Fr

uit

Ani

sald

ehyd

e(8

35),

Asc

orbi

cac

id(

0–75

),B

orny

lace

tate

(35

),d

elta

-3-c

aren

e (2

70),

bet

a-ca

rote

ne(

5),C

arve

ol(

435)

,C

aryo

phyl

lene

(14

0–32

0),1

,8C

ineo

l(40

–135

),C

opae

ne(

30),

p-C

ymen

e(81

0–12

600)

,Far

neso

l(83

0),L

imon

ene

(60–

695)

,L

inal

ool(

30–3

15),

Met

hylC

havi

col(

30),

Myr

cene

(35

–120

),N

iaci

n(4

5),P

inen

e(1

0–66

00),

Pip

erito

ne(

170)

,Te

rpin

ene

(25–

1180

0), T

erpi

nen-

4-0l

(30

),T

erpi

neol

(30

–275

)

Cor

iand

er

(Cor

iand

er s

ativ

um)

Frui

tA

neth

ole

(1–2

), A

scor

bic

acid

(18

0–62

90),

Bor

neol

(2–

50),

Cam

phor

(10

0–13

00),

Car

vone

(20

–25)

,Car

yoph

ylle

ne(

1–8)

,1-

8C

ineo

le,p

cym

ene

(70–

725)

,Fer

rulic

acid

(46

0–13

60),

Ger

anio

l (30

–440

),L

imon

ene

(30–

1230

),L

inal

ool(

4060

–169

00),

Pi

nene

(50

–137

50),

Ter

pine

ol(

30–4

0),V

anill

ica

cid

(220

–960

)

Car

dam

on

(Ele

ttar

ia c

arda

mom

um)

Frui

tB

orne

ol(

30–8

000)

,Cam

phen

e(1

0–30

),C

amph

or(

5–20

),1

,8C

ineo

le(

525–

5600

0),C

itron

ella

l,C

itron

ello

l(10

–40)

, p-

cym

ene

(130

–280

00),

Ger

anio

l(45

–140

),L

imon

ene

(595

–948

0), L

inal

ool(

1285

–800

0),M

yrce

ne(

335–

3000

),

Ner

ol1

0–30

),N

eryl

acet

ate,

Pin

ene

(70–

3000

),T

erpi

nen-

4-ol

(25

0–23

200)

, Ter

pine

ne(

20–1

40)

7089_C011.indd 344 10/8/07 12:19:07 PM


Tur

mer

ic

(Cur

cum

a do

mes

tica

)R

hizo

me

Asc

orbi

cA

cid

(0–2

90),

Bis

desm

etho

xyc

urcu

rmin

(60

–270

00),

Bor

neol

(15

–350

),C

amph

or(

100–

720)

,1,8

,Cin

eole

(30

–720

)C

inna

mic

aci

d,c

urum

in(

10–3

8500

),p

-cym

ene,

Nia

cin

(5–6

0),p

-Tol

met

hyl-

carb

inol

(50

0–17

50),

Tur

mer

one

(180

0–4

3200

)

Bla

ckp

eppe

r(P

iper

nig

rum

)Fr

uit

Asc

orbi

cac

id(

10),

Ben

zoic

Aci

d,B

orne

olC

amph

or,C

arva

crol

,Car

veol

,Car

yoph

ylle

ne,1

,8C

ineo

le,C

inna

mic

aci

d,C

itral

,C

itron

ella

l,p-

cym

ene,

Eug

enol

,Lim

onen

e,L

inal

ool,

Myr

cene

,Myr

istic

in,P

inen

e,P

iper

idin

e,P

iper

ine,

Saf

role

,Te

rpin

en-4

-ol

Bla

ckm

usta

rd

(Bra

ssic

a ni

gra)

Seed

/Lea

fA

llyl i

soth

iocy

anat

e(6

510–

1176

0,s

eed)

,Arg

inin

e(1

810–

2665

7,L

F), A

scor

bic

acid

(23

5–40

00,L

F),β

-car

oten

e(3

0–47

5,L

F),

Eru

cic

acid

(77

0–11

340,

LF)

,Met

hion

ine

(230

–339

0,L

F),N

iaci

ne(

3–48

,LF)

,Try

ptop

han

(270

–397

5,L

F)

Saff

ron

(Cro

cus

sati

vus)

Flow

erβ-

caro

tene

,1-8

Cin

eole

,Cro

cetin

,Cro

cin

(200

00),

Del

phin

idin

,Hen

tria

Con

tane

,Kae

mfe

rol,

Lyco

pene

,Myr

icet

in,

Nap

htha

lene

,Pin

ene,

Que

rcet

in

Lic

oric

e(G

lycr

yrrh

iz g

labr

a)R

oot

Ace

tica

cid

(2),

Ane

thol

e(1

),B

etai

ne,C

holin

e,O

-Cre

sol,

Est

rago

le,E

ugen

ol(

1),F

erul

ica

cid,

Gly

cryr

hizi

cA

cid

(100

000–

2400

00),

Gua

iaco

l,K

aem

fero

l,L

inal

ool,

Man

nito

l,N

iaci

n(7

0)

Mac

ean

dnu

tmeg

(M

yris

tica

frag

ans)

Seed

Bor

neol

(42

00–2

5600

),1

,8-C

ineo

le(

440–

3500

),p

-cym

ene

(120

–960

),E

lem

icin

(20

–350

0),E

ugen

ol(

40–3

20),

Fu

rfur

al(

1500

0),G

eran

iol,

Lim

onen

e(7

20–5

760)

,Lin

aloo

l,M

ethy

lEug

enol

(20

–900

),M

yrce

ne(

740–

5920

),

Myr

istic

in(

800–

1280

0),P

inen

e(3

000–

4000

),S

afro

le(

120–

2720

),T

erpi

nen-

4-ol

(60

0–48

00),

Ter

pine

ol(

120–

9600

)

Thy

me

(Thy

mus

vul

gari

s)Pl

ant

Bor

neol

(15

–146

0),B

orny

lace

tate

(15

–540

),C

affe

icA

cid,

Cam

phen

e(1

5–27

0),d

elta

-3-

Car

nene

(51

0),B

eta-

caro

tene

(2

0–25

),C

arva

rol(

15–1

8720

),C

hlor

ogen

ica

cid,

1,8

Cin

eole

(80

–459

0),p

-cym

ene

(145

–208

00),

Ger

anio

l(0–

1066

0),

Lim

onen

e(1

5–52

00),

Lin

aloo

l(18

0–17

420)

,Met

hion

ine

(137

0–19

80),

Myr

cene

(35

–675

),N

iaci

n(5

0),P

inen

e(1

5–16

00),

R

osm

arin

icA

cid

(500

0–60

00),

Ter

pine

n-4-

ol(

70–8

320)

, Ter

pine

ol(

35–6

500)

,Thy

mol

(15

–240

00),

Try

ptop

han

(186

0–20

00),

Urs

olic

aci

d(1

5000

–188

00)

Red

Pep

per/

Chi

lli

(Cap

sicu

m c

orra

ls)

Frui

tA

rgin

ine

(400

–800

0), A

scor

bic

Aci

d(3

50–2

0000

),A

spar

agin

e,B

etai

ne,C

apsa

icin

(10

0–22

00),

bet

a-ca

rote

ne(

0–46

0),

Chl

orog

enic

aci

d,H

espe

ridi

n,M

ethi

onin

e(1

00–1

900)

,Nia

cin

(5–1

70),

Oxa

lica

cid,

Try

ptop

han

(100

–200

0)

Van

illa

(Van

illa

pla

ntif

olia

)Fr

uit

Ace

tald

ehyd

e,A

cetic

aci

d,A

nisa

ldeh

yde,

Ben

zald

ehyd

e,B

enzo

ic A

cid,

Cre

osol

,Eug

enol

,Fur

fura

l,G

uaia

col,

Van

illic

aci

d,

Van

illin

(13

000–

3000

0), V

anill

ylA

lcoh

ol.

Sour

ce:

Muk

hopa

dhya

y,M

.,N

atur

al E

xtra

cts

Usi

ng S

uper

crit

ical

Car

bon

Dio

xide

,CR

CP

ress

,Boc

aR

aton

,FL

,200

0. W

ithp

erm

issi

on.

7089_C011.indd 345 10/8/07 12:19:08 PM


areparticularlysuitableforsuchsnacksbecausetheycanbeusedveryconveniently(withouthavingtohandleinbulkrawspicessuchasginger,garlic,chili,onion,car-damom,andcinnamon).Forexample,approximately7000kgofonionsareneededtoproduce1kgofhighlyconcentratedonionoil.

Mostspiceextractscanbesuppliedinbothoil-andwater-solubleforms.Asaresult,spiceoleoresinsandessentialoilsarenowshippedindispersionsofedibleoilorotherliquids.Furthermore,dispersionscanbestandardizedwithotheringredients,such asmono-, di-, or triglyceridesor polysorbates.Another technique for easierspice application is to make a liquid emulsion of spice oleoresins, essential oils,andastarchorspraydrytheessentialoiltoapowder.Inthiscase,thespray-dryingprocesshastomakesurethatotherproductsarenotspray-driedonthesameequip-menttoavoidcontamination.Oleoresins,spiceoils,substrates,anddiluentallshouldindividuallymeetreliablestandardization.

Theblendformulationsforspiceseasoningsuseflavorenhancersandotherflavoringredients,suchasmonosodiumglutamate,sodiumerythorbate(usedindelimeats),dextrose,maltodextrin,andhydrolyzedvegetableproteins.Manyoftheseflavoringsandingredientsarecornorsoybased.Otherprocessedingredientsarederivedfromproductsthatgothroughamultistageconversionprocessofenzymolysis,fermenta-tion,andregenerationuntilthefinalproductisachieved.Thequalityandsafetyoftheformulationareassuredbeforemarketing[4].Moreinformationmaybegatheredfromthereferencesprovided[5,6].

11.6 ConventIonal extraCtIon methods

Figure11.1 outlines the various alternative steps involved in the conventionalmethodsforproductionofspiceextracts.Thespicearomaoressentialoilistradi-tionallyproducedbysteamdistillation(SD)ofthegroundspiceorSDoftheextractsobtainedbysolventextraction(SE)oraqueousalkalineextraction(AE)ofthegroundspice.Avarietyofsolvents,suchasalcohols,acetone,andhexane,canbeusedforextractionofspices.However,removaloftheseorganicsolventsleavessomeresidualsolventbehind,whichrequiresthedesolventizationatelevatedtemperatures.Thiscancausechemicalmodificationsoftheoleoresins.

Table11.4listsafewexamplesofspiceextractsthatareproducedcommerciallywiththeirpercentageyieldsofessentialoilsandoleoresinsfromgroundspice,asreportedbyMarionetal.[7].Variationsinyieldandqualityofspiceextractsmayoccurduetovariationsintheoriginandharvestingtimeofspices.

Theyieldandqualityofextractsalsodependonpreprocessingoperations,suchasgrinding,thetechniqueofextraction,andthenatureofthesolventwhich,inturn,are decided based on the desired specification of the end product in terms of itsaroma,flavor,andsolubility.Eachextractplaysaspecificroleintheformulationanditsselectionisthekeytotheproductdevelopment,asperthespecificrequirementofvalueaddition.

Conventionalextractionmethods,suchasSE,AE,anddirectorindirectSD(i.e.,hydrodistillation[HD]),arenotselective.Asaresult,theextractsoftencon-tain color (e.g., chlorophyll) or some other undesirable components. Therefore,further purification is imperative using a number of techniques, such as color

7089_C011.indd 346 10/8/07 12:19:08 PM


adsorptionbyactivatedcharcoal,dryingusingsilicagel,chromatographicsepa-ration,vacuumfractionation,ormoleculardistillation.Owing to thebanon theusageofthechlorinatedsolvents,themostcommonlyusedsolventforSEtodayishexane.Formostspiceoilsandoleoresinsintheinternationalmarket,theresidualhexanecontent inproductshas tobereduced to less than25ppm.This limit isexpected to go down further. Hence, the SE process may be phased out in thenearfuture.Thefoodindustryneedstocombatstrictregulationsandcomplywithmeasuresforsafety,reliability,andstandardizationofnaturalproductstobecon-sumedasnutrientsandfoodadditives.ThismaybeachievedbyadoptingSCFE techniques,asSC-CO2canrecovertheactiveingredientsinnaturalformwithoutdegradationorcontamination.Over thelast twodecades,SCFEhasemergedasasuperioralternative toconventionalprocessessuchasSDandSE in the food,pharmaceutical,andcosmeticsindustries.

11.7 superCrItICal CarBon dIoxIde as the extraCtant

Several spice extracts—such as those from basil, black pepper, cardamom, chili,cinnamon,clove,cumin,fennel,fenugreek,ginger,garlic,nutmeg,paprika,savory,turmeric,andvanilla—arenowcommerciallyproducedusingSC-CO2,asitiscurrentlythemostdesirableSCFsolventforextractionofnaturalproducts.ThesolubilityoftheextractinSC-CO2increaseswithpressureordensityofSC-CO2anddecreaseswith

Spice EmulsionSeasonings

Steam Distillation Solvent/Alkali Extraction SC CO2 Extraction

Essential Oil Steam Distillation Essential Oil Oleoresin

Dispersed in Carrier

Dispersed inVegetable Oil

Spray Dried in EdibleGum or Starch Solution

Dispersed in StarchSolution

Blended with OtherFlavors

Commercial SpiceOil

Liquid Oleoresin EncapsulatedSeasonings

Ground Spice/Herb

fIgure 11.1 Various alternative steps for spice extraction. (From Mukhopadhyay, M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.)

7089_C011.indd 347 10/8/07 12:19:09 PM


temperatureuptoalimitingpressure(termedcross-over pressure),beyondwhichthesolubilityincreaseswithbothpressureandtemperature.ThisphenomenonisutilizedforrecoveringandrecyclingCO2afterextractionbysimplyloweringthepressure,increasingthetemperature,orbothintheseparators.ThereisnosolventresidueintheextractasCO2isinagaseousstateattheambientcondition.

AbroadrangeofselectivityandextractabilitycanbeachievedusingSC-CO2justbymanipulatingtheoperatingconditions,suchaspressureandtemperature,therebytargetingthespecificcompoundsofinterest.BecauseSCFEishighlyselective,theconcentrationofthedesiredactivecompoundinthetotalextractishigherandtheyieldofthedesiredactivecompoundisclosertothetotalyield.ThereisrarelyanyneedforadditionalprocessingstepsforSCFextracts,whereasorganicsolvent–extracted

taBle 11.4Commercial spice extracts [2]

spiceessential oils, min-max (%)

oleoresins (%)

Anise 1.0–4.0

Caraway 3.0–6.0

Cardamon 4.0–10.0

Carrot 0.5–0.8

Cassia 1.0–3.8

Celeryseed 1.5–2.5

Cinnamon 1.6–3.5

Clovebud 14.0–21.0

Coriander 0.1–1.0

Cumin 2.5–5.0

Curcuma 2.0–7.2 7.9–10.4

Dill(seeds) 2.5–4.0

Fennel 4.0–6.0

Garlic 0.1–0.25

Ginger 0.3–3.5 3.5–10.3

Marjoram 0.2–0.3 —

Mace 8.0–13 22.0–32.0

Nutmeg 2.6–12 18.0–37.0

Pepper 1.0–3.5 5.0–15.0

Pimentoberry

3.3–4.5 6.0

Saffron 0.5–1.0

Savory 0.5–1.2 14.0–16.0

Vanilla 29.9–47.0


7089_C011.indd 348 10/8/07 12:19:10 PM


oleoresinsincludeundesirableresinsthatprecipitateandmakethesolutioncloudy,requiringanadditionalstepoffiltration.Evensteam-distilledoil formsan immis-ciblelayerduetothepresenceofmonoterpenehydrocarbons,toalargeextent,whichinhibitthesolubilityofoilinsoftdrinksandbeverages.TheremarkableselectivityofSC-CO2overorganicsolventsfacilitatestherecoveryofspiceextractswithdesirableconstituents and superior blending characteristics. Studies on SCFE with SC-CO2andSEwithalcoholindicatethat,althoughtheoverallyieldobtainedusingalcoholasthesolventishigherduetocoextractionofundesirablecomponents(whichyieldsanaccordinglyhigherquantityoftotalextract),thepercentageofthedesiredactivecompoundinthatextractislower[2].

Inadditiontoselectiveextractionandtheabsenceoforganicsolventresidues,SCFE offers another unique advantage; namely, simultaneous fractionation ofdifferentcompoundsispossibleusingthesamesolvent,SC-CO2.Forexample,theactivecomponentsinblackpeppercanbeextractedwithSC-CO2andseparatedintotwofractionsbychangingpressureand temperature; thefirst fraction isenrichedinoleoresinandthesecondfractioninessentialoil.Accordingly,SC-CO2can,inasingleprocessofSCFE,selectivelyextracttheoleoresinandessentialoilfractions(asopposedtoprocessingofspicesbySE,AE,orSD)andthenseparatethembysequentialdepressurization.Furthermore,mostraffinate(thematerialleftoverafterextraction)isuncontaminatedandhasahighmarketvalueduetothecontentoffiberandprotein,whichremaininsolubleinSC-CO2.

However,inviewofthefactthatSC-CO2isessentiallynonpolar,itisunsuitableforextractingwater-solubleconstituents.Thisseemingdisadvantagemaybeeasilyovercomebyaddingafood-gradepolarcosolvent(typicallyinverysmallquantities,say3to5mole%)toSC-CO2.Thebinaryhomogeneousmixture is thencapableof extractingwater-solubleorhigh-molecular-weight compounds.Thebest candi-datesforsuchcosolvents,especiallyforfoodsandnutraceuticals,areethanol,ethylacetate,andinsomecaseswater.Theremarkablevalue-additionthat theSC-CO2extractsofferasnaturalconcentrates,inadditiontotheiradvantagesfromthestand-pointofenvironmentandhealth,hasgeneratedagreatdealofcommercialinterestsforusingSC-CO2astheextractantinthefoodindustry.

11.8 CommerCIal sCfe proCess

Forsolidfeeds,SCFEisusuallyasemi-batchprocessinwhichCO2flowsinacontin-uousmode,whereasthefeedischargedintheextractorbasketinbatches.However,for better viability on the commercial scale, theprocess ismade semicontinuoususingmultipleextractionandseparationvessels,asschematicallydescribedintheflowdiagramshowninFigure11.2.Extractionandseparationoftheextractareoftencarriedoutinstages,bymaintainingdifferentconditionsofpressureandtempera-tureintheextractorsandseparators.Thisallowseasyfractionationoftheextractforenrichmentofthespecificactivecomponents,whicharesubsequentlyfractionatedineachoftheseparators.Itisthuspossibletoproduceavarietyofproductsusingthesamehardwarebymerelychangingpressure,temperature,andcosolventconcen-trationandmakeaplantformultipleproducts[2].ThecommercialSCFEprocessworksinaclosedloopwithconstantcirculationofCO2inthesystem,withatypical

7089_C011.indd 349 10/8/07 12:19:10 PM


batchtimeof2to4hours.TypicaloperatingconditionsforSCFEareintherangeof100to500barand40°Cto80°C.

ThefeedfortheSCFEprocessneedstohavelowmoisturelevel(lessthan10%)andbe in theformofgroundpowder(100 to300mesh).However,preprocessingshouldbedone in such away that there areminimal lossesof essential oils andactiveingredientsandnegligiblethermalandchemicaldegradationduetotheriseintemperatureorexposuretoatmosphere,respectively.Accordingly,dryingcouldbecarriedoutinafluidizedbeddrierinaninertenvironment,suchasinflowingnitrogenorcarbondioxide.Similarly,grindingcouldbeachievedinaliquidCO2ordry-ice,precooledgrinderwhilecontrollingthehumidityoftheincomingairtoavoidcondensationofmoistureinthefeed.

