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Development of a supercritical fluid chromatographymethod with ultraviolet and mass spectrometry

detection for the characterization of biomass fastpyrolysis bio oils

Julien Crepier, Agnès Le Masle, Nadège Charon, Florian Albrieux, SabineHeinisch

To cite this version:Julien Crepier, Agnès Le Masle, Nadège Charon, Florian Albrieux, Sabine Heinisch. Developmentof a supercritical fluid chromatography method with ultraviolet and mass spectrometry detection forthe characterization of biomass fast pyrolysis bio oils. Journal of Chromatography A, Elsevier, 2017,1510, pp.73-81. �10.1016/j.chroma.2017.06.003�. �hal-01574810�

1IntroductionBecauseofthenecessitytodevelopnewsourcesofenergyforthefuture,theproductionofsecondgeneration(2G)biofuelsfromlignocellulosicbiomassseemstobeapromisingoption,implyingdifferentwaysofconversion

[1].Fastpyrolysisisathermochemicalprocessoperatedwithin400–450°Crange,enablingbiomassliquefaction.Theresultingproduct,alsocalledpyrolysisoilorbio-oil, isveryrichinoxygencompounds,corrosiveandthermally

unstable.Itthereforeneedstobeupgradedtobeusedasbiofuels[1].Tocharacterizebio-oilschemicalcompositionatamolecularlevel,severalanalyticaltechniqueswereinvestigated[2],inparticularchromatographictechniques.

Gaschromatography(GC)permitstocharacterizeapartofbio-oilcomposition(estimatedatabout40%)[3–5].HoweverGCiscurrentlyunsuitableforcompoundswithhighpolarityand/orlowvolatilityand/orpoorthermalstability.In

thosecaseswhereGCcannotbeapplied,liquidchromatography(LC)canbeanalternativeforthecharacterizationofbio-oils[6,7].On-linecomprehensivetwo-dimensionalliquidchromatography(LCxLC)wassuccessfullyappliedto

theaqueousfractionofbio-oils[6–8]andcomparedmorerecentlytoon-lineLC×SFC[8].SFCmaybeapromisingapproachtoanalyzebio-oilsasitcombinestheadvantagesofGC(lowfluidviscosityandhighdiffusivityofsolutes)

with thoseofLC including (i) thepossibility of separatingpolar and/or lowvolatile compoundsand (ii) theavailability of a largepanel of stationaryphasesprovidingverydifferent selectivities.Furthermore,SFCconditionsare

expectedtobesoftwithausualtemperaturerangebetween30and60°C,compatiblewithmostofthecompoundspresentinbio-oils.

Overthelastdecade,anewgenerationofSFCdeviceshasbeencommercializedandtheinterestforSFChasgrowninvariousfields[9](pharmaceuticals,bioanalysis,agrochemicals,foodproducts).Theadditionofanactive

backpressureregulatorassociatedwithanewgenerationofpumps,abletogeneratelowflow-ratesinareproducibleway,leadscurrentlytorobustandreliableanalyses.However,inviewofpublishedresults,itseemsthatSFChas

Developmentofasupercriticalfluidchromatographymethodwithultravioletandmassspectrometrydetectionforthecharacterizationofbiomassfastpyrolysisbiooils

JulienCrepiera

AgnèsLeMaslea,⁎

agnes.le-masle@ifpen.fr

NadègeCharona

FlorianAlbrieuxa

SabineHeinischb

aIFPEnergiesnouvelles,Rond-pointdel'échangeurdeSolaize,BP3,69360Solaize,France

bUniversitédeLyon,CNRS,UniversitéClaudeBernardLyon1,EnsdeLyon,InstitutdesSciencesAnalytiques,UMR5280,5ruedelaDoua,F-69100Villeurbanne,France

⁎Correspondingauthor.

Abstract

Thecharacterizationofcomplexmixturesisachallengingissueforthedevelopmentofinnovativeprocessesdedicatedtobiofuelsandbio-productsproduction.Thehugenumberofcompoundspresentinbiomassfast

pyrolysis oils combined with the large diversity of chemical functions represent a bottleneck as regards analytical technique development. For the extensive characterization of complex samples, supercritical fluid

chromatography(SFC)canbealternativetousualseparationtechniquessuchasgas(GC)orliquidchromatography(LC).Inthisstudy,anapproachisproposedtodefinethebestconditionsfortheSFCseparationofafast

pyrolysisbio-oil.ThisapproachwasbasedonSFCdataobtaineddirectlyfromthebio-oilitselfinsteadofselectingmodelcompoundsasusuallydone.SFCconditionswereoptimizedbyusingthreespecific,easy-to-useand

quantitativecriteriaaimingatmaximizingtheseparationpower.Polarstationaryphases(ethylpyridinebondedsilica)associatedtoamixofacetonitrileandwateraspolaritymodifierprovidedthebestresults,withmorethan

120peaksdetectedinSFC-UV.

Keywords:Supercriticalfluidchromatography;Massspectrometry;Bio-oil;Fastpyrolysis;Complexsample

beenmainlyusedfortheseparationofrelativelysimplesamples.Asregardsbio-oils,amajorissueistofindoutwellrepresentativecompoundsabletomimictheirgreatchemicalcomplexityintermsoffunctionalgroupsandmolecular

weights.Totakeintoaccountthematrixcomplexityintheearlystagesofmethoddevelopment,wechoseinthisstudytoinvestigateSFCconditionsbydirectlyanalyzingabiomassfastpyrolysisoil.Theultimateobjectiveofourwork

wastoachieveadetailedmoleculardescriptionofsuchabio-oil.Todoso,severalstepswererequired,startingfromtheoptimizationofSFC-UVconditions(stationaryphase,mobilephase,temperature,etc…)andgoingtoanin-depth

analysisbasedonhyphenationbetweenSFCandhighresolutionmassspectrometry.ThepresentworkcorrespondstothefirststepofourmethodologyandaimstogetabetterunderstandingonhowSFCretentionisinfluencedbykey

experimentalparameterssuchastemperature,pressureormobilephasecomposition.Asaresult,thepurposeofthispaperwasnottoprovidebio-oilmoleculardatabutrathertoproposearelevantmethodology,basedonquantitative

andobjectivecriteria,allowing to select thebestchromatographicconditions inorder to separate thehighestnumberof compounds thatarepresent incomplexmatrices suchasbiomass fastpyrolysisoils.Accordingly,anovel

approachwasproposedwiththreeoptimizationcriteriadirectedtowardsmaximizingpeakcapacity.Asanoutlook,preliminaryresultsbasedontheresultingoptimizedSFCseparationandhyphenationtomassspectrometrydetection

areillustratedinordertopointoutthepotentialofthistechniqueforbio-oilanalysis.

2Materialsandmethods2.1Chemicalsandsamplespreparation

Solvents(acetonitrile,methanol,water)wereMSgradefromSigmaAldrich(Steinheim,Germany).CarbondioxideSFCgrade(99,97%)waspurchasedfromAirLiquide(B50bottleunderpressure).Tetrahydrofuran(THF)was

purchasedfromVWR(Fontenaysousbois,France).Thefastpyrolysisbio-oilwasobtainedfromconifersawdust,providedbyIFPEnergiesnouvelles.ThesamplewasdilutedinTHF(1/5w/w)beforeanalysis.

2.2InstrumentationandchromatographicconditionsAll experiments were performed on an Acquity UPC2 instrument (Waters, Milford, MA, USA). Key parameters (stationary and mobile phases, back pressure, column temperature and gradient conditions) were optimized

accordingtoaproceduredeveloped inthe ‘Resultsanddiscussion’ section.Thetypeofstationaryphasewasoptimizedaccordingto theproceduredescribed in thenextsection.Thestudiedcolumnsandtheircharacteristicsare

reportedinTable1.Themobilephaseflow-ratewas1.4mL/minforcolumns#1,2,3,5and7,exceptforcolumn#4and6whosegeometrycharacteristicsweretakenintoaccountbyusingaflow-rateat3.5mL/min.