ItisgenerallybelievedthatSCFEiscapital-intensive,duetotherequirementsoftheprocesstobeoperatedathighpressureswithverypreciseprocesscontrol.ThereisalsoageneralconcernthatSCFEtechnologyisenergy-intensive.However,itisinterestingtonotethattheenergyneededtoattainasupercriticalstate(P>73.8bar,T > 31.1°C) is more than compensated for by the negligible energy required forsolventrecoveryfromtheextractbyasimplestepofdepressurization.Asaresult,theoverallenergyconsumptionofSCFEusingCO2islowerthanthatfortraditionalSDorSE,duetosteamgenerationinSDandduetosolventevaporationandblowingoffofsteamforremovalofresidualsolventfromtheresiduesinSE.Forexample,theremovalofresidualsolventfromanextractbySErequiresabout8kWhofenergyperkilogramofplantextract[8],whereasextractionwithSC-CO2requiresone-tenthofthisenergy.Also,thesolventlossinthebatchSEprocessisuptoone-thirdofthefeedsolvent(thoughitissomewhatlower[10%to15%]inthecontinuousSEpro-cess),whereasthelossofCO2intheSCFEprocessisnegligiblebecauseCO2canbe

E1 E2 E3

S1 S2 T

Condenser

Sub-cooler Pre-heater Co2 Pump

CO2 Supply

Depressurisation Line

HE

E1, E2, E3 : Extractors S1, S2 : Separators T : CO2 Day Tank HE : Heat exchanger

I.I.T., Bombay

Entrainer Pump

fIgure 11.2 ProcessflowsheetofSCFEofspices.

7089_C011.indd 350 10/8/07 12:19:12 PM


easilyregeneratedandrecycled.Thesolventrequirementinthebatchprocessis10to20timesthatofthefeedchargedandthatinthecontinuousSEprocessismuchlower,namelyabout3to4timesthefeed,whereasanhourlycirculationrateofSC-CO2isintherangeof16to24timestheamountoffeedcharged.

TherelativelyhigherinvestmentrequiredintheSCFEprocessiswellbalancedbyotherbenefitsofSCFE,suchaslowsolvent(CO2)cost,lowerbatchtimes,higherconcentrations of active desirable components in the extract, and no additionalpurification-andpollution-abatement-relatedcosts.SCFEalsogeneratespracticallynoeffluent.Inaddition,theextractedresidue(cake)doesnotundergoanydegrada-tionorcontamination,unlike inSEandSD.ResiduefromtheSCFEprocesshasamarketvalueas it retainsall theuseful ingredients,suchasedibleproteinsandfibers.Thiscanbesoldasahighvalueby-producttoyieldadditionalrevenue.

ThenormalSCFEprocesssimultaneouslyandseparatelyyieldsbothliquidandsolidproducts,startingwiththesamefeedofspicesinasinglestep,unlikeSDandSE.InSD,thesteamvolatileessentialoil,whichisaliquidproduct,isdistilledout,whereasinSE,theliquid(essentialoil)andsolid(oleoresin)productsareobtainedtogether.Subsequently,SDorSEwithanothersolvent isemployed torecover theessentialoilfromthemixedproduct.Liquidspiceproductsaremorestable,haveamorereproduciblequalitythantheirconventionalforms,andcontainthecharacter-isticaroma,taste,andodor.Duringtheirutilization,asmallerquantityisrequiredforobtainingthesameeffect.Thestandardizationofthesenewliquidspiceproductsimpliescontrollingtheircomposition.Adetailedfeasibilitystudyshowsthatevenattheexistingprice(ofextractsfromSEandSD)ofoilandoleoresins,theinvestmentinSCFEisprofitable,whichjustifiesittobethepreferredroutefromalong-termperspective.TheinstrumentationandcontrolsystemnecessaryfortheSCFEprocessisdesignedtoprovideaccuratecontroloftheparameters,ensuringhighconsistency,reliability,andstandardizationofthefinalproduct.

11.9 ComparIson of spICe extraCts By ConventIonal and sCfe proCesses

AlthoughrecoveryofessentialoilsfromspicesbySDandAEhasbeenpracticedforcenturies,theoilsproducedbytheseprocessesmaycontainartifactsformedduringtheprocessing,inadditiontothefactthattherecoveryoftheoilsisquitelow,assomeofthecomponentsarenotsteamvolatile.WhenSEinvolvestheuseoforganicsol-ventstoextractessentialoilsfromgroundspices,thequalityoftheextractisdecidedbythepresenceofresidualsolvent,artifactsformedduetothermaldegradationdur-ingtherecoveryofthesolvents,orbycoextractionofundesirablecomponentsduetopolarityofthesolvent.Apolarsolventislikelytoextractmostpolarcomponentsfromspices,someofwhichmayevenbeundesirable.ExtractionofspiceswithSC-CO2orsubcriticalliq-CO2ismostfavorableforcommercialproductionofessentialoilsandoleoresins,asCO2isanaturalsolventandisideallysuitableforthermallylabile natural products. The oleoresins are extracted at relatively high pressures,whereastheessentialoilsarerecoveredatrelativelylowpressures,whichcanformaclearsolutionwhenaddeddirectlytosoftdrinks.Butsimultaneousextractionofoleoresinsandessentialoilsataveryhighpressureintherangeof250to350bar,

7089_C011.indd 351 10/8/07 12:19:13 PM


followedbystage-wiseselectivefractionationatsupercriticalandsubcriticalcondi-tions,ispreferredforcommercialproduction.Theoperatingconditionsofpressure,temperature,andcosolventareappropriatelyselectedinordertoobtainaspecificproductprofile.Thecosolventisoftenselectedontheconsiderationthatitcanbeleftbehindintheextractedproductorusedformakingtheformulationwithallow-ancemadefordilutionlevel.Thecosolventselectedisafood-gradeGRASorganicsolvent,suchasethanol,ethylacetate,aceticacid,orwater.TheadvantageofSCFEwithcosolvent-mixedSC-CO2overSEisthattheformerisselectiveandretainsallotheradvantagesoftheSCFEprocessbecausethecosolventaddedtoSC-CO2isinverysmallamount(3to5mole%).MostofthecosolventaftertheSCFEprocessescapeswithCO2intheseparator,ensuringmarginalcontentofresidualsolventinthefinalextract.TheextractionyieldsobtainedbyCalameandSteiner[9]usingSDandSC-CO2aregiveninTable11.5.

ItisnowanestablishedfactthatSC-CO2extractionatoptimizedconditionsyieldsmuchmoreactiveingredientsthanSEorSD.Butotherfactors,suchasparticlesize,preprocessingmethods (e.g.,dryingandgrinding), timeofextractionandstorageafterharvesting,andevengeographicaloriginoftherawspice,responsiblefortherecoveryoftheextractarenotincludedinTable11.5.Accordingtotheexperienceoftheauthor,higheryieldsmaybeobtainedfromsomeofthesespiceswithSC-CO2,evenwithoutacosolvent,ascanbeseenlaterinTable11.7.

Notonlytheyieldsoftheextractsbutalsotheirorganoleptic(sensory)charac-teristicsmaybedifferentforextractsobtainedbydifferentmethods.Accordingly,the criteria for selection of the best process condition are based on the desired

taBle 11.5yields by sd and sC-Co2 extraction with a Cosolvent

steam distillation sC-Co2 extraction

spice yield (%) Cosolventextractor

p/t (bar/°C)separator

p/t (bar/°C) yield (%)

Allspice 2.5 Ethanol 300/40 55/37 5.3

Basil 0.5 Ethanol 200/40 56/15 1.3

Cardamom 4.0 Methylacetate 150/60 50/9 5.8

Coriander 0.6 Ethanol 300/40 54/13 1.3

Ginger 1.1 Ethanol 300/40 52/11 4.6

Juniperberry 1.5 Hexane 300/60 52/11 7.2

Marjoram 2.06 Ethanol 250/40 50/35 1.7

Oregano 3.0 Ethanol 150/40 55/14 5.4

Rosemary 1.44 Ethanol 250/60 53/12 7.5

AU 1.34 Hexane 250/60 53/12 7.5

Sage 1.1 Methylacetate 200/40 53/12 4.3

Thyme 1.85 Hexane 150/46 50/9 2.1


7089_C011.indd 352 10/8/07 12:19:13 PM


quality. Inanycase,CO2extractshavemore topnotes,morebacknotes,nooffnotes,nodegradation,moreshelf life,andbetteraromaandblendingcharacter-istics than steamdistilled andhexane extracts, as canbe seen inTable11.6. Ingeneral, theextractproducedbySEcontainsall the ingredients thataresolublein theorganic solvent, including thevolatileoils and resins.Some triglycerides(lipids)presentinspicesarecoextractedandactasnature’sownfixativeresultingeasyandproperblending.

Theyieldof essentialoilbySDof cumin (2.5%) is less than thatbySC-CO2

extraction(3.5%)at120barand40°C[10].Acomparisonofthecompositionofthe

taBle 11.6spice Constituents (area %) by various methods

Constituents distillation (%) l Co2 (%) sC-Co2 (%) hexane (%)

GingerExtract(byGC)

α-Curcumene 10.0 3.7 2.3

α-Zingiberene 44.0 19.6 12.1

β-Zingiberene 8.0 3.4 2.0

β-Bisabolene 8.3 3.7 2.4

β-Sesquiphellandrene 17.8 7.9 4.9

Zingerone 0.8 0.7 0.3

GingerExtract(byHPLC)

6-gingerol 0.2 16.4 0.9

8-gingerol 0.3 3.1 0.7

10-gingerol — 3.8 0.8

6-shogaol 0.3 2.8 6.3

8-shogaol — — 1.6

CuminExtract2(byGC)

α-pinene — 1.1 —

(EthylEther)-pinene 13.0 21.0

p-cymene 13.0 9.4

γ-terpinene 24.8 20.0

Cuminaldehyde 16.0 20.3[19] 21.0 11.4

Cymol 33.4[20] 26.7[19] 15.2 13.5

CloveExtract(byGC)

(EthylEther)

Eugenol 76.4 77.1 71.8* 73.3

Eugenolacetate 5.6 4.9 11.1* 4.6

β-caryophyllene 5.8 8.5 9.3* 10.4

* PilotplantexperimentatIITBombaySource: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide, CRC Press,

BocaRaton,FL,2000.Withpermission.

7089_C011.indd 353 10/8/07 12:19:14 PM


cloveextractsobtainedbyliquidCO2,SEwithethylether,andSDindicatesthatliquidCO2andethyletherextractsaresimilar,thoughliquidCO2extracthasthecharacter-isticsofbothessentialoilandoleoresin[11].LiquidandSC-CO2extractsarealwaystransparentandcontainmoreactivecomponentsthatareclosertothoseinthefreshornaturalspice,duetolowoperatingtemperatureandinertenvironment.SC-CO2extractionfollowedbyfractionalseparationwascarriedoutforavarietyofspicesusinga10-Lextractorcapacitypilotplantat Indian InstituteofTechnology (IIT),Bombay.Thecompositionoftheactiveingredientsoftheextractsseparatedinthetwoseparators,asanalyzedbyeithergaschromatography(GC)orgaschromatography-massspectrometry(GC-MS),are indicated inTable11.7(except forpepper,wherepiperinewasquantifiedbyultraviolet[UV]method).TheyieldsandthecompositionsoftheSC-CO2extractsarecomparedwiththoseofhexane-extractedproductsfromthesamespices(Table11.7).SC-CO2extractionyieldsarebetterforclove,cumin,andblackpepper.Inmostcases,theconcentrationsoftheactiveingredientswerehigherintheSC-CO2extractedproduct[2].

11.10 proCess analysIs of sCfe from seleCted spICes

Spiceextractsareusuallyacomplexmixtureofvolatileessentialoils,waxes,tri-glycerides,andresinousandothermiscellaneousmaterials,withthecompositionoftheconstituentscontributingtoaroma,flavor,andpungencyselecteddependingonthespecificapplication.Accordingly,forcustomizedapplications,SC-CO2extractionofspicesrequiresfractionalseparationofselectedgroupsofconstituents.Thiscanbeachievedintwoways:bystage-wiseextractionfollowedbydepressurizationoftheextract-ladenSC-CO2orbysingle-stageextractionataveryhighpressurefollowedbystage-wisedepressurizationforfractionalseparation.Intheformermethod,thevolatileoil isfirst extractedat relativelymilder conditions and, subsequently, thenonvolatileoleoresinsareextractedatrelativelymore-severeconditions.Inthelattermethod,thefinelygroundspiceismoreorlesscompletelyextractedatarelativelymore-severeconditiontorecoverbotholeoresinsandvolatileoilsimultaneouslyandefficientlysothatthetimeofextractionisgreatlyreduced.Theextract-ladenSC-CO2is subsequentlydepressurized in twoor three separators at predeterminedcondi-tionssothatspecificproductsareselectivelyfractionatedandcollected.Thesecondmethodofferssignificantadvantages,asthequalityoftheproductisimprovedandthebatchtimeforextractionisreduced,resultinginhigherproductioncapacityandcosteffectivenessoftheSC-CO2extractionprocess.Simultaneousfractionationatpreciselyselectedconditionsallowsproductionofcustomizedqualityfractionsandeliminationofundesirablecontaminantsfromthem.ThespecificadvantagesofthisSC-CO2extractionand fractionationprocessarementioned in the following sub-sectionswithrespecttoafewcommonspices.

11.10.1 Celery Seed (Apium grAveolen)

AcomparisonofchemicalcompositionofceleryseedoilbyHDandSC-CO2extrac-tion [12] at 100bar and40°C indicated that theHDoil containedmostlymono-terpenes,whereastheSC-CO2extractedoilcontainedmostlyphthalides(Table11.8).

7089_C011.indd 354 10/8/07 12:19:14 PM


The SC-CO2 extract contained some additional components, such as fatty acids,whichwerenotpresentintheHDoil.Monoterpenesconstituted57.6%oftheHDoil,whereastheSC-CO2oilcontained56.8%phthalides[12].Thelowlevelofphthalides(15.2%)intheHDoilwasattributedtotheirhighboilingpointsandlowvolatilityinsteam.Morethan10hrofHDwasnecessaryfortheircompleterecovery.Phthalidesare cyclic esters or lactones with outstanding odor characteristics of celery. TheodoroftheSC-CO2-extractedoilismoreintenseandlessterpenic.Therefore,theSC-CO2-extractedoil ispreferred to theHDoil to impart theceleryflavor.With

taBle 11.7yields and Concentrations of active Ingredients in extracts with sC-Co2 and hexane

spices (active Ingredient)

sC-Co2 extraction (200 bar, 40°C) (by wt.)

solvent (hexane) extraction (by wt.)

yield (%) % ess. oil % oleoresin yield (%) % extract

Clove 23.8 16.8

Eugenol 71.8 — 70.7

Eugenolacetate 11.1 — 11.3

(byGC,10%FFAP)

Cumin 21.0 12.2

Cymol 15.2 — 13.5

Cuminaldehyde 15.3 — 11.4

(byGC-MS,DB5)

Coriander 3.6 20.0

Linalylacetate 7.8 — 5.8

D-linalool 13.0 — —

(byGC-MS,DB-5)

Ginger 4.6 4.9

Zingiberene 26.7 1.6 31.6

Gingerol 5.65 10.1 5.4

(byGC-MS,DB-5)

Cinnamon 3.0 5.11

Cinnamicaldehyde 77.5 45.0

(byGC,SPB-1)

Pepper 4.6 5.0

Piperine — 53.0 46.4

(byUVmethod)

Ajwain 4.5 5.18

Thymol 63.6 — 24.6

(byGCOV-101)

Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide, CRC Press,BocaRaton,FL,2000.Withpermission.

7089_C011.indd 355 10/8/07 12:19:15 PM


SC-CO2extractioncarriedoutfromceleryseedsatarelativelymoderatepressureof100barand40°C,theyieldofessentialoilwasmerely2.03%ofthechargedmate-rial[13].TheyieldofessentialoilfromceleryleavesbySC-CO2extractionat90barand40°Cwasevenlower(0.04%).ThecompositionsoftheessentialoilsfromceleryseedsandceleryleavesbySC-CO2extractionwerefoundtobesignificantlydiffer-ent,ascanbeseeninTable11.9.Theceleryseedextractscontainedmoreparaffinandfattyacidmethylestersthantheceleryleafextract.

11.10.2 red Chili

SC-CO2extractionofredchiliiscarriedoutinthepressurerangeof300to500barand80°C to100°C,withsimultaneousfractionationof theextracts into lightandheavyfractions.Thelightfractioncontainsmostofthecapsaicin(i.e.,thecompoundresponsible for the hotness of the spice), in addition to the essential oil, whereastheheavy fractioncontains triglyceridesand thecolorcompounds, inaddition to

taBle 11.8Composition (%) of Celery seed essential oil by sC-Co2 extraction and hydrodistillation

Class of Compounds sC-Co2 extraction hd

Monoterpenes 16.1 57.6

Oxygenatedmonoterpenes 0.2 0.6

Sesquiterpenes 19.7 23.3

Phthalides 56.8 15.2

Others 4.8 0.3

Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.

taBle 11.9Composition of essential oils from Celery seeds and leaves by sC-Co2

ComponentCelery seeds

(100 bar, 40°C)Chinese Celery seed

(100 bar, 40°C)Celery leaves (90 bar, 40°C)

Limonene 3.7 14.9 33.4

β-Selinene 33.8 17.6 3.0

α-Selinene 5.3 1.8 0.5

Butylphthalide 19.8 5.5 2.8

Sedanenclide — 22.4 —

Bedanolid — 28.8 —

Germacrone 21.0 — 45.4


7089_C011.indd 356 10/8/07 12:19:15 PM


a small quantity of capsaicin [14], as shown in Table11.10a. The distribution ofcapsaicininthetwofractionsisadjustedbyselectingtheconditionsinthesepara-torsforfractionation.SCFEofamorepungentvarietywasperformed[28]overalowertemperaturerange(35°Cto70°C)andawiderpressurerange(100to550bar).Itwasshownthatthecapsaicinoid(capsaicin,dihydrocapsaicinandthelike)couldberaisedto75%inthe0.2%to0.3%oleoresinifextractionwascarriedoutfromthedriedmaterialandupto99%fromfreshmaterial.Itwasobservedthecapsaicinoidcontent increasedwithtimeofextractionandadditionofacosolvent,suchas5%aceticacid,anditincreasedfurtherbysuccessivelyincreasingthetemperature,aspresentedinTable11.10b.

11.10.3 PaPrika

Paprikaisusefulinindustryforitsnaturalcolor.ForSC-CO2extractionofpaprika,mostof the color compounds are collected in theheavy fraction,while aroma iscollectedinthelightfraction.Researchindicates[14]thatthecolorvalueofSC-CO2-extractedproductcouldreachashighas7200ASTA,whereasanormalcommercialproductischaracterizedtohaveacolorvalueintherangeof1000to2000ASTA.

taBle 11.10(a)Composition in light and heavy fractions of Chili extract

products % Capsaicin% dihydro- Capsaicin

total % Capsaicinoid

Rawmaterial 0.21 0.14 0.39

Lightfraction 8.10 4.05 13.50

Commercialproduct 1.83 1.52 3.93

Heavyfraction 0.57 0.31 0.95

RatioL/H 14.2 13.1 14.2

taBle 11.10(B)sCfe from fresh Chili with successive Increase in temperature and pressure with 5% acetic acid (by wt.) of feed Chargedpressure

(bar)temperature

(°C) time (min)yield

(% wt. of feed)Capsaicinoid (% of extract)

100 40–50 60 0.065 47.0

130 50–60 45 0.040 57.5

150 60–70 45 0.010 87.4

175 70–75 45 0.003 98.8

250 75 45 0.106 0.8


7089_C011.indd 357 10/8/07 12:19:15 PM


11.10.4 GinGer

Ginger extract usingSC-CO2 is fractionated into two fractions: the essential oil–enrichedlightfractionandtheoleoresin-enrichedheavyfraction,thecompositionsofwhicharecomparedinTable11.11aandTable11.11b.Gingerols(G)andshogaols(S)arethecompoundsresponsibleforthepungencyofginger,andtheyaremostlycollectedintheheavyfractionoftheSC-CO2extract,ascanbeseeninTable11.11a.Shogaols,beingtheoxidationproductsofgingerols,arepresentinverylessquanti-ties in theSC-CO2-extracted fractions.Aproduct of desired specification canbeformulatedbycombiningthetwofractionsinasuitableproportion[14].