Table1Characteristicsofthecolumnsinvestigatedinthisstudy.

alt-text:Table1

Columnnumber Columnname Manufacturer Stationaryphasechemistry dca(mm) Lc

b(cm) dpc(μm) Lc/dp

1 AcquityUPC2BEH Waters Unbondedsilica 3.0 10 1.7 5.88

2 AcquityUPC2HSSC18SB Waters C18 3.0 10 1.8 5.55

3 AcquityUPC2BEH2-EP Waters EthylPyridine 3.0 10 1.7 5.88

4 LunaCyano Phenomenex Cyanopropyl 4.6 15 3.0 5.00

5 AcquityUPC2BEHRP18Shield Waters PolarembeddedC18 3.0 10 1.7 5.88

6 NucleodurPolartech Macherey-Nagel PolarembeddedC18 3.0 10 2.5 4.00

7 AcquityUPC2CSHFluorophényl Waters Fluorophenyl 3.0 10 1.7 5.88

a Columninternaldiameter.b Columnlength.c Particlediameter.

Theinjectionvolumewas1μLforallexperiments.Theinjectorneedlewaswashedwith600μLofmethanolaftereachinjection.Thevarianceduetoextracolumnbandbroadeningwasmeasuredunderliquidchromatographic

conditionsbythemethodofstatisticalsecondmoment[10]andestimatedat85μL2.Themeasureddwellvolume,correspondingtotheinstrumentvolumebetweenthemeetingpointofthesolventsandthecolumninlet,was425μL.

ThisvalueishigherthanUHPLCones(typicallyabout100μL)whichisrelatedtothelargemixingchamber(approximately300μL)usedinthisstudytoavoidanydemixingphenomenon.Thecolumnoutletwasconnectedtoaphoto-

diodearraydetector(PDA)equippedwithahighpressureUVcell(400bar)withavolumeof8μLandapathlengthof10mm.Thedetectionwavelengthsvariedbetween210and400nmwithabandwidthof1.2nm.Thesamplingrate

wassetat40Hz.TheinstrumentwascontrolledbyEmpower3software(Waters).

MassspectrawereobtainedusingaLCMS2020instrument(Shimadzu,Kyoto,Japan)equippedwitheitherelectrosprayionization(ESI)oratmosphericpressurechemicalionization(APCI)sourcesbothworkinginnegativeand

positivemodes.Asimplequadrupoleensuredthemassmeasurementintherange80–800m/z.MSparameterswereoptimizedtofavorthedetectionofpseudo-molecularions.Suchanoptimizationwasbasedonmodelcompounds

detection.Nebulizinggasflowanddrygasflowwere0.5and10L/minrespectively.Theinterfacetemperaturewassetat400°Candthedesolvationlinetemperaturewas250°C.ForESIsource,theinterfacevoltagewas+3.5kVand

−5kVinpositiveandnegativeionmodesrespectively.ForAPCIsource,thecoronacurrentinnegativemodeandpositivemodeswererespectively80μAand70μA.

3Resultsanddiscussion3.1MethodologyforoptimizingkeySFCparameters

Thisworkaimsatdevelopinganefficientmethodfor theSFCseparationof fastpyrolysisbio-oils.The lackofextensiveknowledgeonthecomponentsofsuchverycomplexmixturespreventedus fromconsideringmodel

compoundstodeveloptheseparationmethodasitwasdoneinthepastforcloselyrelatedsamples[6].Thedevelopmentoftheseparationmethodwasthereforecarriedoutonthebio-oil.Inthisway,astrategywasimplementedto

optimizekeyparametersfromafewpreliminaryexperiments.KeyparametersforanySFCseparation,includethetypeofstationaryphase,thetypeofco-solvent,thegradientconditions,thecolumngeometry,thecolumntemperature

andthebackpressureoftheSFCsystem.Theflow-ratewasselectedsothatthemaximumpressureandhencetheminimumanalysistimewereattained.Thechoiceofoptimizationcriteriawasbasedonsamplepeakcapacity.This

latterisknowntobeapowerfuldescriptortoassesstheseparationpowerofagivenchromatographicsystemforagivensampleundergivengradientconditionsandfurthermoretocomparetheabilityofdifferentchromatographic

systemstoseparatethecomponentsofthissample.Thesamplepeakcapacity,ngrad,ingradientelutionwasdefinedbyDolanetal.[11]as

wheretnandt1 are the retention timesof themostand the least retained solutesundergradient conditionsandw, theaverage4σ peakwidth.Eq. (1) canbeuseful to comparedifferent chromatographic systemsprovided that

thenumberofcompoundsinthesampleislimited.However,gradientseparationsofverycomplexsamplessuchasbio-oilsleadtomultiplepeakcoelutionswhichmakeitimpossibletodeterminetheaveragepeakwidthandhencethe

sample peak capacity from Eq. (1). Furthermore, an additional peak capacity, due to the isocratic initial step, has to be taken into account for the determination of the total sample peak capacity. Under isocratic conditions

(correspondingtotheinitialcomposition),thesamplepeakcapacity,niso,isgivenbyEq.(2)definedas[11]

whereNisthecolumnplatenumber,k1,isoandkn,isoaretheretentionfactorsoftheleastandmostretainedcompoundsunderinitialisocraticstep.Nisrelatedtothecolumnlength,Lcandtheparticlediameter,dp,byEq.(3):

wherehisthereducedcolumnplateheight.

35yearsago,Snyderandco-workersdevelopedthesemi-empiricalLinearSolventStrengthTheory(LSST)basedonalinearrelationshipbetweenthelogarithmofthesoluteretentionfactorandthetime:

ki is theretention factor in initialgradientconditions; t0 is thecolumndeadtime; tdelay is thedelay timecomposedof thedwell time, tD,andofapossibleprogrammed initial isocratichold, tini(tdelay=tD + tini); b is the gradient

steepnessgivenby

s is the normalized gradient slope ( , Δ φ being the gradient range expressed in volume fraction of the strong solvent and tG, the gradient time) and S is the slope of the relationship between the logarithm of the

soluteretentionfactorandthevolumefractionofthestrongsolvent:

(1)

(2)

(3)

(4)

(5)

j

(6)

k0beingthesoluteretentionfactorintheweaksolventandφ,thevolumefractionofthestrongsolvent,B.

AccordingtoLSSTandconsidering(i)anaverageSvalueand(ii)highkivaluesforallsolutes,thefollowingequationcanbederivedfromEq.(1)[11]:

whereφn,gradandφ1,gradarethecompositionatelutionofthemostandleastretainedcompoundsundergradientconditions.

Incaseofverycomplexsamples,itcanbeassumedthattheleastretainedcompoundiselutedinthevoidvolume(k1,iso=0)whilethelastelutedpeakintheinitialisocraticstepiselutedatthebeginningofthegradientwhich

meansthatφ1,gradcorrespondstotheinitialcompositionφiniandthat

FromEqs.(2),(3),(8)and(9),theresultingequationforthetotalsamplepeakcapacitycanbeexpressedas

ItwasfirstnecessarytoverifythatEq.(7)wasvalid,namelythatLSSTcouldbeappliedtolineargradientsinSFCascanbedoneinRPLC(reversedphaseliquidchromatography).LSSparameters,k0andS(Eq.(6))canbe

calculatedfromtheretentiondataoftwopreliminarygradientruns[12,13]whichallowsthentheaccuratepredictionofanygradientretentiontimesprovidedthatLSSTisvalid.Experimentallyweperformed,ontheAcquityBEH-2EP

column,twolineargradientruns,runningfrom1%to40%ACNandusingtwodifferentnormalizedgradientslopes,(0.01and0.03)sothatk0andScouldbecalculatedbyOSIRISV4.1(Datalys,Grenoble,France).Thiswasdonefor6

peaksselectedinsuchawaythat(i)theycouldcoveralargeretentionrangeand(2)theycouldbeeasilytrackedfromthetwopreliminaryexperimentalchromatograms.Athirdlineargradientwithadifferentnormalizedgradient