11.10.5 nutmeG

SC-CO2extractionandfractionationofnutmegcanyieldgoodqualitynutmegbutterastheheavyfractionwithverylittlevolatileoilandnutmegoilasthelightfraction,

taBle 11.11(a)light and heavy fractions of sC-Co2-extracted ginger oleoresin

product % 6-g% 8-g+ 6-s

% 10-g+ 8-s

(8g+6s) % total

total % extract

Rawmaterial 0.87 0.14 0.27 0.11 1.28

Heavyfraction 13.95 2.58 4.37 0.12 20.90

Commercialproduct 2.81 5.83 1.19 0.52 11.12

Lightfraction 1.43 0.61 0.36 0.25 2.40

RatioH/L 9.8 4.2 12.1 0.5 8.7

G:Gingerol;S:Shogaol

taBle 11.11(B)Compositions (%) light and heavy fractions of sC-Co2-extracted ginger essential oil

productraw

materialheavy

fractionlight

fractionratio l/h

Essentialoil(ml/100g)

2.0 4.4 98.8 22.5

β-pinene 2.5 0.5 2.6 5.2

Camphene 7.0 1.6 7.3 4.6

Cineole 8.4 2.3 8.6 3.7

Limonene 1.2 0.3 1.2 4.0

Zingiberene 21.8 17.4 22.7 1.3

Bisabolene 8.7 8.6 8.8 1.0

Sesquiphellandrene 11.9 12.7 11.9 0.9


7089_C011.indd 358 10/8/07 12:19:16 PM


inwhichtheundesirablehallucinatorycompoundmyristicinispresentinnegligibleconcentration[14].ItispossibletouseSC-CO2toproducenutmegoildevoidofthiscompound.ThisisanimportantadvantageoftheSCFEprocess,asthepresenceofthiscompoundinnutmegoilisbannedinsomecountries.

11.10.6 BlaCk PePPer

WhenblackpepperisextractedandfractionatedintotwofractionsusingSC-CO2,thelightfractionmaybecompletelyfreefrompiperine,theactiveingredientofpepper,whereastheheavyfractionmaybeenrichedwithupto60%piperine[14].Besidestheconcentrationofthespecificcomponent,allSC-CO2-fractionatedproductsareofsuperiorquality.Therateofextractionat500barisalmostdoubletherateat300barand60°C.Theproductioncapacityofthefractionsmaybeenhancedfourtimesat500barusingacascadeoffourextractors.Thus,theoperatingcostofextractioncanbereducedtoone-fourthofthatobtainedbythetraditionalSCFEplant.ThecurrentcommercialpracticeistofollowthistechniquetoimprovetheefficiencyandcosteffectivenessofSC-CO2extractionofmajorspices.

11.10.7 Vanilla

Naturalvanillafragranceisextractedfromcuredvanillabeans.Greenvanillabeansarecuredtobringabouthydrolysisoftheglucosidespresentinthebeanstogeneratevanillinandotherflavorandfragrancecomponents.Thecuringprocesschangesthegreenvanillabeansintodark,brownish,softbeans.Thecurrentcommercialextrac-tionmethodusesaqueousalcoholof35to40vol.%inconcentrationatatemperatureashighas87°Cinanumberofsteps,makingtheextractthermallydegraded.SC-CO2extractionofcryogenicallyground,driedbeansresulted10.6%yieldofoleoresinat110barand36°C,whichisevenhigherthan5.3–8.4%yieldsbyalcoholextraction[15].Thevanillaoleoresincontainedashighas16%to36%vanillinbySC-CO2extraction,whichamountedto74%to97%recoveryof the totalvanillincontent,respectively.Otherflavorand fragranceconstituents in thenaturalvanillaextractarep-hydroxybenzaldehyde,vanillicacid,andp-hydroxybenzoicacid.Thequalityof theextract is,however,characterizedbyitsvanillincontent.ThecompositionsofthenaturalvanillaextractsbytraditionalalcoholextractionandSC-CO2extrac-tionarecomparedinTable11.12.ThehighestpurityvanillincouldbeobtainedbySC-CO2extractionofwater-presoakedbeans,thoughtheyieldwasonly3%.Ontheotherhand, cryo-grinding apparently releasesmore compounds and, accordingly,theyieldwasalsohigh(10.6%).Thepurityofthealcoholextractaswellaspercentrecoveryofvanillin(61%)islowerthanthatoftheSC-CO2extract.Eventhecoloroftheextract,whichisyellowcomparedtothedarkbrowncolorofthealcoholicextract,issuperiorinthecaseofSC-CO2extraction.

11.10.8 Cardamom

SC-CO2 extraction of cardamom requires much higher pressure (100 bar) thansubcriticalpropane,whichrequiresas lowas20bar toyield thesameamountof

7089_C011.indd 359 10/8/07 12:19:16 PM


essentialoil.AdditionofethanoltoSC-CO2doesnotgreatlyincreasetheyield,butincreasesthecoextractionofpigments,ascanbeseeninTable11.13.Reductioninpressure of SC-CO2 usually reduces the contents ofβ-carotene, chlorophyll, andpheophytinintheextract.

Theamountofpigmentextractedissignificantlymorewhensubcriticalpropaneisusedastheextractant.However,betterrecoveryofaroma(Table11.14)ispossiblewithSC-CO2at100barand35°C,asreportedbyIllesetal.[16].

11.10.9 Fennel, Caraway, and Coriander

Recovery of active components from fennel, caraway, and coriander by differentmethodsofextractioniscomparedinTable11.15.ItisclearthatSC-CO2extractsarericherinactivecomponents,owingtobetterselectivityoftheextractant[17].

taBle 11.12Comparison of Compositions of vanilla extracts

solvent(Beans)

sC-Co2 (120 bar, 33°C) ethanol + h2o(Water soaked)(dry) (ground) (Water soaked)

p-hydroxybenzoicacid(area%) 0.2 0.1 0.1 1.1

Vanillicacid(area%) 0.1 1.3 0.1 1.1

p-hydroxybenzaldehyde(area%) 0.6 1.9 0.9 2.7

Vanillin(mass%) 21.0 16.1 36.3 20.0a

Unknown 0.0 2.4 0.0 8.0a: Water-freebasis.


taBle 11.13yield of Cardamom oil and pigment by sC-Co2 and propane

process Conditionsyield % (g/g oil)

β-Carotene (g/g oil)

Chlorophyll (g/g oil) pheophytin

SC-CO2(80bar,25°C) 5.65 0.8 0.65 —

SC-CO2(100bar,35°C) 5.45 2.1 0.30 —

SC-CO2(200bar,35°C) 5.95 3.9 0.36 0.33

SC-CO2(300bar,35°C) 6.65 5.8 4.53 2.36

CO2+ethanol(100bar,25°C) 5.28 1.64 9.65 2.10

Ethanol — 0.80 11.95 2.60

Propane(50bar,25°C) 7.24 18.6 10.80 4.80

Propane(20bar,25°C) 6.85 16.2 3.40 2.10


7089_C011.indd 360 10/8/07 12:19:17 PM


11.9.10 GarliC

SC-CO2extractionofvaluableingredientsfromgarliciscomparabletothatbyhexane[18].Themajorcomponentsofgarlicoilarediallyldisulfide(30%),diallyltrisulfide(30%),anddiallylsulfide(15%).Alliin,amajorgarlicactiveingredient,isknowntodegrade toallicinbyanenzymatic reaction,andothergarliccomponentsarealsosusceptibletooxidationwithtemperature.Acomparisonofhigh-performanceliquidchromatography(HPLC)andGCanalysisofextractsobtainedbySEwithavarietyofsolventswithvaryingpolaritywiththatbySC-CO2indicatedthattheformercontained

taBle 11.14peak area (× 103) of aroma Constituents of Cardamom oil by sCf

β-pinene Cineole linalool α-terpinol Borneole

CO2(80bar,25°C) 16.1 295 34.8 47.8 356

CO2(100bar,35°C) 27.6 450 73.5 91.2 579

CO2(300bar,35°C) 17.4 341 32.7 46.4 340

Propane(20bar,25°C) 15.5 286 25.6 36.9 304

Propane(50bar,25°C) 26.9 386 72.1 82.7 521

CO2+ethanol(100bar,25°C) 6.5 198 5.8 8.9 112


taBle 11.15recovery of active Components from fennel, Caraway, and Coriander by various methods

active Component

sC-Co2

ultrasound water hexane steam

p (bar) 80 100 200–300

t (°C) 28 30 35

fennel

Fenchon 10.7 13.1 9.2 21.9 16.3 0.3

Estragol 1.6 0.5 1.5 6.6 3.1 1.7

Transanethole 68.2 50.8 72.5 70 70 77.6

Caraway

Limonene 33.5 32.0 33.3 30.1

D-carvone 56.9 54.0 54.3 50.2

Coriander

Linalool 20–30 15 80–85 67 80 79


7089_C011.indd 361 10/8/07 12:19:17 PM


morecomponents.ThisisattributedtodegradationofthecomponentsinSE.ClinicaltestsalsoindicatedthattheSC-CO2garlicextracthasmorepotentbioactivity,closetothatofrawgarlic[18].

11.10.11 Cinnamon

Twotypesofessentialoilnamely,leafandbarkoil,areproducedfromtwodifferentpartsofacinnamontree.CinnamonleafoilismainlyproducedinSriLanka.ItisalsoproducedinIndiaandSeychelles.Mostcinnamonoilisproducedfromleaves.Barkoilamountstoonly15%oftotalproduction.Leavesyield1%oil.However,therootbarkyields3%oil[19].ThecomparisonofthecompositionsofextractsfromSrilankancinnamonbarkand leaves isgiven in Table11.16.SDof thecinnamonbarkyields1.4%oil,whereasSC-CO2extractionat200barand60°Cresults1.5%yield.However,additionofethanolasacosolvent increasestheyieldto2.6%[9].Theleafoilrichineugenolmakesitasubstituteforcloveoilandmaybeusedforconversiontovanillin.Barkoilismorevaluablethantheleafoil,althoughbothfindwideusesinflavoringandpharmaceuticalindustries.

11.11 CorrelatIon for spICe oIl soluBIlIty In sC-Co2

Solubility of spice oils in SC-CO2 is an important process parameter needed fordesignandscale-upofthecommercialSCFEplant.SolubilitydepictsthemaximumpossiblesolventcapacityofSC-CO2atagiventemperature,pressure,orcosolventconcentration in SC-CO2, though the actual loading or dissolution of the soluteis much less than this solubility in the presence of the solid substrate. However,theneatsolubility(withoutthepresenceofthesubstrate)behaviorofspiceoilcansufficeforselectionoftheprocessconditionsforthemostefficientperformanceoftheSCFEprocess.Becauseexperimentalmeasurementofsolubilityistediousand

taBle 11.16Compositions of Cinnamon leaf oil and Cinnamon Bark oil

% leaf oila % Bark oil

Componentsteam

distillatesteam

distillatesC-Co2 extract (200 bar, 60°C)

sC-Co2 + ethanol extract (200 bar, 60°C)

Eugenol 85–95 3.3 2.0 2.8

Caryophyllene 6 Traces 2.1 1.6

Cinnamicaldehyde 38 7.88 1.98 6.8

Isoeugenol 21.9 1.2 0.4

Linalool 20.1 0.9 0.8

Cinnamylacetate 23.6 5.1 1.8

o-methroxycinnamic

aldehyde 1.3 1.6 2.6a: Fromreference(Wright,1994)Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide, CRC Press,

BocaRaton,FL,2000.Withpermission.

7089_C011.indd 362 10/8/07 12:19:17 PM


timeconsumingatdifferentconditions,areliablecorrelationcanservethepurpose,as itcanbeutilizedforestimationofsolubilityspiceoil inSC-CO2.Chrastil [21]relatedtheequilibriumsolubilityofasoluteinSC-CO2byalinearrelationshipintermsofitsdensityas:

lny*=klnρ+aT

+b (11.1)

where,y*isthesolutesolubility(g/L),T(K)isthetemperature,ρisthedensityofSC-CO2(g/L),anda,b,andkareadjustableconstantsthatcanbeevaluatedfromthelimitedexperimentaldata.

DeValleandAguilera [22]modified theChrastil’scorrelationbyaddingonemoreregressableconstanttowidenitsvalidityforthetemperaturerangefrom20°Cto80°Candforpressuresvaryingfrom150to280baras:

lny*=klnρ+aT

bT

+2

+C (11.2)

Silva et al. [23] correlated the experimental data (as reported in Table11.17)intermsofthedensityofSC-CO2asreportedbyAngus[24].TheconstantsinthecorrelationsarepresentedinTable11.18.Ferreiraetal.[25]reportedSCFEofblackpepperessentialoilfromwhichthesolubilitydataweregeneratedandwerecorre-latedintermsofvaporpressures(Ps),consideringoilasapseudo-purecomponent:

y*=PP

S

exp[A+Bρ] (11.3)

taBle 11.17Black pepper oil solubility in sC-Co2

t (°C) p (bar)density of sC-Co2

(g/cm3)oil solubility (g/cm3 Co2)

30 150 0.8478 0.0755

40 150 0.7812 0.0728

50 150 0.7010 0.06015

30 200 0.8909 0.1006

40 200 0.8404 0.08774

50 200 0.7851 0.7812

30 300 0.9486 0.13698

40 300 0.9106 0.1243

55 300 0.8712 0.1093

Source: Silva,D.C.M.N.etal.,Correlatingsolubilityvaluesofblackpepperoilin supercritical CO2 using empirical models, in Proceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,279.Withpermission.

7089_C011.indd 363 10/8/07 12:19:20 PM


where A and B are empirical constants evaluated from the experimental data.However,thisrequiresvaporpressureofspiceoilasafunctionoftemperature.

Essentialoilobtainedfromturmeric(Curcuma longa)containsalargenumberofcomponents,suchascurcumin,ar-turmeric(42%),turmerone(12%),andtherest(<5%).Consideringit tobeasinglecomponentbyresearchers[26]thesolubilityofturmericoilinSC-CO2wasmodeledusingthesteady-stateextractiondataattheinitialperiodaswellasusingNaik’scorrelation[27]as:

Y= Y t

B t∞

+ (11.4)

where Y=extractionyield(kgextract/kgcurcumin)×100 t=CO2mass(kgCO2/kgcurcumin) Y∞=extractionyieldatequilibrium B=CO2massneededtoreachthehalfofY∞.

AcomparisonofthepredictedsolubilitieswiththecorrespondingexperimentaldataisgiveninTable11.19.Itmaybenotedherethatthefraction[0.5Y∞/B]issimilartotheslopeoftheextractioncurveattheinitialstage(i.e.,whentheextractionofspiceoil is controlledby its solubility).Bothmethods result similaragreementwith theexperimentaldataandmaybeconsideredforascertainingthesolubilitybehavior.

11.12 ConClusIons

SCFEofspicesisconsideredasuperioralternativetotheconventionaltechniquesofSD,SE, andASE for simultaneousproductionof essential oils andoleoresinsinasinglestep.SCFEensureshighconsistencyandreliability in thequalityandsafetyofthebioactivenaturalmolecules.SC-CO2isGRASandyieldscontaminant-free, tailor-made extracts of superior organoleptic profile, with high potency ofactive ingredientswithoutanyresidualorganicsolventandartifacts.Theextractsareveryclosetothatinnatureinsmellandtasteandhavelongershelflivesduetocoextractionofantioxidantsandbetterblendingcharacteristicsduetocoextractionof triglycerides. SCFE is known to be commercially viable for high-value, low-volumeextracts,andifmultipleproductsareobtainedoperatingthesameplantattherespectiveoptimizedprocessconditions.

taBle 11.18parameters for the solubility Correlations of Black pepper oil

Correlation a b c k

Chrastil –14807.9 26.123 — 3.84

DeValle&Aguilera 70207.45 –13500128.15 –107.409 3.84

Source: Silva,D.C.M.N.etal.,CorrelatingsolubilityvaluesofblackpepperoilinsupercriticalCO2usingempiricalmodels,in Proceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,279.Withpermission.

7089_C011.indd 364 10/8/07 12:19:21 PM


referenCes

1. Darling,M.Louis,BiomedicalLibrary,UCLA,Spices: Exotic Flavors & Medicines, Availableathttp://unitproj.library.ucla.edu/biomed/spice/

2. Mukhopadhyay,M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.

3. Duke, J.A., Biologically active compounds in important spices, in Spices, Herbs, and Edible Fungi, Charalambous,G.,Ed.,ElsevierSciencePublishers,Netherlands,225–250,1994.

4. Rosen,R.T.,Ta’am Tov B’Tuv Ta’am: A Flavorful Blend of Kashrus and Spices, Avail-ableathttp://www.kashrut.com/articles/spices/September12,2006.

5. Tainter,D.R.andGrenis,A.T.,Spices and Seasonings: A Food Technology Handbook,SecondEdition,CulinaryandHospitalityIndustryPublicationsService,1997.

6. Raghavan Uhl, S., Handbook of Spices, Seasonings, and Flavorings, Culinary andHospitalityIndustryPublicationsService,1996.

7. Marion, J.P., Audrin, A., Maignial, L. and Brevard, H., Spices and their extracts:Utilization, selection,quality control, andnewdevelopments, inSpices, Herbs, and Edible Fungi,Charalambous,G.,Ed.,71–95,1994.

8. Pellerin,P.,Comparingextractionbytraditionalsolventswithsupercriticalextractionfrom an economic point and environmental standpoint, in Proceedings of the Sixth International Symposium on SCFs, France,2003,Tome1,13.

taBle 11.19solubility of essential oils of Curcuma longa

t (°C) p (bar)solubility (g/100 g Co2)

By naik’s modelsolubility (g/100 g Co2)

from extraction data

30 100 0.39 0.67

150 0.82 1.00

200 0.87 1.12

250 0.95 1.20

280 1.59 —

40 100 0.17 0.20

150 0.58 0.63

200 1.24 1.34

250 1.67 1.51

280 1.88 1.74

50 100 0.19 0.31

155 0.77 0.89

200 1.51 1.53

250 2.54 1.96

280 2.80 2.16

35 280 1.59 1.54

Source: Blasco,M.etal.,SCFEofCuruma longa:Solubilityofessentialoil,in Proceedings of the Sixth International Symposium on Supercritical Fluids, Nice, France, 2003,Tome 1, 279.Withpermission.

7089_C011.indd 365 10/8/07 12:19:21 PM


9. Calame,J.P.andSteiner,R.,Supercriticalextractionofflavors,inTheory and Practice of Supercritical Fluid Technology,Hirata,M.andIshikawa,T.,Eds.,TokyoMetropolitanUniv.,275–318,1987.

10. GangadharaRao,V.S.G.andMukhopadhyay,M.,Selectiveextractionofspiceoilcon-stituents by supercritical carbon dioxide, Proceedings of the Annual Convention of Indian Institute of Chemical Engineers, Baroda,India,1988.

11. Meireles, M.A.A. and Nikolov, Z.L., Extraction and fractionation of essential oilswithliquidCO2,inSpices, Herbs, and Edible Fungi,Charalambous,G.,Ed.,ElsevierSciencePublishers,Netherlands,171–199,1994.

12. Zhang, J. et al., Volatile compounds of a SCF extract of Chinese celery seed, inProceedings of the Fourth International Symposium on Supercritical Fluids,Sendai,Japan,1994,235–237.

13. DellaPorta,G.,Reverchon,E.andAmbrousi,A.,PilotplantforisolationofceleryandparsleyessentialoilbySC-CO2,inProceedings of the Fifth Meeting of Supercritical Fluids,Nice,France,1998,Tome2,613–618.

14. Nguyen,U.Y.,Anstee,M.andEvans,D.A.,ExtractionandfractionationofspicesusingSCFCO2,inProceedings of the Fifth Meeting of Supercritical Fluids,Nice,France,1998,Tome2,523–528.

15. Nguyen,K.,Barton,P. andSpencer, J.S.,Supercritical carbondioxide extractionofvanilla,J. Supercrit. Fluids,4,40–46,1991.