slopeof0.005 wasperformedexperimentally.InordertodemonstratewhetherLSSTcanbeappliedtolineargradientsinSFC,asimulatedchromatogramwasobtainedfromOSIRISforthe6peaks(Fig.1a)andcomparedtothe

experimentalchromatogramofthebio-oil.(Fig.1b).Asshownthesimulatedandexperimentalchromatogramsareinverygoodagreement,therebyvalidatingLSSTforlineargradientsinSFC.ResultingSandlog(k0)valuesareshown

insupplementarymaterial.Itisinterestingtonoticethat,unlikeRPLCseparationswithmethanolorTHFasstrongsolvent[14],thedependenceoflog(k0)vsSisdecreasing,suggestingthataslightlyconvexgradientshouldbemore

suitableinSFCthanalinearone.However,consideringthelowvariationofS(valuesbetween4and7withanaveragevalueofabout5),lineargradientsremainsthemostsuitablefortheanalysisofbio-oils.Onlylineargradientswere

thereforeevaluatedintherestofthisstudy.Asimilartrend(notshown)wasobservedwithchromatographicsystemswhichwerestudiedinthiswork.

Atfirst,somekeyparametersofEq.(9)werefixed.Thoseincludeφi(5%),tdelay/t0(15)andthegradientsteepness,b(svalueof0.01,seeEq.(5).Theseparameterswereoptimizedinthesecondsteponcethechromatographic

system(mobileandstationaryphases)hadbeenselectedthankstoascreeningofsevencolumns(seeTable1)andthreeco-solvents(acetonitrile,methanolandamixofacetonitrileandmethanol(50/50v/v)).Forsakeofconsistency,the

(7)

(8)

(9)

Fig1Comparisonof(a)predictedand(b)experimentalseparationofafastpyrolysisbio-oilwithanormalizedgradientslopeof0.005(stationaryphase:AcquityBEH2-EP,modifier:ACN/H2O(98/2),temperature:30°C,BPRpressure:150bar).Appliedgradientinred.

alt-text:Fig1

ratioLc/dpwasapproximatelythesameforallstudiedcolumns(seeTable1).Bothcolumntemperature(30°C)andbackpressure(150bar)werefixedinthefirststepandfurtherconsideredinthesecondstep.

Since itwasnotpossible todirectlydeterminethesamplepeakcapacity fromchromatogramscomposedofmore thanonehundredpeaks, theconceptofpeakcapacitywasevaluatedthroughthreecriteria.The firstone

(CriterionA)representsthetotalnumberofobservedpeaks.Thechoiceofthiscriterionisbasedontheassumptionthatpeaksarerandomlyspacedonthechromatogramincaseofverycomplexsamples.Inthiscase,thestatistical

theoryofpeakoverlap,developedbyGiddingsandDavis[15]canbeapplied.Accordingtothistheoreticalapproach,foragivensample(i.e.agivennumberofcompounds),thepeakcapacityisexpectedtoberelatedtothenumberof

observedpeaksinthechromatogram.Thesecondcriterion(CriterionB)measuresthesizeoftheelutionwindow.Itcorrespondstothecompositionatelutionofthemostretainedcompound,φn,grad,whichisakeyfactorinEq.(9).The

elutionwindowsofSFC-UVchromatograms(detectionat210nm)weredividedintotwoequalparts.Thethirdcriterion(CriterionC)isthenumberofobservedpeaksinthesecondpartofthechromatogram.Thiscriterionaimsat

limitingtheoccurrenceofpeakclusters,especiallyinthefirstpartoftheseparation,andhencetopromoteagood distributionofthepeaks.Thecountofpeakswascarriedoutbymeansofahome-madesoftwareINDIGO

basedonarobustdetectionalgorithmfrommathematicalmorphology.Thisapproachcandetectall localmaximawithaminimumintensitywhichwassetata levelequalto3timesthesignal tonoiseratiowithoutcalculationof

derivatives.Touseefficientlythethreecriteriawepropose,eachofthemhasbeennormalizedbythemaximumandtheminimumvaluesencounteredforalltheexperiments.Therelevanceofthethreechosencriteriaishighlightedin

Fig.2,showingthedependenceofCriterionAvsCriterionB(Fig.2a)andofCriterionBvsCriterionC(Fig.2b)forthedifferentstudiedsystems.Itisclearthatthereisapoorcorrelationbetweenthethreecriteria,therebyunderlining

their complementarity. Finally a response function, corresponding to the geometrical mean of the three criteria, was calculated to assess the performance of the different chromatographic systems. The different steps of our

methodologyareillustratedinFig.3.

repartition

Fig.2Dependenceof(a)criterionBvscriterionAand(b)criterionCvscriterionBforthestudiedchromatographicsystemsidentifiedbycombinationofnumber(seeTable1)andletter(A:ACN,B:mixACN/MeOH50/50,C:MeOH).

alt-text:Fig.2

Fig.3MethodologyforoptimizingSFCkeyparameters.

alt-text:Fig.3

3.2ScreeningofmobileandstationaryphasesThefirststeptodeterminethebestconditionsconsistedinascreeningofbothstationaryandmobilephases.BecauseSFCmobilephasesarecomposedofapressurizedmixtureofnon-polarCO2andpolarorganicmodifier,

thereisnorestrictioninthechoiceofstationaryphases.AccordingtoWestetal.[16,17],itispossibletocoveralmostallinteractionpossibilitiesfromasetoffivestationaryphasesinvolvingdipole–dipoleinteractions,interactionsdue

tonandπelectrons,acid-baseinteractionsandsteric interactions [17]. Inourwork,wetherefore investigatedfivesilica-basedstationaryphases, includinganunboundedoneandfourbondedones(C18,C18withpolarembedded

groups,ethylpyridineandcyanopropyl).TypicalSFCcolumnsdimensions (3mmx100mm)and typical sizeparticles (1.7or1.8μm) for UHPSFC were tested as listed in Table1.Two additional stationary phases were chosen to

completethisstudy:LunaCN(#4inTable1)andNucleodurPolartech(#6inTable1).AccordingtotheclassificationofWestetal.[9]theircharacteristicsareclosetoBEH-2EP(#3inTable1)andBEHRP18(#5inTable1)respectively.

Thiswasdone inorder tohighlightanydifferenceofperformancewithinagivengroupofstationaryphases.At thesametime,severalmobilephaseswere investigated.SFCmobilephasesbeingmixturesofCO2andanorganic

modifier,theirpolarityandhencetheireluentstrengtharedependentonthenatureofthemodifier[18].Asaresult,differentstrongsolvents(B)wereinvestigatedinthisstudy,includingmethanol(MeOH),mostlyusedinSFCdueto

itshighpolarity,acetonitrile(ACN)andamix(50/50v/v)ofboth.Fig.4showstheresponsefunctionvalueforeachchromatographicsystem.Ascanbeseen,thebeststationaryphasesareeitherunbondedsilica(#1inTable1)orpolar

bonded silica (Ethyl pyridine and Cyanopropyl, #3 and #4 respectively). Those promote dipole–dipole and H donor-acceptor interactions according to LSER (Linear Solvation Energy Relationship) classification [19]. Bio-oils are

composedofalargenumberofpolarcompounds(alcohols,phenolics,organicacids,furans,ketones)[20].Thepresenceofsuchoxygenfunctionalgroupsseemstobeinaccordancewithobservedinteractions.

TheBEH2EPcolumn(#3)appearstobethemostattractive.HoweveritisinterestingtonotethatMeOHassolventBleadstoalowervaluefortheresponsefunctioncomparedtoMeOH/ACN(50/50v/v)or,evenbetter,topure