16. Illes,V.,Daood,H.,Karsai,E.andSzalai,O.,Oilextractionfromcardamomcropbysubandsupercriticalcarbondioxideandpropane, in Proceedings of the Fifth Meeting of Supercritical Fluids, Nice,France,1998,Tome2,533–538.

17. Then, M., Daood, H., Illes, V. and Bertalan, L., Investigation of biologically activecompoundsinplantoilsextractedbydifferentextractionmethods,inProceedings of the Fifth Meeting of Supercritical Fluids,Nice,France,1998,Tome2,555–560.

18. Nawrot, N. and Wenclawiak, B., Supercritical fluid extraction of garlic followed bychromatography, in Proceedings of the Second International Symposium on Super-critical Fluids,Boston,1991,451–455.

19. Mahindru,S.N.,Indian plant perfumes, Metropolitan,NewDelhi,India,1992. 20. GangadharaRao,V.S.G.,Studies on Supercritical Extraction of Spices, Ph.D.Disser-

tation,IndianInstituteofTechnology,Bombay,1990. 21. Chrastil,J.,Solubilityofsolidsandliquidsinsupercriticalgases,J. Phys. Chem,86,

3016–3021,1982. 22. DeValle,J.M.andAguilera,J.M.,Animprovedequationforpredictingthesolubility

ofvegetableoilsinsupercriticalCO2,Ind. Eng. Chem. Res.,27(8),1551–1553,1988. 23. Silva,D.C.M.N.,Ferreira,S.R.S.andMeireles,M.A.,Correlatingsolubilityvaluesof

blackpepperoilinsupercriticalCO2usingempiricalmodels,in Proceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,279.

24. Angus,S.,Ramstrong,B.andDeReuck,K.M.,International Thermodynamic Tables of the Fluid State:Carbon Dioxide, NewYork,PergamonPress,3,1976.

25. Ferreira,S.R.S.etal.,SCFEofblackpepperessentialoil,J. Supercrit. Fluids,14(3),235–245,1999.

26. Blasco,M.etal.,SCFEofCuruma longa:Solubilityofessentialoil,inProceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,341.

27. Naik,S.N.,Lentz,H.andMaheshawari,R.C.,Extractionofperfumesandflavourfromplant materials with liquid carbon dioxide at liquid–vapour equilibrium conditions,Fluid Phase Equilibria,49,115–126,1989.

28. McGaw,D.R.,Holder,R.,Commissiong,E.andMaxwell,A.,Extractionofvolatileand fixed oil products from hot pepper, in Proceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,111.

7089_C011.indd 366 10/8/07 12:19:21 PM

367

12 Preparation and Processing of Micro- and Nano-Scale Materials by Supercritical Fluid Technology

Eckhard Weidner and Marcus Petermann

Contents

12.1 Introduction................................................................................................. 36712.2 ParticleGenerationbyHigh-PressureSprayProcesses.............................. 368

12.2.1 RapidExpansionofaSupercriticalSolution................................ 37012.2.2 AntisolventProcesses................................................................... 37212.2.3 SprayingofGasSaturatedLiquids............................................... 37312.2.4 Economics.................................................................................... 375

12.3 CompositeswithHigh-PressureSprayProcesses....................................... 37612.3.1 SprayAgglomerationwithaHigh-PressureSprayProcess......... 37612.3.2 Liquid-FilledCompositeswithaHigh-PressureSpray

Technology.................................................................................... 37812.4 ProcessingofNutraceuticalswithSupercriticalFluidTechnology............38012.5 Conclusions.................................................................................................384References..............................................................................................................384

12.1 IntroduCtIon

Thegenerationofnano-andmicro-particlesandtheformationofparticulatecom-positeshavebecomemoreandmore important inmany industrialareas. Infood,pharmaceutical,material,andlifescienceindustries,existingandnewproductsinnewapplicationformswith tailor-madepropertiesarebeingdevelopedfasterandfaster.Toformparticulateproducts,differentwell-establishedprocessesareavail-able.Powderscanbeobtainedbycrystallization,grinding,orspraydryingprocesses.However, all these techniques have drawbacks, especially if sensitive substancessuchasnutraceuticalsorbioactivesystemshavetobeprocessed.Inclassicalcrystal-lizationtechniques,solvents—inmanycasesorganicsolvents—havetobeusedasauxiliarymedia.Resultantresiduesofthesesolventsmaybefoundintheproductsand have to be removed by time-consuming and expensive technologies. Similar

7089_C012.indd 367 10/8/07 12:21:35 PM


drawbacksappearinmostspraydryingprocesses.Inaddition,thehightemperaturesnecessarytoevaporatesolventsmaycauseproductdegradation.Grindingprocesses,whicharetypicallysolventfree,canonlybeappliedforbrittlesubstances.Toachievesufficient brittleness, deep-freeze conditions are sometimes required. But even ifmillingispossible,onlyparticleswithsharpedgesareavailable(Figure12.1).

Theincreasingdemandfornewproductpropertiesandthedrawbacksofexist-ingprocessesarecausingasteadysearchfornewtechnologicalpossibilitiesfortheformationofparticulatesystems.Somepromisingtechniquesincludeusingsuper-criticalfluids(SCFs)togeneratenano-andmicro-scaledparticlesystemswithwell-definedmorphologiesand,therefore,productbehavior.Inadditiontobeingusedforpureparticleformation,thesetechniquesarebeingusedmoreandmoretogeneratecompositesconsistingoftwoormoresubstances,eventhoseindifferentstatesofaggregate(liquid/solid).Thisallowsmanufacturingofhigh-qualityproductsoffer-ingtailor-madeproperties,suchascontrolledreleaseofactivesubstances[1–5].

12.2 PartICle GeneratIon by HIGH-Pressure sPray ProCesses

Generatingparticlesfrompuresubstancesorcompositesbyhigh-pressuretechnolo-giesrequiresunitoperationssimilartothoseusedforclassicallow-pressureprocesses.Thoseunitoperationscomprise,forinstance,melting,dissolving,mixing,spraying,separating,andpumping.PerformingsuchunitoperationsunderhighpressuresandinthepresenceofSCFsrequiresspecificadaptationsinplantdesignandprocess-ing. Due to extensive R&D work and industrial experience, those adaptations ofmachinesandapparatusesaremeanwhileknownquitewell.Inspiteofthefactthatmajortechnicalproblemsaresolvedforhigh-pressureapplications,itisundisputedthatthoseprocessesaremorechallengingfromthetechnicalandeconomicalpointsof view than are low-pressure processes. New possibilities to create value-addedproductsthatarenotaccessiblewithclassicaltechnologiesareastrongdrivingforcefor the industrial use of high-pressure processes. A series of such processes andprocessmodificationsthatallowsgeneratingnewproductformshasbeendeveloped

FIGure 12.1 Morphologyofgrindedparticles.

7089_C012.indd 368 10/8/07 12:21:36 PM

Preparation and Processing of Micro- and Nano-Scale Materials 369

inthepastyears.Anextensivenumberofacronymsareusedtocharacterizethoseprocesses.Thoseacronymswillnotbe listedhere,but theprocessesaregroupedaccordingtothermophysicalprinciplestheyuse.

Inrapidexpansionofsupercriticalsolution(RESS)processes,thesubstancetobepowderizedisdissolvedintheSCF. Intheparticlesfromgas-saturatedsolutions(PGSS)processes,thesubcriticalfluidorSCFisadmixed,dispersed,anddissolvedin the substance to be powderized. In both types of processes, the particles aregeneratedbyexpansionincapillariesornozzles[6–9].Intheso-called“antisolventprocesses,”suchasthegasanti-solvent(GAS)process,theproducttobepowderizedisfirstdissolvedinaclassicalorganicsolvent.Afterward,thissolutionisadmixedwith aSCF.This causes adecreaseoffluiddensity and leads to reduced solventpowerof theorganic solvent.Resultantparticles areprecipitatedwithin themix-ture of organic solvent and SCF [10–13]. Compared to traditional crystallizationprocesses, in which organic solvents are evaporated at high temperatures or in avacuum,GASprocessesworkatlowtemperaturesandachievesupersaturationmuchfaster.Therefore,GASprocessesareadvantageouscomparedtotraditionalcrystal-lizationprocesses,eveniftheresidualsolventproblemhastobesolved.

Allprocesseshave incommonthat thesubstance tobepowderizedhas tobebroughtintoaliquidordispersedform.Thisisachievedbymeltingthesubstance,bydissolvingtheproductinclassicalsolvents,orbydispersingtheproductinaliquid.Inmanycases,thisstepisperformedatambientorslightlyelevatedpressures.Thentheproduct is compressedbycontinuouslyoperatedplungerpumps,gearpumps,orextruders.Thetypeofdosingsystemuseddependsmainlyonthepropertiesofthe product (e.g., melting point and viscosity�). Dosing of substances with highviscositiesathighpressuresrequiresspecialtechnicalsolutions,someofwhichhavebeenelaboratedinthepastyears[14].Manyresearchactivitiesdemonstratedthathighpressure,inconnectionwiththeuniquepropertiesofSCFs,opensthechancetogeneratepowdersfromhighlyviscousliquids,whichcannotbesprayedbyclassicaltechnologiessuchasspraydrying.Sprayabilityisachievedbyaconsiderablereduc-tionofviscosityandsurface tension ifsuchhighlyviscous liquidsareeffectivelyadmixedwithaSCFthatissufficientlysoluble[15,16].Admixingisachievedviastirrers,dispersers, impinging jets, staticmixers,ormembranes thatareoperatedunderhighpressure.

ImprovementofsprayabilityisnottheonlyadvantageofSCFs.AfteraSCFisadmixed,thephysicalpropertiesofthesubstancesarechangeddramatically[17–19].Themostimportanteffectsarereductionofviscosityandmeltingpointdepressionduetodissolvedgas.Botheffectsallowhandlingsubstancesnearorevenbelowtheirmeltingpointunderambientpressure.DuetocoolingoftheSCFthatoccursdur-ingexpansion,thetemperaturesareeven(much)loweraftertheparticleshavebeenformedviaexpansion.Astheheatofsolidificationisremovedbydirectheattransferfromtheparticlestothecoexpandedgas(inthecaseofRESSandPGSS),solidifica-tionoccursmuchfaster(some10milliseconds)[20–22]andthetemperaturestressontheparticlesislowerthanthatduringclassicalairdryingprocesses,wherebythe

�In discontinuous processes, products are dosed either as liquid or solid into an autoclave, whereadditionallycompressedgasisaddedandadmixed.Theelevatedpressureintheautoclaveisusedtotransporttheproductsintothenextprocesssteps.

7089_C012.indd 369 10/8/07 12:21:36 PM


solventisevaporatedbyheattransferbetweenliquiddropletsandthesurroundinghotairorgas[23].Furthermore,unlikemanytraditionalspraydryingprocesses,thesupercriticaltechnologiesareintrinsicallyfreefromcontactingtheproductswithairoroxygen.Sothesetechnologiesaresuitableforsensitivesubstances[24].Anotherpositiveeffectisthataslongasthecontactbetweenthegeneratedpowdersandairiscarefullyavoided,dustexplosionsmaynotoccur.

Animportant—andsometimesunderestimated—stepofallhigh-pressurepro-cessesisthecollectionoftheparticles.UsingsprayprocesseslikeRESS,PGSS,andconcentratedpowderform(CPF),theparticleshavetobeseparatedfromagasstream.Dependingonparticlesizeandparticleconcentrationinthegasstream,differentseparation techniquesmightbesuitable.Coarse fractionscanbesepa-ratedjustbysettlingtheparticlesinspraytowersorbyenlargingtheseparationforcesincyclones.Forfinerparticlesandlowerparticleconcentrationfilters,mem-branesor sinter plates canbeused to collect themanufacturedproduct.For theantisolventprocesses,inwhichparticlesareformedbyprecipitationinaliquid,asolid-liquidseparationmustbeused.Thisseparationisachievedbysettlingorbydifferentfiltersystems.

After the particles are separated from the SCF or the solvent, postprocessingmightbenecessary.Inthecaseofantisolventprecipitation,solvent-wetparticlesareobtained,whichhavetobedried.Thiscouldbedone,forexample,byflushingwithheated gas or by additional SCF extraction steps [25]. Depending on the applica-tionoftheproduct,awholerangeoffurtherposttreatmentstepsmightbeconsidered(coating, sieving, agglomeration, sifting, size fractionation, dispersion, and soon),whichcouldeitherbeappliedaloneorusedincombinationwiththeupstreamparticlegenerationprocess.Someofthesecombinationshavealreadybeenstudied[26–28].

Anoften-discussed issue forallprocesses isgas recycling. If liquidsolventsareinthesystem,solventremovalfromthegasisrequiredinordertoavoidenrich-mentintherecyclegas.Insolvent-freeprocesses,theparticleshavetoberemovedcarefullybeforerecompressingthegasinordertoavoidpluggingoftherecyclingsystem.Bothpurificationmethodscantechnicallybeapplied,buteachisconnectedwith additional costs for equipment andoperation.On a case-by-casebasis, onemustconsiderwhetherrecyclingofthegasisfeasibleandreasonableaccordingtoeconomic and environmental aspects. Recycling might be a disadvantage of theuseofSCFforpowdergeneration,asthepressuredifferencesbetweenpreexpan-sionandpostexpansionrequirehighenergiesforrecompressingthegas.Therefore,reducingthegasdemandforparticlegenerationasfaraspossibleisrecommended.Someprocesses(e.g.,thePGSS-orCPF-method)allowgenerating1kgofpowderwith0.1to1kgofgas.IfacheapSCF,suchascarbondioxide(CO2),isused,gasrecyclingforsmall-andmedium-sizedplantsmightbemoreexpensivethanusingfreshgas.

12.2.1 Rapid Expansion of a supERcRitical solution

Oneoftheoldestprocessesthatusesthespecialpropertiesofcompressedgasesistheso-calledRESSprocess [29–37].Aflowscheme for thisprocess ispresentedinFigure12.2. The substance tobepowderized is stored in an extraction vessel.

7089_C012.indd 370 10/8/07 12:21:37 PM


Compressedgas(inmostcasesCO2)isledthroughthevessel.Underhighpressures,sometimesupto800bar,theproductis(partly)solubleinthegas.Theso-formedsolution is dosed through a heat exchanger and finally is expanded via a nozzle.Causedbytherapiddepressurization,thedissolutionpowerofthegasisreduced,supersaturationoccurs,andaprecipitationoffineparticlesisinduced.Particlefor-mationcanbeinfluencedbythepressureduringextraction,theconcentrationofthedissolvedsubstance, the temperaturebeforedepressurization, thegeometryof thenozzle,andconditionsinthespraychamberaftertheexpansion.Veryfinepowdersintherangeof0.1to10µmwithnarrowparticledistributionsareobtainedbyprop-erlyadjustingtheprocessparameters.

The RESS process is characterized by a rather simple setup in laboratory orsmallproductionscale,butitislimitedbythepoorsolubilityofmanysubstancesinCO2.Sometimesmorethan100kgofCO2wouldbenecessarytomanufacture1kgoftheparticulateproduct.Subsequently,theparticleshavetobeseparatedfromveryhighlydilutedgasstreams,whichisaproceduralchallenge.

Therefore, the RESS process offers a high potential for high value-addedproductssuchaspharmaceuticalsandcosmetics.Investigationswiththemodelsub-stancegriseofulvinshowedthat,comparedwithproductsmicronizedwithclassicalprocesses,anaccelerateddissolvingbehaviorcanbeachievedwiththeRESSproduct.Inaddition,researchersobservedbetterabsorptionbehaviorinanin-vitrotestsystem[38].Inallnano-scaledprocesses,theposttreatmentoftheparticlesafterparticlefor-mation isachallenging task.Nano-particles tend toagglomerate,andredispersingsuch systems is very difficult or sometimes nearly impossible. To stabilize RESSparticlesintheirnano-scale,researchersproposedtocollecttheminliquid-containingemulsifiers[39].Newlypublishedpapersshowedthat,usingthistechnique,along-termstabilityofnanosuspensionsisobtainedwithparticlessmallerthan100nmandconcentrationsofup to11g/dm³ [40].Other researchers sprayed ternarymixturesconsisting of a SCF, the active substance, and a polymer; they obtained resultantpowderswithencapsulatedactivesubstancesindifferentconcentrations[41–43].

Powder

Gas

Powder

FIGure 12.2 RESSprocessscheme.

7089_C012.indd 371 10/8/07 12:21:38 PM


12.2.2 antisolvEnt pRocEssEs

Anotherimportantgroupofhigh-pressureprocessesaretheso-called“antisolventprocesses.”SimilartotheRESSprocess,thesetechniquesaresuitableformanufac-turingpowdersofpuresubstancesandcompositesfromnanotomicroscale.Alotofdifferentprocessmodificationsaredescribed[44–51],forexample:

GAS: gasantisolventSAS: supercriticalfluidantisolventPCA: precipitationwithacompressedantisolventASES: aerosolsolventextractionsystemSEDS: solution-enhanceddispersionbysupercriticalfluids.

Alltheprocessesbehindthesedifferentacronymsmakeuseofaneffectwellknowninclassicalcrystallizationtechniques.Byaddingathirdcomponent(antisolvent)toasolution,thesolubilityofthedissolvedcomponentisreducedandfinallythesub-stanceprecipitates.Inhigh-pressuretechnology,theantisolventisasupercriticalornear-criticalfluid.Theuseofcompressedgasesinsteadofotherantisolventsopensthewaytonewparticlemorphologiesandnewcomposites[52,53].

Thefirstthreeprocesses(GAS,SAS,andPCA)aretypicallyoperateddiscon-tinuously. A simplified process scheme is shown in Figure12.3. The substanceormixture tobepowderizedisdissolvedina liquid(mostlyorganic)solvent.Toprecipitateparticles,thesolutionhastobecontactedwithaSCF.Thisisachievedindifferentways:

IftheliquidsolutionisprovidedinavesselandafterwardtheSCFisdosedintothatvessel,theprocessiscalledtheGAS process.In the SAS and PCA processes, the SCF is provided in a high-pressurevesselandthesolutionissprayedintothatsupercriticalsolution.IntheSEDSandASESprocesses,bothfluids(thesolutionandtheSCF)aremixedinnozzlesandsprayedintoautoclaves.

•••••

•

•

•

Precipitation

Filtration Drying

FIGure 12.3 Antisolvent-process(GAS)processscheme.

7089_C012.indd 372 10/8/07 12:21:39 PM


Inalloftheseprocesses,admixingofthegascausesaprecipitationofthedissolvedproductintheliquidsolution.Inthenextprocessstep,theso-formedparticleshavetobeseparatedfromtheliquid.Inlaboratoryscale,filtersintegratedintothehigh-pressurevessels typicallyperform this step. Ina thirdprocess step, thecollectedparticleshavetobedried.Inanelegantway,thisisachievedbyaddingfreshSCFtothepressurevessel.Theliquidisextractedfromtheprecipitatedpowderbythegas.Oneadvantageofthisprocessingisthetemperaturerequiredinthesupercriticaldryingstep,whichismoderatecomparedwiththetemperaturerequiredforconven-tionaldryingprocedures.

The antisolvent processes have been successfully tested with many differentproducts. Beside the particle generation from explosives likeβ-HMX and nitro-guanidin,polymers(polyacrylnitril,polycaprolacton)andotherorganicsubstances(hydroquinoneandphenanthrene)havebeenpowderized.AsintheRESSprocess,themainfocusofresearchisaddressingpharmaceuticalslikeascorbicacid,insulin,andparacetamol.

12.2.3 spRaying of gas satuRatEd liquids

TheRESSprocessandtheantisolventprocessesaretypicallycarriedoutindiscon-tinuousorsemicontinuousmode.ThePGSSprocessmayrathereasilybeoperatedinacontinuousmodeand,therefore,isalsosuitableforproductsmanufacturedinlargerquantities[54–57].

Figure12.4illustratesaprincipleflowschemeofthePGSSprocess.Tosprayagas-saturatedliquid,theSCFhastobeadmixedwiththeproducttobepowderizedunder elevated pressures. Typically, the product has to be melted or liquefied byaddinga solvent inadvanceat low-pressureconditions.Subsequently thisfluid ispumpedviahigh-pressurepumpstoamixingdevice(mostlystaticmixers),wheretheSCFisadmixed.Underhigh-pressureconditionstheSCFispartlysolubleinthemelt,dispersionor solution.The solubility causesa reductionofviscosityandof

Gas

Powder

Gas

FIGure 12.4 PGSSprocessscheme.

7089_C012.indd 373 10/8/07 12:21:41 PM


interfacialtension.Botheffectsresultinimprovedsprayability,evenforsubstancesthatnormallycannotbeatomizedbysprayprocesses.Afterward,thegas-enrichedfluidsaredepressurizedviaanozzleintoaspraytower.Normally,thespraytowerisoperatedatambientpressure.Duetothevolumeincreaseoftheexpandinggas,theproductisdisintegratedintofinedroplets.Simultaneously,thegascoolsdownimmediatelyduringexpansion.Althoughthetemperatureinspraytowercaninprin-ciplebeadjustedbyadditionalheatingor cooling,most applicationsdonotneedthis temperaturecontrol.The temperature in thespray tower ismostly setby thepreexpansionconditionsinstaticmixer.Typicaltemperaturesinthespraytowerliein the range of –20°C to 100°C. If the resultant temperature is low enough, theliquid/melt reaches the solidification point and the droplets freeze. Particle sizeandparticlesizedistributionoftheobtainedpowderscanbeadjustedbychangingtheSCF,thepressureinthemixingdevice,thetemperaturebeforeexpansion,andthegeometryofthenozzle.

Asanexample,differentmorphologiesandparticlesizesofpowdersareillus-trated in Figure12.5. The technique can be used for the powderization of manydifferent systems. In addition to organic substances such as citric acid and poly-ethylene glycol (PEG), certain pharmaceuticals (e.g., nifedipine and tobramycin)weresuccessfullymicronized.Moreover,compositesandevenreactivesystemslikepowdercoatingscanbehandledwiththistechnique[58].

Oneadvantageofthisprocess,comparedwithotherhigh-pressuretechniques,isthelowtomoderateconsumptionofSCF.Typically,0.5to5kgofSCFarenecessary

FIGure 12.5 ParticlemorphologiesofPGSSparticles.

7089_C012.indd 374 10/8/07 12:21:41 PM


toproduce1kgofpowder.ThelowgasdemandtogetherwiththerelativelysimpleconstructionofthePGSSplantsallowstheproductionofhugequantitiesofproducts.Thedesignoftheprocessandthecontroloftheproductpropertiesdependsonthethermodynamicandfluiddynamicbehavior,forexample,thesolubilityofthecom-pressedgasesintheproducttobepowderized,theviscosity,andflowbehaviorofthegas-containingmelts.

12.2.4 Economics

Figure12.6presentsestimatedcostsforindustrialPGSS(non-GoodManufacturingPractice [GMP])production facilities.Thediagramgives the total costs for 1kgofproduct,includingcostsforinvestment,personnel,energy,andgasconsumption[57,59].Dependingonthehourlycapacityandtheannualproductionhours,costsrangefromthirtycentsto1€(about$0.75)perkilogramofpowder.Theestimatedcosts aremainlydue topersonnel (40%)andcarbondioxideconsumption (40%).Comparedwiththecostsofclassicalmicronizationtechniques,suchasmillingorspraydrying,thecostsareonthesameorderofmagnitude.

Coststudieshavebeenpublishedforotherhigh-pressureprocesses,likeRESSandGAS.RantakyläanalyzedtheantisolventprocessSAS[60].Estimatedmanufac-turingcostsforanewGMPplantarearound50to300€/kg(38to230$/kg)productwithoutafeedstockprice.Thisisfora4000to8000kg/yearproductionrateand5to10wt%feedconcentrationofthestartingmaterialinanorganicsolvent.Aneffectivewaytodecreasethemanufacturingcostsistoincreasetherawmaterialconcentrationinsolvent.Weberetal.[61]providedataforanon-GMPPCAprocess.Foraninitialsolventconcentrationof10wt%andaproductionof11.25kg/hr(correspondingto87MT/year),thecostsperkilogramofpowderarearound8€($6).Ifthecapacityisdoubled,thespecificcostsarereducedtoapproximately5€/kg($3.80/kg).

0.54

0.390.33

0.0

0.2

0.4

0.6

0.8

1.0

2000 3000 4000 5000 6000

Annual Production (hours/year)

Cos

t (E

UR

/kg

Pow

der)

200 kg/h

350 kg/h

500 kg/h

FIGure 12.6 Production cost for PGSS process (gas consumption 2 kg/kg product).DiagramcourtesyofNatex,Austria.

7089_C012.indd 375 10/8/07 12:21:42 PM


Türk[62]hasgivenvaluesforRESSplantswithafixedCO2flowof2.35MT/hr.Inthecaseofasubstancewithlowsolubility,theannualproductionis1.78MTandthespecificcostsarebetween100and140€/kg(dependingonthetimeofdepre-ciation).Forhighlysolublesubstances,theproductioncapacityinthesameplantisconsiderablyhigher(uptosomehundredtons),leadingtoreducedspecificcoststhatmightreachtherangeof1€/kgorevenlower.NeverthelessithastobenotedthatonlyalimitednumberofsubstanceshaveahighsolubilityinCO2.

The costs for RESS and antisolvent processes are dominated by relativelyhigh investmentcosts for largepressurevesselsandconsiderablegasconsumption.Therefore,thesetechniquesarepreferablyappliedforhigh-pricedproducts,suchaspharmaceuticals.PGSSisalreadyappliedindustriallyinplantsizesofsomehundredkg/hr.Fats,fatderivatives,polymers,andchocolatearealreadyprocessedinindustry.

Amainfocusofthepastyearsofresearchanddevelopmentinthefieldofsuper-criticalmicronizationhasbeenonthegenerationoftailor-madeparticlesfromsinglecomponents.Insomecases,thesetechnologieshavealreadybeentransferredsuc-cessfullyintoindustrialscale.Recently,thefocushaswidenedtowardtheformationofcomposites.TechnologieswithSCFsofferanincreasednumberofpossibilitiestogeneratecompositeswithnewfunctionalities.ThefollowingsectionhighlightstwoexampleshowSCFcanbeusedtoproducesuchproducts.

12.3 ComPosItes wItH HIGH-Pressure sPray ProCesses

Forcommercialsuccess, ithasbecomemoreandmoreattractivetodesigntailor-madeparticlesystemsthatallow,forexample,thecontrolledreleaseofactiveagentsorofferdurableprotectionof sensitive ingredients.Classicalprocesses like spraydrying,crystallization,andin-situpolymerizationprocessesareinprincipleabletoproducesuchcomposites.Inspraydrying,thehightemperaturelevellimitsthetech-niquetoinsensiblesubstances.Incrystallizationprocesses,completeencapsulationishardtoachieveandtheparticleshapeisdifficulttocontrol.Forpolymerizationprocesses,onlyafewmaterialcombinationsaresuitable.Inthisfield,afewtech-niquesusingSCFsareestablishedandtheresultsareverypromising.TheseSCFtechnologies allow thegenerationofpowderswithproperties that aredifficult orevenimpossibletoachievebyclassicalmethods.

12.3.1 spRay agglomERation with a high-pREssuRE spRay pRocEss

The processes described above lead to reduced particle size of the raw material.SCFprocessesarenot limited toparticle size reduction.Different shape-formingmethodshavebeenestablished in the last fewyears (e.g.,coating,agglomeration,impregnation,anddispersionprocesses)[63,64].

TheCPFtechniqueisasprayagglomerationtechniquethatallowstheproduc-tionofliquid-loadedcompositeswithloadingsofupto90wt%.Theagglomeratesobtained have a high mechanical stability and good flow behavior. Figure12.7illustratestheflowschemeoftheCPFprocess.

The liquid to be powderized is dosed with a high-pressure pump from thestoragevesseltoastaticmixer.Here,asecondstreamofSCF,mostlyCO2,isadded.

7089_C012.indd 376 10/8/07 12:21:43 PM


Underpressuresofupto200bar,thegasandliquidaremixedandsubsequentlydepressurizedviaanozzletoatmosphericpressure.Thevolumeincreaseofthegasleads to theformationofveryfinedroplets.Temperatures in thespray towerarecontrolledbythepreexpansionconditionsinthestaticmixerandaretypicallyintherangeof–20°Cto60°C.Therefore,thistechniqueisespeciallysuitableforprocess-ingoftemperature-sensitiveorvolatilesubstances.Byaddingasolidcarrierwithapneumaticconveyingsystemintothespraytower,theliquiddropletsarebound.Solid,free-flowingagglomeratesareformedthatcanhaveamaximumof90wt%liquidcontent.Theliquidisboundbyadsorptiontothesurfaceofthecarrierandbycapillaryforcesbetweenthesingleparticlesintheagglomeratesoreveninporousstructuresofthesingleparticles(Figure12.8).Theformationofsuchagglomerateswastestedwithmanydifferentsubstances(e.g.,naturalextractsofbasil,pepper,lemonoil,α-tocopherol,whiskey).Silicicacid,celluloses,andstarcheswereusedascarriers.Inalloftheseexperiments,thefirsttaskwastogetfree-flowingpowdersthatcanbeeasilyhandledinpostprocessing.Inadditiontotheflowabilityoftheproducts,thereleaseoftheboundliquidisofimportance.Byvaryingthecarriermaterial, products with defined release behaviors can be achieved for the food,pharmaceutical,andcosmeticindustries.

Figure12.9 illustrates the controlled release of a CPF product [65]. For thisproduct, vitamin B2 was sprayed on a potato starch using the CPF technique.

Powder

Carrier

Gas

Powder

FIGure 12.7 CPFprocessscheme.

Adsorption Agglomeration Impregnation

FIGure 12.8 BindingmechanismofCPFproducts.

7089_C012.indd 377 10/8/07 12:21:45 PM


Afterward,theproductwaspouredwithwaterandwasmixedwithamagneticstirrerbarwithandwithoutheating.Thereleaseofthecoloredvitaminwasmeasuredbytheextinctionoftheaqueoussolution.Thelowerflatgraphindicatesthereleaseofthevitaminbymixingwithcoldwater.Onlysmallamountsofthecoloredvitaminare released. If thewater isheated, the releaseoccurs (as indicatedby theuppercurve).At thebeginning, it is comparable to theexperimentwithcoldwater,butwhenthetemperaturefinallyreaches80°C,anearlycompletereleaseisobvious.

12.3.2 liquid-fillEd compositEs with a high-pREssuRE spRay tEchnology

BasedonthePGSSprocess,ahigh-pressurespraytechniquewasinvestigatedthatallowstomanufacturecompositesconsistingofacorematerial,whichcouldbealiquidorasoliddispersedinaliquid,andashellmaterialthatmustbesolidunderstorage conditions [66–68].Aprincipleflowschemeof thisprocess is illustratedinFigure12.10.Bothcomponentshavetobeprovidedinapumpableform.High-pressurepumpsareusedtofeedtheshellmaterialsandtheliquidcorematerialstothestaticmixer,wherethecomponentsaredispersed.Inaddition,aSCFispumpedintothemixer.Dependingonthesystemandthemixersize,amoreorlessstabledispersion isobtained.Subsequently, thisdispersion isdepressurized intoaspraytower.Theshellmaterialsolidifiesduetotemperaturereductionoftheexpandinggas.Thecorematerialisencapsulatedintheshell.Figure12.11showsinprinciplethemorphologiesthatcanbeobtainedontheonesidewithaliquiddispersedinameltandontheothersidewithadispersionoftwoimmisciblemelts.Oneadvantageofthistechniqueisthatthedispersionoremulsionsformedinthestaticmixercanbeeitherstableorunstable.Theresidencetimeaftermixingisextremelyshort(somemillisecondstoseconds)sothataphasesplitdoesnotoccurbeforeexpansion.Aftertheexpansion,solidificationoftheshellmaterialhappensinstantly;thedispersionisstabilizedbysolidification.

As an example for the manufactured composites, three scanning electronmicroscope (SEM) pictures of a solid wax/liquid PEG composites are shown inFigure12.12.Thelightergrayregionsconsistofwax;thedarkregionsinthepicturesshowtheboundliquidPEG.Intherangeof50to60wt.-%,achangefromclosedtoopencompositesisobserved.

0

20

40

60

80

100

0 20 40 60 80 100 120t(min)

Cont

rol R

elea

se (%

)

Complete Release at 80°C By Mixingand Heating

Release at 25°C byMixing

FIGure 12.9 Temperature-triggeredreleaseofaCPFproduct.

7089_C012.indd 378 10/8/07 12:21:46 PM


Components

Gas

PowderousComposites

A B

FIGure 12.10 Processschemeofcompositeprocess.

Solid-liquid DispersionEncapsulated Microdroplets

Solid-solid Dispersion

FIGure 12.11 Morphologiesofcomposites.

54 wt.–% 20 µm 20 µm 20 µm

57 wt.–% 64 wt.–%

FIGure 12.12 SEMpicturesofcompositeparticles(wax/PEGsystem).

7089_C012.indd 379 10/8/07 12:21:49 PM


Thesurfaceofthesphericalcompositeswith54wt.-%ofPEGontheleft-handside of Figure12.12 is completely closed and uniform. No darker regions, whichwould indicate the presence of liquid PEG on the surface, can be detected. Byincreasingtheamountof liquidPEG,whichmightnotsufficientlybeadmixedinthestaticmixer,thesurfaceofthesphericalcompositesisstillclosedanduniform.Nevertheless, in thatcase, free liquidPEGmaycoexistwithdroplets thatconsistofwaxanddispersedPEG.Thefreeliquidisboundbycapillaryforcesinbetweenthesolidifiedwaxparticles.AgglomeratesareformedwithdispersedPEGencapsu-latedinthewaxandPEGascapillaryliquidbetweenthewaxparticles.Byfurtherincreaseoftheliquidcontent,thevolumefractionoftheshellmaterialistoolowtoallowcompleteencapsulation.Thephotographontheright-handsideshowsapar-ticlewithanopenstructure,wheretheliquidPEGisboundinporesofthewax.

The morphologies of the composites show that agglomerates, single particlesas well as closed and open-structured composites, can be produced. The highestconcentrationofPEGthatstillallowstheformationofcompletelyclosedcompositeswasapproximately60wt.-%.Arisingconcentrationoftheliquidfavorsthegenera-tionofopencomposites.Thiscanbeunderstoodbyfocusingonthebasicsofparticleformation.Toformacomposite,theliquidhastobeadmixedtotheshellmaterialasthedispersedphaseofanemulsion.Subsequently,themixtureissprayedandsolidi-fiedusinganexpandinggas.Themainfactorforthegenerationofclosedoropencompositesisthedifferencebetweenthespeedofsolidificationandphasesepara-tionoftheemulsion.Anincreasingamountofliquidleadstoarisingdropdiameterofthedispersedphaseorarisingnumberofdisperseddroplets.Arisingnumberofdisperseddropletsleadstoanacceleratedbreakageoftheemulsion.Withconstantprocessparameters(i.e.,temperatureandgastoproductratio),thesolidificationtimewillbecomparablebuttheseparationoftheemulsionismuchfaster.Thisresultsinthegenerationofopencomposites.

Theprocessdescribedabovehasalreadybeensuccessfullyappliedtodifferentproductsinthechemical,food,andcosmeticindustries.Waterhasbeenencapsulatedinfat;liquidaromasandantioxidantshavebeenboundinafatmatrixtoreducethelosses during storage, different vegetable oils have been encapsulated in PEGS, aparaffinwaxhasbeenboundinpolyester,andkirschwasencapsulatedinachocolatematrix[69,70].Themicronizedchocolatewiththeencapsulatedkirscharomacouldbeused,forexample,toenhancetheflavorofhotcocoaortobringadditionalaromaintoanychocolateproduct.Thereleaseofthearomaforthesediffersfromproductsonthemarketwhereliquorsareencapsulatedinmacroscopicstructures,likepralines.Theverysmallsizeofthechocolateparticles(some10tosome100microns)leadstoimmediatemeltinginthemouth.Thereby,thearomasofchocolateandkirscharereleasedtogethertoformaflavorthatcombinesthebestofboth(Figure12.13).

12.4 ProCessInG oF nutraCeutICals wItH suPerCrItICal FluId teCHnoloGy

Manyoftheprocessesdescribedabovearedesignedandoperatedtosubstituteoneclassicalprocesstask(e.g.,milling).Togainlargerbenefits,differentprocesstasks

7089_C012.indd 380 10/8/07 12:21:49 PM


canbecombinedandsolvedinoneSCF-assistedprocess[71,72].Table12.1givesanoverviewofsomenutraceuticalsprocessedwithSCFs.

SCFtechnologyistypicallyalow-temperaturetechniqueandisnormallycarriedout in completely inert atmospheres. Therefore, these techniques are especiallysuitableforthermo-andoxygen-sensitivesubstances.Greenteaandespeciallypoly-phenolextractsfromgreentealeavesarewidelyusedinnutraceuticalapplications.Polyphenols,substancesknowntostabilizeoilandfatproducts,areantioxidantsthathavebeendiscussedforcancerpreventionandfordentalcariesprevention,tonamejusttwopositiveeffects.Toisolatethesepolyphenolsfromgreentealeaves,awaterextractionismade.Afterfiltration,thisaqueousextractisdriedwithclassicalspraydrying techniques. Inspraydrying,high temperaturesarenecessary toevaporatewater.Inaddition,mostspraydryersworkwithheatedairandthereforethepoly-phenolsmaysufferfromthermalandoxidativestressduringprocessing.Resultantgreen teaproductsmaycontain lower concentrationsof antioxidants thancanbeachievedwithgentlerprocessing.

OnepossibilityforobtainingsolidgreenteaproductswithoutdegradationoftheantioxidantsistouseSCFtechnology.Therefore,aprocessthatcombinesthedryingstepofaqueousgreenteaextractswithparticleformationwasdesigned.TheflowschemeofthisprocessispresentedinFigure12.14.Thegreenteaextractusedforthedryingandpulverizationexperimentswasobtainedbyanextractionperformedat 60°C, by mixing 1 kg of extract in 10 kg of deionized water for 15 minutes.Thisextract isdosed toavesselbyahigh-pressurepumpthroughastaticmixer.Here,preheatedCO2isaddedunderelevatedpressures.Theresidencetimeinthemixerisextremelyshort(<1sec);therefore,itispossibletoraisethetemperatureabove100°C,evensometimesashighas180°C,withoutdegradationoftheproduct.Subsequently, themixture isdepressurized intoa spray towerand thusquenched

FIGure 12.13 Chocolate–kirschcomposite.

7089_C012.indd 381 10/8/07 12:21:50 PM


immediatelytolowtemperatures.Finedropletswithlargesurfacesareformed.ThewaterisextractedalreadyinthestaticmixerathightemperaturesoristakenupbythedryCO2afterexpansionand,finally,solidgreenteaextractisprecipitatedinthespraytower.TheobtainedgreenteapowdersareshowninFigure12.15.Theevapo-ratedwatercanbewithdrawnwiththeexpandedCO2.

Table12.2showsthepolyphenolconcentrationsandwatercontentofthediffer-entproducts.Therawmaterial(tealeaves)hashadawatercontentof2.97weight%.Thegroundleaveswereextractedwithwater(leaves:water/1:10[g/g]),andthewaterextractwasdriedwithalow-temperaturevacuumevaporation(40°C)andwiththeSCFprocess.ThemainprocessparametersofthesupercriticaldryingprocessareshowninTable12.3.Theresidualwatercontentinthevacuumprocessafter6hourswasdeterminedto8.82weight%.InSCFprocessing,5.09weight%wasobtained.Thepolyphenolconcentrationafterwaterextractioncouldbeincreasedforallthreetypesofpolyphenolscomparedwiththerawmaterial.ThePGSSdryingstepshowsthesameorslightlyhigherconcentrationsofpolyphenolsthanthewaterextractdried

table 12.1nutraceuticals Processed with supercritical FluidsProcess substance reference

RESS Benzoicacid [76]

Ibuprofen [77,78]

Aspirin [79]

Caffeine [76]

Griseofulvin [38,80]

Lidocaine [81]

GAS Mefenamicacid [82]

Copper-Inomethacin [78,83]

Insulin [84–86]

Paracetamolandascorbicacid [47]

β-carotene [87]

SAS Amoxycilin [25,88]

Dextran,cholesterol [89–91]

Inulin [92]

Lecithin [93]

Organicpigments [94]

PGSS Felodipine [55]

Glucose [79]

Albuterolsulphate [79,95]

Cromolynsodium [79,95]

Glucoseoxidase [96]

CPF Flavourextracts [24,97]

Emulsions [98]

7089_C012.indd 382 10/8/07 12:21:50 PM


FIGure 12.15 PictureandSEMofpowderousgreenteaextract.

table 12.2Polyphenol Content at different Process steps

Process step

water Content (%) Polyphenols (g/100 g dry raw material)

residue (%)epicatechin

(eC) epigallocatechin-Gallate (eGCG)

epicatechin-Gallate (eCG)

Rawmaterial 2.97 0.97 3.92 1.41

Waterextract(1:10)vacuumdriedforanalyses

8.82 2.31 4.07 1.50

Supercriticalfluiddried

5.09 2.16 4.90 1.70

Gas

Green Tea

Gas + Solvent

Green Tea Solution

FIGure 12.14 Greenteaprocessingwithsupercriticalfluids.

7089_C012.indd 383 10/8/07 12:21:51 PM


invacuum.ThisdemonstratesthatduringprocessingwiththeSCF,nopolyphenols(exceptasmalldecreaseoftheepicatechinconcentration)weredegradedcomparedwithavacuumdryingprocess.Inaddition,SCF-assistedtechnologyallowsthepro-ductionofparticulatesystemswithlargesurfacesthatcanbeeasilyredissolvedinwater.Particlemorphologyandparticlesizecanbeadjustedbyvaryingtheprocessparameters[73–75].Invacuumdrying,abulkyproductisobtainedthathastobegrindedtogetfineparticles.

12.5 ConClusIons

In the last15 to20years,numerousprocessesforparticlegenerationusingSCFshavebeenproposed and applied for substances from the food, polymer, pharma-ceutical,lifescience,andnutraceuticalindustries.Themainfocusoftheseapplica-tionshasbeenon themicronizationofpure substances.The thermodynamicandfluid-dynamic properties of certain single-component model systems (e.g., PEGs,triglycerides,naphthalene)inthepresenceofcompressedgases,mostlyCO2,havebeenstudiedintensively.Thisfundamentalresearchhasledtoanimprovedunder-standingoftheprocessesforparticlegeneration.Asaresult,high-pressuretechnologybecomesmoreandmoreestablished in industry.Meanwhile,high-pressureplantswithcapacitiesofsomegramsperhour tosomehundredkilogramsperhourcanbedesignedandbuiltbyseveralspecializedplantconstructors.Costanalysisshowsthatthesetechniquescanbecompetitivetoclassicalmicronizationtechniques.SCFsnotonlyopennewpossibilitiesforprocessingsubstancesthataredifficulttohandle(e.g., substanceswith lowmeltingpoints, highviscosities, or sticky surfaces) butalsoallowgenerationofcompositeswithcustomizedproperties.Thesenewchancesmotivateresearchforimprovedunderstandingoftheprocessesandthedevelopmentofnewproducts.

reFerenCes

1. Reverchon, E., Micro- and nano-particles produced by supercritical fluids assistedtechniques:Presentstatusandperspectives,Chem. Eng. Trans.,2,1,2002.

2. Jung,J.andPerrut,M.,Particledesignusingsupercriticalfluids:Literatureandpatentsurvey,J. Supercrit. Fluids,20,179,2001.

3. Knez,Z. andWeidner,E.,Precipitationof solidswithdensegases, inHigh Pressure Process Technology: Fundamentals and Application,Bertucco,A.andVetter,G.,Eds.,Elsevier,2002.

table 12.3Conditions during High-Pressure spray ProcessMassflowofgas(kg/hr) 54.5 Textract(°C) 23

Massflowofsol.(kg/hr) 1.4 Tbeforeexpansion(°C) 125

GSR(gastosolutionratio) 38.9 pbeforeexpansion(bar) 73

Nozzlediameter[mm] 1.4 Ttower(°C) 64

msprayedsolution(kg) 5.1 ptower(bar) 1.0

7089_C012.indd 384 10/8/07 12:21:52 PM


4. Weidner,E.,Powderouscompositesbyhighpressuresprayprocesses,inProceedings of the Sixth International Symposium on Supercritical Fluids,Versailles,France,2003,3,1483.

5. Gamse,T.,Schwinghammer,S.andMarr,R.,Erzeugungfeinsterpartikeldurcheinsatzvonüberkritischenfluiden,Chem. Ing. Techn.,77,669,2005.

6. Türk,M.etal.,Stabilizationofpharmaceuticalsubstancesbytherapidexpansionofsupercriticalsolutions(RESS),inProceedings of the Sixth International Symposium on Supercritical Fluids,Versailles,France,2003,3,1747.

7. Reverchon, E., Supercritical-assisted atomization to produce micro- and/or nano-particlesofcontrolledsizeanddistribution,Ind. Eng. Chem. Res.,41,2405,2002.

8. Reverchon,E.andDellaPorta,G.,Micronizationofantibioticsbysupercriticalassistedatomization,J. Supercrit. Fluids,26,243,2003.

9. Shariati, A. and Peters, C.J., Measurements and modeling of the phase behavior ofternary systems of interest for the GAS process: I. The system carbon dioxide+1-propanol+salicylicacid,J. Supercrit. Fluids,23,195,2002.

10. Yeo,S.,Kim,M.andLee,J.,Recrystallisationofsulfathiazoleandchlorpropamideusingthesupercriticalantisolventprocess,J. Supercrit. Fluids,25,143,2003.

11. Ventosa,N.,Sala,S.andVeciana,J.,DELOSprocess:Acrystallisationtechniqueusingcompressedfluids:1.Comparison to theGAScrystallizationmethode,J. Supercrit. Fluids,26,33,2003.

12. Sun,X.etal.,Thecharacteristicsofcoherentstructuresintherapidexpansionflowofthesupercriticalcarbondioxide,J. Supercrit. Fluids,24,231,2002.

13. Hanna,M.andYork,P.,PatentWO95/01221,1994. 14. Weidner,E.etal.,Manufactureofparticlesfromhighviscousmeltsusingsupercritical

fluids, reviewed.Proc.“HighPressureinVenice”,ISBN88-900775-1-4,2,735,2002. 15. Peter,S.etal.,Interfacialtensioninbinarysystemscontainingadensegas,inSupercrit-

ical Fluids: Fundamentals for Application,Kiran,E.andLevelt-Sengers,J.M.H.,Eds.,Boston,KluwerAcademicPublishers,731,1994.ProceedingsoftheNATOAdvancedStudyInstituteonSupercriticalFluid—FundamentalsforApplication,Kemer,Antalya,Turkey,July18–31,1993;NATODordrecht,KluwerNATOASISeries:SeriesE,273.

16. Jakob,H.,Peter,S.andWeidner,E.,DieViskositaetkoexistierenderPhasenbeiderueberkritischenFluidextraktion,Chem. Ing. Techn.,59,37,1987.

17. Jaeger,P.,Eggers,R.andBaumgartl,H.,Interfacialpropertiesofhighviscousliquidsinasupercriticalcarbondioxideatmosphere,J. Supercrit. Fluids,24,203,2002.

18. Gulari,E.etal.,Rheologyofmoltenpolystyrenewithdissolvedsupercriticalandnear-criticalgases,J. Polym. Sci.,37,2771,1999.

19. DeSimone,J.M.etal.,Viscosityeffectsonthethermaldecompositionofbis(perfluoro-2N-propoxypropionyl) peroxide in dense carbon dioxide and fluorinated solvents,J. Am. Chem. Soc.,123,30,7199,2001.

20. Petermann,M.,Herstellung von Pulverlacken durch Verspruehung gashaltiger Schmel-zen, Ph.D.Thesis,Erlangen,1999.

21. Kayrak, D., Akman, U. and Hortacsu, Ö., Micronization of Ibuprofen by RESS,J. Supercrit. Fluids,26,17,2003.

22. Kappler,P. et al.,Sizeandmorphologyofparticlesgeneratedby sprayingpolymer-meltswithcarbondioxide, inProceedings of the Sixth International Symposium on Supercritical Fluids,Versailles,France,2003,1891.

23. Sievers,R.E.etal.,Micronizationofwater-solubleoralcohol-solublepharmaceuticalsandmodelcompoundswithalow-temperaturebubbledryer,J. Supercrit. Fluids,26,9,2003.

24. Grüner,S.,Otto,F.andWeinreich,B.,CPF-technology:Anewcryogenicsprayingpro-cessforpulverizationofliquid,inProceedings of the Sixth International Symposium on Supercritical Fluids,Versailles,France,2003,1935.

7089_C012.indd 385 10/8/07 12:21:52 PM


25. Reverchon, E. et al., Pilot scale micronization of amoxicillin by supercritical anti-solventprecipitation,J. Supercrit. Fluids,26,1,2003.

26. Best,W.etal.,GermanPatent2943267,BASF AG,1979. 27. Subramaniam,B.etal.,PatentWO97/31691. 28. Krause,H.,Niehaus,M.andTeipel,U.,GermanPatentDE19711393,1998. 29. Türk, M., Erzeugung von organischen Nanopartikeln mit ueberkritischen Fluiden,

Habilitationsschrift,UniversityKarlsruhe,2001. 30. Türk, M. et al., Micronization of pharmaceutical substances by rapid expansion of

supercriticalsolutions(RESS):Experimentsandmodelling,Part. Syst. Charact.,19,327,2002.

31. Reverchon,E.andDonsi,G.,SalycylicacidsolubilizationinsupercriticalCO2anditsmicronisationbyRESS,J. Supercrit. Fluids,6,241,1993.

32. Türk, M. et al., Micronisation of pharmaceutical substances by rapid expansion ofsupercriticalsolutions(RESS):Apromisingmethodtoimprovethebioavailabilityofpoorlysolublepharmaceuticalagents,J. Supercrit. Fluids,22,75,2002.

33. Helfgen,B.,Türk,M.andSchaber,K.,Hydrodynamicandaerosolmodellingof therapidexpansionofsupercriticalsolutions(RESS),J. Supercrit. Fluids,26,225,2003.

34. Diefenbacher,A.andTürk,M., Phaseequilibriaoforganicsolidsolutesandsupercriti-calfluidswithrespecttotheRESSprocess,J. Supercrit. Fluids,22,175,2002.

35. Orlovic,A.andSkala,D.,Materialsprocessing,usingsupercriticalfluids,Hem. Ind.,59,213,2005.

36. Sun, Y.P. et al., Polymeric nanoparticles from rapid expansion of supercritical fluidsolution,Chem. Eur. J.,11,1366,2005.

37. Moribe,K.,MicronisationofphenylbutazonebyrapidexpansionofsupercriticalCO2solution,Chem. Pharm. Bull.,53,1025,2005.

38. Türk,M.et al.,Micronizationofpharmaceutical substancesby the rapidexpansionofsupercriticalsolutions(RESS):Apromisingmethodto improvebioavailabilityofpoorlysolublepharmaceuticalagents,J. Supercrit. Fluids,22,75,2002.

39. Sane,A. andThies,M.C.,The formationoffluorinated tetraphenylporphyrinnano-particlesviarapidexpansionprocess:RESSvsRESOLV,J. Phys. Chem. B,109,19688,2005.

40. Türk,M.,Herstellungorganischernanopartikelnundderenstabilisierunginwässrigenlösungen(RESSAS),Chem. Ing. Tech.,75,792,2003.

41. Türk,M.andWahl,M.,Utilizationofsupercriticalfluidtechnologyforthepreparationofinnovativecarriersloadedwithnanoparticulardrugs,2004 International Congress for Particle Technology (PARTEC).Ed.S.E.Pratsinis,Nueremberg,2004.

42. Türk,M.,UntersuchungenzumCoatingvonsubmikronenPartikelnmitdemCORESS-Verfahren,Chem. Ing. Techn., 76,3,2004.

43. Thakur,R.andGupta,R.B.,Formationofphenytoinnanoparticlesusingrapidexpan-sionofsupercriticalsolutionwithsolidcosolvent(RESS-SC)process,Int. J. Pharm.,308,190,2006.

44. Kümmel,R.,Weiß,C.andBertling,J.,Smartmaterials:Newwaystomicrostructuredparticlesofenvironmentalconcern,Adv. Environ. Mat.,II,175,2001.

45. Weber, A. and Kümmel, R., Nanoparticles by precipitation in supercritical fluids –possibilitiesandlimitations,inProceedings of the Seventh Meeting on Supercritical Fluids,Antibes,France,2001,49.

46. Weber,A.etal.,Aproductionplantforgasantisolventcrystallization,inProceedings of the Fifth Meeting on Supercritical Fluids,Nice,France,1998,281.

47. Weber, A., Tschernjaew, J. and Kümmel, R., Coprecipitation with compressed anti-solventsforthemanufactureofmicrocomposites,inProceedings of the Fifth Meeting on Supercritical Fluids, I,1999,243.

48. Krause,H.,Niehaus,M.andTeipel,U.,GermanPatent19711393,1998.

7089_C012.indd 386 10/8/07 12:21:53 PM


49. Yeo,S.etal.,Supercriticalanti-solventprocessforaseriesofsubstitutedpara-linkedaromatic polyamides: phase equilibria and morphology, Macromolecules, 26, 6207,1993.

50. Berends,E.M.etal.,CrystallizationofphenanthrenefromtoluenewithcarbondioxidebytheGASprocess,AIChE J.,42,431,1996.

51. PerezdeDiego,Y.et al.,Openingnewoperatingwindows forpolymerandproteinmicronisationusingthePCAprocess,J. Supercrit. Fluids,36,216,2006.

52. Thiering, R., Dehghani, F. and Foster, N.R., Current issues relating to anti-solventmicronisationtechniquesandtheirextensiontoindustrialscales,J. Supercrit. Fluids,21,159,2001.

53. Weiß,C.etal.,Modelingmasstransferandcrystallizationindispersesystems,Chem. Eng. Technol.,23,485,2000.

54. Weidner, E., Powder generation by high pressure spray processes, in Proceedings of the International Meeting on High Pressure Chemmical Engineering,Karlsruhe,WissenschaftlicheBerichteFZKA6271,1999,217.

55. Knez,Z.,Micronisationofpharmaceuticalsusingsupercriticalfluids,inProceedings of the Seventh Meeting on Supercritical Fluids,Antibes,France,1,21,2000.

56. Weidner,E.,Petermann,M.andBlatter,K.,Dieherstellungvonpulverlackendurchversprühengashaltigerschmelzen,Chem. Ing. Techn.,72,743,2000.

57. Münüklü,P.,Particle Formation of Ductile Materials using the PGSS Technology with Supercritical Carbon Dioxide, Ph.D.Thesis,UniversityDelft,Netherlands,2005.

58. Weidner,E.,Petermann,M.andKnez,Z.,Multifunctionalcompositesbyhighpressuresprayprocesses,in Current Opinion in Solid State and Material Science,Netherlands,Elsevier,385,2004.

59. Weidner,E.,Powderouscompositesbyhighpressuresprayprocesses,inProceedings of the Sixth International Symposium on Supercritical Fluids,Versailles,France,1483,2003.

60. Rantakylä,M.,Particle Production by Supercritical Antisolvent Processing Techniques, PhDThesis,HelsinkiUniversityofTechnology,Helsinki,2004.

61. Weber,A.etal.,Aproductionplantforgasantisolventcrystallization,inProceedings of the Fifth Meeting on Supercritical Fluids,Nice,France,1998,3,281.

62. Tuerk, M., Erzeugung von organischen Nanopartikeln mit überkritischen Fluiden,Habilitationsschrift,Karlsruhe,2001.

63. Grüner, S., Entwicklung eines Hochdrucksprühverfahrens zur Herstellung hoch-konzentrierter, flüssigkeitsbeladener Pulver, Ph.D. Thesis, University Erlangen,Germany,1999.

64. Grüner,S.etal.,CPF-Processforpowdergenerationfromliquids,inProceedings of the Fifth Conference on Supercritical Fluids and Their Applications, Garda, Italy,1999,471.

65. Grüner, S. and Otto, F., Adalbert Raps Forschungszentrum, Unpublished Data,Weihenstephan,2005.

66. Brandin, G. et al., Tailor-made composites by high pressure spray processes, inProceedings of the Fifth World Congress on Particle Technology,Orlando,FL,2006.

67. Weidner,E.etal.,Fluid-filledmicroparticlesusingPGSStechnology,inProceedings of the Third International Meeting on High Pressure Chemical Engineering,Erlangen,Germany,2006.

68. Brandin,G.,Herstellung pulverförmiger Komposite mittels Hochdrucksprühverfahren, Ph.D.Thesis,Ruhr-University,Bochum,2005.

69. Brandin, G. et al., Fluid-filled micro particles by the use of the PGSS technology,Advanced Powder Technology,acceptedforpublication,2006.

70. Weidner, E. et al., Production of powdery phase change materials using the PGSSprocess,J. Supercrit. Fluids, notyetpublished,2006.

7089_C012.indd 387 10/8/07 12:21:53 PM


71. Weidner,E.etal.,Manufactureofparticlesfromhighviscousmeltsusingsupercriticalfluids,rev. Proc. “High Pressure in Venice”,ISBN88-900775-1-4,2,735,2002.

72. Petermann,M.etal.,MicronizationofpolymersolutionsbyPGSS-drying,inProceed-ings of the Eighth Conference on Supercritical Fluids and Their Applications,Ischia,Italy,2006.

73. Weidner, E., Final report: Product engineering with neutraceuticals with superiorquality,PRONUTRA,EU-Project,GRD1-1999-10212,2003.

74. Meterc, D. et al., Extraction of natural substances and drying with PGSS process,Slovenian Chemical Days 2006,Maribor,2006.

75. Meterc,D.,Ph.DThesis,Ruhr-UniversityBochum,inpreparation. 76. Reverchon,E.,DellaPorta,G.andFalivene,M.G.,Processparameterscontrollingthe

supercriticalanti-solventmicronizationofsomeantibiotics,inProceedings of the Sixth Meeting on Supercritical Fluids, Chemistry and Materials, Nottingham,UK,1999.

77. Chroenchaitrakool,M.etal.,MicronizationbyRESStoenhancethedissolutionratesof poorly water soluble pharmaceuticals, in Proceedings of the Fifth International Symposium on Supercritical Fluids,Atlanta,GA,2000.

78. Foster, N.R. et al., Application of dense gas techniques for the production of fineparticles, AAPS PharmSci,5,2,105–111,2003.

79. Manning,M.C.etal.,U.S.Patent5770559,1998. 80. Martin,H.-J.etal.,Nanoscaleparticlesforpharmaceuticalpurposebyrapidexpansion

ofsupercriticalsolutions(RESS).PartII:Characterizationoftheproductanduse,in Proceedings of the Seventh Meeting on Supercritical Fluids,Antibes,France,2000,1,53.

81. Frank, S.G. and Ye, C., Small particle formation and dissolution rate enhancementof relatively insolubledrugsusingrapidexpansionofsupercritical solutions (RESS)processing, in Proceedings of the Fifth International Symposium on Supercritical Fluids (CD-ROM),2000.

82. Foster,N.R.etal.,Processingpharmaceuticalsusingdensegastechnology,Proceed-ings of the Fifth International Symposium on Supercritical Fluids (CD-ROM),2000.

83. Warwick, B. et al., Micronization of copper-indomethacin using gas anti-solventprocesses,Ind. Eng. Chem. Res.,41,8,1993–2004,2002.

84. Thiering,R.,Dehghani,F. andFoster,N.R.,Micronizationofmodel proteinsusingcompressedcarbondioxide,inProceedings of the Fifth International Symposium on Supercritical Fluids,Atlanta,2000.

85. Thiering,R.etal.,Theinfluenceofoperatingconditionsonthedensegasprecipitationofmodelproteins,J. Chem. Technol. Biotechnol.,75,29–41,2000.

86. Thiering,R.etal.,Solventeffectsonthecontrolleddensegasprecipitationofmodelproteins,J. Chem. Technol. Biotechnol.,75,42–53,2000.

87. Cocero,M.J.,Ferrero,S.andVicente,S.,GAScrystallizationofβ-carotenefromethylacetatesolutionsusingCO2asantisolvent, inProceedings of the Fifth International Symposium on Supercritical Fluids,Atlanta,2000.

88. Reverchon,E.etal., inProceedings of the Sixth Conference on Supercritical Fluids and Their Applications,Maiori,Italy,2001,301.

89. Subra,P.andVega,A.,inProceedings of the 15th International Congress on Chemical and Process Engineering,CHISA2002,Prague,CzechRepublic,2002.

90. Pellikaan,H.C.andWubbolts,F.E.,Nozzelconstruction forparticle formationusingsupercriticalantisolventprecipitation,in Proceedings of the Sixth International Sympo-sium on Supercritical Fluids,Versailles,France,2003,1765.

91. Reverchon,E.etal.,Supercriticalfluidantisolventmicronisationofsomebiopolymers,J. Supercrit. Fluids,18,239,2000.

7089_C012.indd 388 10/8/07 12:21:54 PM


92. Jung,J.,Clavier,J.Y.andPerrut,M.,Gramtokilogramscale-upofsupercriticalanti-solventprocess,inProceedings of the Sixth International Symposium on Supercritical Fluids,Versailles,France,2003,1683.

93. Magnan, C. et al., Soy lectin micronisation with a compressed fluid antisolvent—influenceofprocessparameters, J. Supercrit. Fluids,19,69,2000.

94. Nagahama,K.andKamoshita,C.,inProceedings of the Fourth International Sympo-siom on High Pressure Process Technology and Chemical Engineering,Venice,Italy,2002.

95. Sloan,R.etal.,Supercriticalfluidprocessing:Preparationofstableproteinparticles,in Proceedings of the Fifth Meeting on Supercritical Fluids,Nice,France,1998,1,301.

96. Reverchon,E.,Supercriticalanti-solventprecipitation:Itsapplicationtomicroparticlegenerationandproductsfractionation,inProceedings of the Fifth Meeting on Super-critical Fluids,Nice,France,1998,1,221.

97. Petermann, M. et al., CPF—Concentrated powder form—A high pressure sprayagglomerationtechnique,in Proceedings of Spray Drying 01 Conference,Dortmund,Germany,2001,143.

98. Wehowski,M.andWeidner,E.,Water-containingagglomeratesbyhighpressurespray-ingaccording to theconcentratedpowder form(CPF)process, CIT,77,3,274–278,2005.

7089_C012.indd 389 10/8/07 12:21:54 PM

7089_C012.indd 390 10/8/07 12:21:54 PM

391

Indexα-Carotene, sources of, 281α-Linolenic acid, 57, 78α-Tocopherol, 113, 115β-Carotene Chrastil parameters of from fish oil, 160 fruit and vegetable oil extraction and, 86 overview of, 56, 196–197 physical properties of, 53 separation of isomers of, 196–198 sources of, 281β-Cryptoxanthin, 281

AAbsorption, 26Acetone, 34Acorns, 63, 70Activity coefficients, 13Adlay seed oil, 223–224Adsorption chromatographic separations and, 156 concentrated powder form process and, 377 extraction process and, 26, 327–328 liquid feed extraction and, 321 procyanidin extraction and, 233 solute separation and, 218Aerosol solvent extraction systems (ASES),

372–373Agglomeration, 371, 376–378, 380Aging, free radical theory of, 276–277Aglycons, 280Aguaribay, 245Ajwain, 355Algae, 192. See also MicroalgaeAlkaloids, 342Almonds, 52, 57, 59, 62, 63, 66, 69–71Amaranthus grain, 80, 81, 85Ammonia, 3Andreadoxa, 249Anethole, 261, 314Angelica sinensis, 228–230Anise, 261Annatto, 245Anthocyanadins, 280Anthocyanins, 342Anticaking agents, 343Antimicrobial activity, 285Antioxidants A. sinensis, L. chuanxiong hort and, 228 carotenoids as, 281, 284 conventional solvent extraction of, 292–293

determination of activity of, 285–286 effect of pressure and temperature on

extraction of, 289–292 lycopene as, 56 overview of, 275–276 overview of natural, 277–282 phenolics as, 280, 282–283 SC-CO2 extraction of, 286–292 spices and, 342 terpenoids as, 280–281, 283 tocols and, 59–60 types and regulation of, 276–277 vitamin E as, 281–282, 284Antisolvent extraction. See Supercritical

antisolvent extractionAntisolvent processes, 369, 372–375.

See also Specific processesApricots, 73, 79Aqueous alkaline extraction, 346Arachidonic acid, 57Arnica, 249Aroeira, 249Aromatic compounds, 11, 245–247Arruda de serra, 249Artemisia, 249Artemisinin, 312Arteriosclerosis, 142Arthritis, 142–143Arthrospira (Spirulina) spp., 205–209ASES. See Aerosol solvent extraction systemsAstaxanthin, 193, 195, 198–201ATBC study, 56Atherosclerosis, 56, 60, 228Atomization, 33–34Avocado, 249Ayurveda, 338

BBaccharia, 249Bacuri, 244, 248Bamboo piper, 245Basil, 245, 249Batch reactors, 20Bergamot peel oil, 328BHA. See Butylated hydroxyanisoleBHC. See HexachlorocyclohexanesBHT. See Butylated hydroxytolueneBinaries behavior, 15–16Binding, 377Binodal curves, 19

7089_Index.indd 391 10/8/07 11:48:22 AM

392 SupercriticalFluidExtractionofNutraceuticalsandBioactiveCompounds

Bioethanol, 15Bioflavonoids, 342Biofuels, 126, 192Black mustard, 345Black oreo, 147, 148Black pepper bioactive compounds from, 244, 245 biologically active constituents of, 345 extraction of bioactive compounds from, 359 solubility of oils in SC-CO2 and, 363, 364Black wattle, 249Blends, 346Blowout discs, 41Boldo, 249Borage, 73, 79Botrycoccus braunii, 191–193Brazil, 244Brazilian ginseng, 249, 261Breakage, 324Broken-intact cells, 324, 325–326Buriti fruit, 87, 248Bushy lippia, 245Butylated hydroxyanisole (BHA), 276, 277Butylated hydroxytoluene (BHT), 276, 277

CCacao, 249Caking, 343Calcium stearate, 343Campesterol, 58Camphor, 313Cancers adlay seed oil and, 223–224 carotenoids and, 284 lycopene and, 56–57 omega-3 fatty acids and, 142–144 phenolics and, 282, 283 sitosterol and, 59 squalene and, 58 terpenoids and, 283 tocols and, 60 Vitamin A and, 146 vitamin E and, 284Candida antarctica lipase, 158Canthaxanthin, 193, 195, 200, 281Cap automation mechanisms, 29–30Caprylic acid methyl ester, 78Capsaicin, 252, 342, 356–357Capsules, 52Caraway, 360–361Cardamon, 344, 359–360, 361Cardiovascular disease carotenoids and, 284 phenolics and, 282 polyunsaturated fatty acids and, 57 terpenoids and, 283

tocols and, 60 vitamin E and, 284CARET study, 56Carnahan-Starling equation, 7Carotenes, 56, 193. See also β-CaroteneCarotenoids algae and, 193–195, 198–201, 206–209 as antioxidants, 280, 281 biological properties of, 284 cosolvents and, 89 fruit and vegetable oil extraction and, 86 rice germ extraction and, 84 solubility of in SC-CO2, 290 in specialty oils, 56–57 spices and, 342Carrier materials, extraction process and, 26Carrots, 87, 90Caryophyllene, 313, 316Cascading extraction vessels, 29Cashews, 250Cassia, 344Catalysts, 105–106Catechins, 280Celery seed extraction, 354–356Cell cycle, 284Cellular location, 324Cellulosic structure, 252–254Cereal oils, 80–84Chamazulene, 314Chamomile bioactive compounds from, 245 composition of essential oil from, 315–316 essential oil extraction from, 314 overall extraction curve for, 259Charge time, 29–30Cherry, 58, 72, 73, 79, 80Chile, 244Chilean hops, 250Chili extracts, 356–357Chili peppers, 346Chlorella vulgaris, 192, 193–196, 202Chocolate-kirsch composites, 380, 381Cholesterol, 58, 59, 145, 338Chrastil correlation, 159, 160, 363–364Chromatography gas chromatograph with electrical conductivity

detector analysis, 233–234 high-speed countercurrent, 224 overview of, 156 supercritical fluid, 36–37, 173–175Chromobacterium viscosum lipase, 157Cinnamon, 341, 344, 355, 362Citronella, 245Cleaning-in-place, 35Cloudberries, 87Clove bud oils, 225–228, 244, 245, 314–316

7089_Index.indd 392 10/8/07 11:48:22 AM

Index 393

Cloves biologically active constituents of, 344 cost of manufacturing and, 261 extraction of by various methods, 353 therapeutic benefits of, 341 yields and concentrations of active

ingredients from, 355CMC-Na, 222–223CO2 (SC) advantages of processing with, 52, 158, 276,

338 cereal oil extraction in, 80–84 cosolvents, TCM processing and, 220–221,

222 fruit and vegetable oil extraction in, 84–90 liquid-liquid immiscibility and, 11 nut oil extraction in, 62–72 processing of TCM and, 220 seed oil extraction in, 72–80 solvent properties of, 3 specialty oil extraction in, 61–62 TCM processing and, 217–219Coca, 250Cod, 143, 153Codex Alimentarius, 277Coffee, 26, 246Cold processing, specialty oils and, 52Collection, high-pressure spray processes and, 370Color algae and, 193 fruit and vegetable oil extraction and, 90 nut oil extraction and, 71 paprika extraction and, 357 seed oil extraction and, 78, 80 spices and, 343Composites, 376–380Compounds, 28–30, 31–34Concentrated powder form (CPF) process, 370,

376–378, 382Concretes, 310, 312, 316–318Conjugated double bonds, 56Constant extraction rate periods, 257–260Control systems, 41–42Conventional solvent extraction of antioxidants, 292–293 cereal oil extraction and, 84 fruit and vegetable oil extraction and, 90 nut oil extraction and, 72 seed oil extraction and, 80 specialty oils and, 52Copaiba, 250Copper Reduction Assay (CUPRAC), 286Coriander, 246, 344, 355, 360–361Coronaridine, 261Cosolvents A. sinensis, L. chuanxiong hort and, 229–230 algal extraction and, 200–201

BHC extraction from radix ginseng and, 235–236

cereal oil extraction and, 83 essential oil extraction and, 308 fruit and vegetable oil extraction and, 86, 89 heat treatment for removal of, 91 nut oil extraction and, 69, 71 polarity and, 6, 349 processing of TCM and, 220–221, 222 procyanidin extraction and, 231–232 SC-CO2 extraction and, 166–168 seed oil extraction and, 77–78 solubility and, 123 vegetable oils as, 89 vitamin E and, 352Cost estimates cellulosic structure and, 252–254 industrial process implementation and, 44–48 for PGSS, 375–376 for selected Latin American plants, 260–261 selection of parameters for, 254–260 SFE for Latin American plants and, 243,

254–262 spice oil extraction and, 350–351Cost of manufacturing (COM). See Cost estimatesCountercurrent extraction columns, 18, 31–32,

36, 328Couplings. See Drive couplingsCPF (concentrated powder form) process, 370,

376–378, 382Critical curves, 115Critical point, 2Critical pressure, 115Critical properties, 3, 5, 30Crossover effects, 30, 348Croton, 246Cryoprotection, 58Crystallization, 151–152, 152–156, 372–373Cumin, 341, 344, 353, 355CUPRAC. See Copper Reduction AssayCupuassu, 248Curcuma longa, 365Curcumin, 343Cuticular waxes, 310, 315–316

DDecaffeination, 26Degradation fish oil extraction and, 148–149, 151 green tea leaves and, 381 hydrodistillation and, 309 lipid oxidation and, 276–277 molecular distillation of tocopherols and, 105Degree of extraction, 71Degree of saturation, 152–156Dehydration, 11–15

7089_Index.indd 393 10/8/07 11:48:23 AM


Dense-gas fractionation, 17–18Density fog phenomenon and, 126 nut oil extraction and, 71 overview of, 4 phase equilibrium and, 2–3 solvent power and, 1Deodorizer distillate (DOD), 104, 108.

See also TocopherolsDepressurization rate, 27–29, 67–68Deterpenation, 320DHA. See Docosahexaenoic acidDiacyl glycerol ethers, 144–146, 176–178Diallyl sulfides, 361–362Diffusion coefficients, 1, 4Diffusion-controlled rate periods, 257–260Dilophus ligulatus, 192Diphtheria, 338Discharge time, 29–30Dispersal, 369Dissolution, 369Distillation celery seed essential oil and, 356 essential oils and, 309 of fish oils, 149–151 of menhaden oil, 150 problems with tocopherol concentration using,

104–105 SFE and for TCM processing, 223–224 spice constituent extraction and, 353 spice extraction and, 346–347Distribution coefficients, 113, 115Diterpenes, 341Docosahexaenoic acid (DHA), 57, 142–144,

168–169Docosapentanoic acid (DPA), 57DOD. See deodorizer distillate.

See also TocopherolsDPA. See Docosapentanoic acidDPPH radicals, 286Drive couplings, 32, 33DSS, 222–223Dunaliella spp., 191, 192, 196–198Dynamic axial columns, 36–37

EEbers Papyrus, 338Ecdysterone, 261Echium, 73, 79Economics. See Cost estimatesEDTA. See Ethylenediamenetetraacetic acidEicosanoids, 57Eicosapentanoic acid (EPA) algae and, 201–202, 202–205 extraction of from fish oils, 142–144 overview of, 57

SC-CO2 extraction of, 168–169 structure of, 144Emulsions, 346Encapsulation, 380Entrainment, 105, 201, 209Enzymatic transformation, 156–158, 175–176Ephedrine, 222Equilibration time, 66, 76Equilibria, 151–152Equilibrium calculations, 8–9Equipment, 34–35, 219–220Erva baleeira, 246Essential oils antisolvent extraction and, 322–323 celery seed extraction and, 356 examples of, 320–322 extraction of from flowers, 314, 316–318 extraction of from leaves, 312–313 extraction of from seeds, 314 flower concretes fractionation and, 316–318 ginger extraction and, 358 liquid feed extraction and, 318–319 mathematical modeling of extraction of,

324–328 operating parameter selection for, 319–340 overview of, 305–307, 343, 346 solids processing and, 307–312 sources of, 311 spices and, 341, 342Esterification, 105–106, 156–157Estragole, 314Ethane, 3, 11Ethanol. See also Cosolvents cereal oil extraction and, 83 as cosolvent, 91 fruit and vegetable oil extraction and, 89 nut oil extraction and, 69 processing of TCM and, 220–221 seed oil extraction and, 77–78 squalene extraction and, 168 urea inclusion complexation and, 155–156Ethylenediamenetetraacetic acid (EDTA), 277Eucalyptus, 246Eugenia carophyllata, 225–228Eugenol, 225–227, 316Evening primrose, 73, 79Expansion. See Rapid expansion of supercritical

solution (RESS) processExplosives, 373Extract materials, 26Extraction. See also Conventional solvent

extraction of compounds from liquid feed, 31–34 of compounds from solid matrix, 28–30 control systems for, 41–42 equipment design and, 34–35 heat exchangers for, 38–39

7089_Index.indd 394 10/8/07 11:48:23 AM

Index 395

industrial process implementation for, 42–48 overview of, 48–49 of oxychemicals, 11–15 piping, valves and, 39–41 process development and, 218 process overview, 26–28 processing parameters for solids extraction,

30–31, 308–309 pumps and compressors for, 37–38 from vegetable matrices, 18–19 vessels for, 34, 35–37Extraction time, 68–69, 77, 83Extraction vessels, 29, 34, 35–37

FFalling extraction rate periods, 257–260FAME. See Fatty acid methyl estersFanshensu, 225Fatty acid methyl esters (FAME), 105–106,

106–107, 126–136Fatty acids cereal oil extraction and, 83–84, 85 Chrastil parameters of from fish oil, 160 of fish oils, 143 fruit and vegetable oil extraction and, 89–90 nut oil extraction and, 70 seed oil extraction and, 78, 79Feed materials, 26Fenchone, 314Fennel, 246, 261, 314, 360–361Fermenters, algae and, 191Ferric Reducing Antioxidant Power (FRAP), 286Ferulic acid, 228–230Fish oils chromatographic separations of, 156 distillation of, 149–151 enzymatic transformation of, 156–158 low-temperature crystallization of, 151–152 omega-3 fatty acids of, 142–144 overview of, 141–142 overview of separation and fractionation

technologies for, 147–149 phase equilibria of in SC-CO2, 158–168 polyunsaturated fatty acids and, 57, 168–176 squalene and diacyl glycerol ethers of,

144–146, 176–178 urea crystallization of, 152–156 vitamin A and, 146, 178–181 wax esters of, 146–147, 181Fixed costs, 260–261Flammability, 18–19Flavanones, 280Flavones, 280, 313Flavonoids, 280Flavonols, 280Flax, 74, 79

Flow control valving, 39–40Flow rates and directions cereal oil extraction and, 83 fruit and vegetable oil extraction and, 86, 89 nut oil extraction and, 68 seed oil extraction and, 77 solids extraction processing parameters and,

31Flowers, 251, 313, 314, 316–318Fog phenomenon, 126Fractional extraction processes, 26–27, 150–151Fractionation of essential oil extracts, 306, 310 FAME removal during tocopherol

concentration and, 126–136 of oils, 17–18 of rose concrete, 317–318 spice extraction and, 354Fragrances, 316–318, 358, 359, 361FRAP. See Ferric Reducing Antioxidant PowerFree fatty acid esters, 163Free fatty acids, 105–106Free radicals, 276–277, 285–286French paradox, 282Fruit oils, 84–90Fugacity, 4–6Fugacity coefficients, 6

GGallates, 276, 277–278Gamma-linoleic acid (GLA), 57 (all-cis-6,9,12-octadecatrienoic acid), 205–206Garlic, 338–340, 344, 361–362GAS (gas antisolvent) process, 15, 372–374, 382Gas Chromatograph with Electrical Conductivity

Detector (GC-ECD) analysis, 233–234Gas recycling, 370Gas salting out effect, 4Gas saturated liquids (PGSS), 369, 373–375, 382Gases, physical properties of, 4Gas-liquid alternating circulation system, 107–109General expenses, 260–261Genetic engineering, 191Ginger bioactive compounds from, 244, 246, 344 cost of manufacturing and, 261 extraction of bioactive compounds from, 358 extraction of by various methods, 353 overall extraction curve for, 259, 260 return on investment and, 47, 48 therapeutic benefits of, 341 yields and concentrations of active ingredients

from, 355Ginseng, 233–236, 249, 261GLA. See Gamma-linoleic acidGlycerides, 105–106

7089_Index.indd 395 10/8/07 11:48:23 AM


Glycosides, 342Good Manufacturing Practice (GMP)

compliance, 47–48, 375Grapefruit, 250Grapes, 74, 79, 230–233, 250Green pepper basil, 246Green tea leaves, 381–384Green-lipped mussel oil, 181Grinding, 76, 86, 368Group contribution equation of state (GC-EOS)

model, 7Guaco, 250Guarana, 250

HHaematococus pluvialis, 192, 198–201Halibut, 143HAT. See Hydrogen atom transferHazard and Operatability (HAZOP) studies, 38Hazelnuts, 63, 66, 69, 70HD. See HydrodistillationHeat exchangers, 38–39Heat treatment, 91Helmholtz residual energy, 7Hemicellulose, 69Herring, 143Hexachlorocyclohexanes (BHC), 233–236Hexane, 80, 347, 353, 355Hibiscus, 74, 79High-critical temperature (high Tc) fluids, 3High-pressure spray processes, 368–376, 376–380,

384High-speed countercurrent chromatography

(HSCCC), 224Hiprose, 74, 87Hoki liver oil, 172Horsetail (giant), 246HSCCC. See High-speed countercurrent

chromatographyHybrid hibiscus, 74, 79Hydrocarbons, 55, 160, 192–193Hydrodistillation (HD), 309, 346–347, 356Hydrogen atom transfer (HAT), 286Hydrolysis, 151, 156–157, 309Hyperforin, 312Hypertension, 228, 338Hypnea charoides, 192, 201–202

IImpregnation, 377Industrial process implementation, 42–48Inflammation, 57, 282Interactions, 324Interfacial tension, 4

Isochrisis galbana, 192Isoflavones, 280Isofugacity criterion, 4–5Isolation valving, 39–40Isomerization, 151Isoprenoids. See TerpenoidsIsopropanol, 15

JJackfruit, 250Jalapeno peppers, 250Jojoba, 248, 341

KKanglaite Injection, 223–224Khoa, 246Kinetics, 257–258Kirsch, 380, 381Koenen and Gaube diagrams, 12

LLabor, 47Latin American plants cost estimates and, 243, 254–262 examples of SFE from, 244–252 overview of SFE of bioactive compounds

from, 243–244 SFE process for, 252–254Lavender, 314Leaves, 312–313Lecithin, 33–34Lemon verbena, 247Lemongrass, 247Licorice, 346Limonene, 314, 316, 320, 327Linalool, 314, 321, 327, 361Lingusticum chuanxiong hort, 228–230Linoleic acid, 55, 57Linolenic acid, 69, 78, 90, 201–202, 230–233Lipases, 156–158Lipids, 18–19, 190, 248, 276–277, 281–282Lippia sidoides, 247Liquid feeds, 31–34, 306Liquid-filled composites, 378–380Liquid-liquid equilibrium, 7Liquid-liquid immiscibility, 9–11Liquids, 4Lobenzarit preparation, 16Low-critical temperature (low Tc) fluids, 3Low-temperature crystallization, 151–152Lunaria, 79Lutein, 53, 84, 86, 200

7089_Index.indd 396 10/8/07 11:48:24 AM

Index 397

Lycopene, 53, 56–57, 86, 89, 281Lyprinol, 181

MMace, 346Macela, 247Mackerel, 143Macroalgae, 190Macroporous resin adsorption technology, 233Magnesium stearate, 343Mangos, 250Manufacturing costs. See Cost estimatesMarigolds, 247, 250Marine macroalgae, 190Mass transfer models, 255, 325–327Mastranto, 247Matricine, 314MD extraction, 224Mechanical mixing, 32–33ME-DOD. See Methyl esterified DODMelting, 369Menhaden, 143, 150Mesityl oxide, 34Methanol, 3, 155–156Methanolysis, 105–106Methyl esterified DOD (ME-DOD).

See also Tocopherols composition of, 130 FAME removal from, 126–129 phase behavior of, 106–124, 124–126 pressure and, 131–134 pretreatment and, 105–106, 129–131Methyl oleate distribution coefficients of, 113, 115 FAME removal and, 106–107, 110–111 gas-liquid interface and, 117 separation factor of in SC-CO2 fractionation,

122–123Microalgae Botrycoccus braunii, 191–193 Chlorella vulgaris, 193–196 Dunaliella spp., 196–198 Haematococus pluvialis, 198–201 Hypnea charoides, 201–202 Nannochloropsis spp., 202–205 overview of, 189–191, 209, 244 Spirulina spp., 205–209Microemulsion. See SurfactantsMicronization, 13, 15–17. See also Supercritical

antisolvent micronizationMicro-particles, 367–368. See also High-pressure

spray processesMigration rates, 156Milk thistle, 74Minerals, 69Miscibility, 9–11, 115

Mixtures, 43Modeling, 7, 255, 324–327Modified Huron-Vidal 2 (HHV2) model, 7Modifiers, 290–291. See also CosolventsMoisture content cereal oil extraction yield and, 80 fruit and vegetable oil extraction and, 86 nut oil extraction and, 66 seed oil extraction and, 76 solids extraction processing parameters and, 31 spice extraction and, 350Molecular distillation, 104–105, 150Mongolia mushrooms, 225Monoterpenes, 341, 355Morphology, 31Mortierella sp., 192mRNA, 284Mullet, 143Multicomponent fluids, 8Multiplunger pumps, 37–38Multistage extraction, 308Munch, 72, 74, 79Myristicin, 359

NNannochloropsis spp., 192, 202–205Nano-particles, 367–368, 371. See also

High-pressure spray processesNanosuspensions, 371Natural products. See Traditional Chinese

medicines and natural productsNear-critical region, 2, 7, 8–9Nebuilizing, 32Neem, 79n-Hexane, 3, 109, 119, 168, 291Nonclassical supercritical effects, 13–14Non-Random Two Liquids (NRTL) model, 7Nut oils, 62–72Nutmeg, 346, 358–359Nutraceuticals, 342, 380–384

OOats, 81, 85Ochronomas danica, 192Odor, 208. See also FragrancesOEC. See Overall extraction curvesOils, 17–19Oleic acids, 69, 78Oleoresins cost of manufacturing of, 261 defined, 244, 342 ginger extraction and, 358 from Latin American plants, 245–247, 252 liquid solvent extraction and, 310

7089_Index.indd 397 10/8/07 11:48:24 AM


overview of, 343, 346 sources of, 311 vanilla extraction and, 359Olives, 87, 248Omega-3 fatty acids, 142–144Onions, 341Operating costs. See Cost estimatesOptimization, 9ORAC. See Oxygen Radical Absorbance CapacityOrange (sweet), 247Orange roughy, 147, 148Oregano, 247, 327Organic acids, 277Organochlorine pesticide, 233–236Osteoarthritis, 60Overall extraction curves (OEC), 256, 257–260Oxidation. See also Antioxidants CO2 processing and, 52 distillation of fish oils and, 151 fish oil extraction and, 149 nut oils and, 62, 72 seed oil extraction and, 80 squalene and, 145Oxychemicals, 11–15Oxygen Radical Absorbance Capacity (ORAC),

286Oxygenated compounds, 341

PPalmarosa, 247Palmitic acid, 69, 90Palms, 248Paprika powder, 248, 357–358Paraffins, 11, 310Paragual, 244Particle shape, 37, 324–325, 368, 379Particle size A. sinensis, L. chuanxiong hort and, 228–229 cereal oil extraction yield and, 80 clove bud oils and, 226 fruit and vegetable oil extraction and, 86 nut oil extraction and, 62, 66 seed oil extraction and, 76 solids extraction processing parameters and, 31Particulates, 367–368, 368–376Passion flower, 251Passion fruit, 248PCA. See Precipitation with compressed

antisolventPeanuts, 64, 66Pecans, 64, 66, 69, 70Pectins, 69Peel oils, 320PEG, 380Pejibaye, 248Pepper, 355

Percolation method, 230Peroxidation, 281–282Pesticides, 233–236PGSS (gas saturated liquids), 369, 373–375, 382Phaffia rodozyma, 192Phase equilibria chromatographic separations and, 156 of fish oils in SC-CO2, 158–168 for Latin American bioactive compounds, 253 of methyl oleate-DOD, 124–126 multiple, 6–8 overview of, 9–11 phase equilibrium analyzers and, 43 solid solubilities and, 4–6 tocopherol concentration and, 110–113,

117–119Phase equilibrium analyzers, 43Phase equilibrium diagrams, 2Phase equilibrium engineering, 8–11, 20Phenolics as antioxidants, 280 biological properties of, 282–283 effect of pressure and temperature on yield of,

291 as primary antioxidants, 277 solubility of in SC-CO2, 290Phenylpropanoids, 342Photobioreactors, 191Photosynthetic capacity, 190–191Phthalides, 355Physical properties, overview of, 4Phytochemicals, defined, 342Phytoplankton, 189Phytyl chains, 59Pigments, 56, 193, 202–205, 281, 360Pilayella littorallis, 192Pink trumpet tree, 251Piperine, 359Piping, 39–41Piprioca, 247Pistachios, 64, 69Pitanga, 251Plant polyphenols, 230–233Polarity, 6, 217, 229, 349Polyethylene Terephthalate (PET) films, 252Polymerization, 151Polyphenols, 381–384Polysaccharides, 225Polyunsaturated fatty acids algae and, 201–202 extraction of from fish oils, 168–176 of fish oils, 142 in specialty oils, 57–58 vitamin E and, 60Potassium stearate, 343Pravastatin, 58

7089_Index.indd 398 10/8/07 11:48:24 AM

Index 399

Precipitation with compressed antisolvent (PCA), 372–373

Preservation, antioxidants and, 276Pressure. See also Depressurization rate CO2 extraction and, 218, 289–290 FAME removal during tocopherol

concentration and, 131–134 fruit and vegetable oil extraction and, 86 nut oil extraction and, 66–68 phase equilibrium and, 2 seed oil extraction and, 76–77 selection of cost estimate parameters and,

254–257 solvent recycle and, 45, 46 tocopherol SC-CO2 concentration and,

117–119Pressure reduction, 218Pressure vs. temperature diagrams, overview of, 8Pressured fractional distillation, 223–224Pretreatments, 105–106, 116–118, 129Preventative antioxidants, 277Primary antioxidants, 277Primrose, 73, 79Process design extraction from vegetable matrices and, 18–19 fractionation of oils and, 17–18 overview of, 26–28 oxychemical extraction, dehydration and,

11–15 particle micronization and, 15–17 supercritical reactions and, 19–20Processing parameters, 30–31Procyanidins, 230–233Propane, 3, 13–14, 18–19, 359–360Prostaglandins, 57Protocatechualdehyde, 225Provitamin A, 342. See also β-CarotenePseudomonas sp., 157, 158Psoralen, 224Pumpkins, 75, 79Pumps and compressors, 37–38Pupunha, 248

QQuinones, 277

RRadix ginseng, 233–236Raffinate, 31–32Rapeseed, 248Rapid expansion of supercritical solution (RESS)

process cost studies of, 375–376 nutraceuticals produced by, 382

overview of, 369, 370–371 solubility and, 15 spraying of gas saturated liquids and, 373–375Recycling, 9, 44–47, 174, 370Red chili extraction, 346, 356–357Redundancy, control systems and, 41Regulation of antioxidants, 277–278Relative volatility, 18Residence time, 31Residual oils, 48Resins, 342. See also OleoresinsResistance temperature detector (RTD) sensors, 41Respiratory tract, 338RESS. See Rapid expansion of supercritical

solution processRetinol, 146Retrograde behavior, 5Revenue estimates, 47Rice bran, 81–82, 84, 85, 248Roasting, 62Rose concrete, 317Rose hips, 75, 79, 248Rosemary, 247, 261

SSAE. See Supercritical antisolvent extractionSafety, 41, 233–236Saffron, 244, 346Sage, 312–313Salmon, 143Saponification values, 107Saponins, 342Saprolegnia parasitica, 192, 195–196Sardine oil, 171SAS. See Supercritical antisolvent micronizationSaturation degree of, 152–156SC-CO2. See CO2-SCScenedesmus obliquus, 191, 192SDS, 222–223Sea buckthorn, 75Sebum, 144Secondary antioxidants, 277SEDS. See Solution-enhanced dispersion by

supercritical fluidsSeed oils celery seed extraction and, 354–356 characteristics of products extracted from,

78–80 cosolvents and, 77–78 essential oil extraction and, 313, 314 extraction time and, 77 flow rate and direction and, 77 modeling extraction of, 327 moisture, equilibration time and, 76 overview of, 72–76 particle size and, 76

7089_Index.indd 399 10/8/07 11:48:25 AM


temperature, pressure and, 76–77Selectivity, 3–4, 26–27Sensors, 41Sensory properties, 280, 352–353Separability, 3Separation, 26, 218Sequential depressurization, 27–29Sesame, 75, 79Sesquiterpenes, 314, 321, 341SET. See Single electron transferSFC. See Supercritical fluid chromatographySharks, 145Shea nut oil, 72Shell-and-tube heat exchangers, 39, 40Shogaols, 358Short-path distillation, 104–105, 150SHS, 222–223Sidda, 338Silanol, 321Silybum marianum, 72, 77Single electron transfer (SET), 286Sitosterol, 58Sitosterolemia, 58–59Skeletonema costatum, 192Small spined oreo, 147, 148Solid matrices, 28–30Solids, 30–31Solubility of β-carotene isomers, 197–198 Chrastil correlation and, 159, 160 CO2 extraction and, 61, 289–290 cosolvents and, 123 crystallization separations and, 151–152 of fatty acids from fish oils, 159–162 hydrodistillation and, 309–310 for Latin American bioactive compounds,

253, 256–257 of methyl oleate and α-tocopherol in SC-CO2,

113, 114 nut oil extraction and, 67 phase equilibrium analyzers and, 43 solids extraction processing parameters and, 30 spice oils and, 362–364, 365Solution-enhanced dispersion by supercritical

fluids (SEDS), 372–373Solvent loading, 29Solvent power, 3, 15, 26Solvent recycle, 9, 44–47, 174Solvent-feed ratios, 30–31Solvents. See also Cosolvents chlorinated, 347 crystallization and, 367 density-dependent nature of, 1 high-pressure spray processes and, 369 reaction with piping surfaces and, 39Span-80, 223

Specialty oils. See also Specific oils bioactives in, 52–55 carotenoids in, 56–57 extraction of, 61–62 overview of, 52, 90–91 polyunsaturated fatty acids in, 57–58 squalene in, 58 sterols in, 58–59 tocols in, 59–61Sperm whales, 147, 148Spice extracts, 343, 346, 348Spices antioxidants from, 277 beneficial aspects of, 339–341 bioactive compounds from, 341–343 black pepper extraction and, 359 cardamom extraction and, 359–360 celery seed extraction and, 354–356 cinnamon extraction and, 362 commercial SCFE process for, 349–351 conventional extraction of, 346–347, 351–354 defined, 338 fennel, caraway, coriander extraction and,

360–361 garlic extraction and, 361–362 ginger extraction and, 358 importance of, 338–339 nutmeg extraction and, 358–359 overview of, 337–338, 364–365 paprika extraction and, 357–358 red chili extraction and, 356–357 saleable products from, 343–346 SC-CO2 extraction of, 347–349, 351–354 solubility of oils in SC-CO2 and, 362–364 specific therapeutic benefits of, 340–341 vanilla extraction and, 359Spiny Dogfish, 143Spirulina maxima, 205–207, 244Spirulina platensis, 207–209Spirulina sp., 192Splinefitting, 258–260Spray agglomeration, 376–378Squalene amaranth oil and, 83, 84 from fish oils, 144–146, 176–178 oil fractionation and, 17 physical properties of, 55 SC-CO2 extraction of, 165–168, 176–178 in specialty oils, 58Standardization, 285Steam distillation, 346, 352–353Sterols, 54–55, 58–59, 342Steroptens, 317Stevia, 244, 247, 251Stigmasterol, 58Stripping, 18Structure, 324

7089_Index.indd 400 10/8/07 11:48:25 AM

Index 401

Sucrose, 244Sugars, 280Supercritical reactions, 19–20Supercritical antisolvent extraction (SAE),

306–307, 322–323Supercritical antisolvent micronization (SAS),

306–307, 322, 372–373, 375–376, 382Supercritical fluid antisolvent. See Supercritical

antisolvent micronizationSupercritical fluid chromatography (SFC), 36–37,

173–175Supercritical fluids, defined, 217, 338Supercritical region, 2Surface tension, 1Surfactants, 221–223Symposia, 219

TTabernaemontana, 251, 261TAG form of polyunsaturated fatty acids, 157Tannins, 342Tanshinone, 225TBHQ. See Tertiary butyl hydroquinoneTEAC. See Trolox equivalent antioxidant capacityTecanalysis software, 262Temperature clove bud oils and, 227 CO2 extraction and, 218, 289–290 distillation of fish oils and, 151 enzymatic transformation of fatty acids and,

156–157 fruit and vegetable oil extraction and, 86 nut oil extraction and, 66–68 phase equilibrium and, 2 seed oil extraction and, 76–77 selection of cost estimate parameters and,

254–257 tocopherol SC-CO2 concentration and, 117–119Terpenes, 280, 320–321, 342Terpenoids (isoprenoids), 144, 280–281, 283, 290,

291Tertiary butyl hydroquinone (TBHQ), 276Texture, 71, 290Thar Technologies, 32, 33Thermal conductivities, 4Thermowell isolation, 41Thrombosis, 142Thyme, 346Tobacco, 323Tocols, 53–54, 59–61Tocopherols binary phase equilibria of, 107–113 conventional extraction and, 72 distribution coefficients of, 113–118 effect of pressure and temperature on yield of,

293

equilibrium lines for, 123–124 fundamental research on concentration of,

106–107 molecular distillation and, 104–105 molecular structure of, 104 nut oil extraction and, 69 oil fractionation and, 17 overview of, 104, 136–138 phase behavior of ME-DOD system and,

124–126 pretreatment before concentration of, 105–106 propane solvents and, 72 regulation of, 277 rice bran oil and, 84 separation factor of with methyl oleate,

122–123 separation of with SC-CO2 fractionation,

126–136 solubilities of, 113, 290 ternary phase equilibria of, 118–122 wheat germ extraction and, 83Tocotrienols, 284Tomatoes, 75, 79, 88, 90Torulaspora delbrueckii, 192Total Peroxyl Radical-Trapping Antioxidant

Parameter (TRAP), 286Traditional Chinese medicines and

natural products of edible and medicinal ingredients from

grape seeds, 230–233 equipment made in China for, 219–220 essential oil from clove buds and, 225–228 of medical ingredients from A. sinensis and

L.chuanxiong hort and, 228–230 of organochloride pesticide from ginseng,

233–236 overview of, 216–217, 236–237 overview of active compounds extracted from,

221, 222 SFE and enhanced separation methods and,

223–224 SFE and ultrasound-enhanced extraction and,

223 SFE with CO2 in presence of solvent and,

220–221 SFE with CO2 in presence of surfactant and,

221–223 SFE with pure supercritical CO2 and, 220 supercritical fluid processing of, 217–219 use of combinations of extraction methods

and, 224–225Traditional processing methods.

See Conventional solvent extractionTRAP. See Total Peroxyl Radical-Trapping

Antioxidant ParameterTree tea oil, 341Triacylglycerols, 52, 160

7089_Index.indd 401 10/8/07 11:48:25 AM


Triglycerides, 69Trilinolein, 89–90Triple point, 2Trolox Equivalent Antioxidant Capacity (TEAC),

286Trout, 143Tuberose volatile oil, 318Tucuman, 248Tuna oil, 172, 175Turbines, 46–47Turmeric bioactive compounds from, 244, 251, 343, 345 solubility of essential oils from, 364 therapeutic benefits of, 341Tween-80, 223Type V phase behavior, 9–10

UUcuuba, 248Ultrasound, 223Ultraviolet radiation, 145Unani, 338Urea crystallization, 152–156Urea inclusion complexation, 155–156Urea-fatty acid ratio, 152–154

VValves, 39–41Vanilla, 346, 359, 360Vanillin, 359Vapor pressure, 149–150Vapor-liquid equilibrium (VLE), 7Vegetable matrices, 18–19Vegetable oils, 17, 84–90Vessels, 29, 34, 35–37

Vetivergrass, 247Vinca, 251Viscosity, 1, 4, 32, 134–136Vitamin A, 146, 160, 178–181Vitamin E, 277, 281–282, 284.

See also TocopherolsVLE. See Vapor-liquid equilibriumVoacangine, 261Volatile oils cost of manufacturing of, 261 from Latin American plants, 245–247, 252 liquid solvent extraction and, 310 sources of, 311–312 spices and, 341Volatility, 18, 67Volume, 2, 71

WW3 fatty acids, 201–202, 223Walnuts, 65, 70Water, 3, 13–14Wax ester oils, 146–147, 181Waxes, 146–147, 181, 310, 315–316Wertheim’s statistical association fluid theory, 7Wheat germ, 82, 85Wheat plumule, 82Workflows, 42–43

XXanthophylls, 56, 193Xylopia aromatica, 247

ZZeaxanthin, 84, 281

7089_Index.indd 402 10/8/07 11:48:26 AM

Super Critical Fluid Extraction......

Documents

Transcript of Super Critical Fluid Extraction......