ACN.Thiscanbeeasilyexplainedbyalargerelutionwindow(criterionB)incaseofACNduetoitslowereluentstrengthasalsoshowninFig.5.AlthoughACNallowstoenlargetheelutionwindowresultinginanincreaseinpeak

capacity,thissolventisnotpronetohydrogen-bondinginteraction,yetofgreatinterestwithoxygenatedcompounds.Furthermore,thisinteractionalsoexistsbetweenthecompoundsandthesilanolsofthestationaryphaseandmay

haveanegativeeffectonpeakshapes.Toaddresstheseissues,theuseof2%waterinACNwasalsoinvestigated.Themoleculesofwatercanstronglyinteractwiththesilanolstherebyleadingtoacompetitiveadsorptiononthese

sites.AsshowninFig.5,theeffectofwaterisnotsignificantasregardsCriteriaBandCwhileCriterionA(numberofpeakdetected)isslightlyincreased.BecausecriterionCremainsthesamewithandwithoutwater,wecansuppose

thatadditionofwateraffectsmoresignificantlycompoundsthatarepoorlyretained.Asaconclusion,additionofwater isbeneficialsinceit increasesthepeakcapacity intheisocraticpartofthechromatogrambyreducingpeak

broadening.

Fig.4Responsefunctionvaluesforthedifferentstudiedchromatographicsystems.ThestudiedcolumnsarenumberedasinTable1.

alt-text:Fig.4

Similarly,othersadditivesweretested(i.e.trifluoroaceticacid,ammoniumacetate,diethanolamineandformicacid)butnoneofthemhadasignificantimpactonthenumberofpeaksdetected.

Finally,thebestchromatographicsystemwascomposedofanAcquityUPC2BEH2EPcolumn(3×100mm,1.7μm)andamixofacetonitrileandwater(98/2v/v)asstrongsolventB.Thiscombinationwasselectedtooptimize

theotherkeyparameters.

3.3OptimizationofmobilephasecompositionThevariationofmobilephasecomposition issplitted into twoparts: isocraticandgradientparts.For the isocraticpart, the initialcomposition (φini)wassetat5% in theprecedingscreening,according to theusualvalue

reportedintheliteratureforSFCseparations.HoweveralowerinitialcompositionwasexpectedtoprovideahigherpeakcapacityaccordingtoEq.(9)(φn,grad-φinilarger).Consequently,φiniwasvariedfrom5%to0%whilemaintaining

thesameratiotdelay/t0andchangingthegradienttimesothatthenormalizedgradientslopewaskeptconstant.CriteriaBandCwereexpectedtoremainunchangedregardlessoftheinitialcomposition.Wethereforefocusedonthe

numberofpeaksdetected(CriterionA).Asshown inFig.6a, the initialcompositionhad little impacton thenumberofpeaks (less than10%variation)during the isocraticpart, suggesting that thepeakcapacitywasmaintained

constantascouldbeexpectedfromEq.(9)providedthatthereducedplateheight,h,wasnotaffected.Conversely,thereisasignificantincrease(upto30%)inthenumberofpeaksdetectedduringthegradientpartwhentheinitial

compositionisdecreased.Thatisrelatedtotheincreaseinthecompositionrange.Thebestinitialcompositionshouldbe0%(106peaks)or1%(104peaks).However,withaninitialcompositionof0%,asmallpressurebumpoccurred

atthegradientstartresultinginbaselinedisruption.Wethereforepreferredtoselectaninitialcompositionof1%fortherestofthisstudy.Retentiontimereproducibilitymightbeanissuewithsuchalowsolventcontentduebothto

theneedforlongerequilibrationtimesandtothefactthatPumpBmustbeabletodeliverlowflow-ratesinareliableway.Toassessretentiontimereproducibilityintheseconditions,threedifferentrunswereperformedunderthe

sameconditionsoverthreedifferentdays.Therelativestandarddeviation(RSD)wascalculatedfromthethreeretentiontimesforfivereferencepeakswhichwerewelldistributedalongtheentirechromatogramandcouldbeeasily

recognizedfromruntorun(Table2).AsshowninTable2,RSDwasalwaysbelow0.6%therebydemonstratingthat1%wassuitableasinitialcompositionforgradientrunswiththeselectedchromatographicsystem.

Fig.5EvolutionofCriteriaA,BandCusingtheAcquityBEH-2EPcolumn(#3inTable1)dependingonthestrongsolventB:Acetonitrile( ),Methanol( ),Acetonitrile/Methanol(50/50;v/v)( ),Acetonitrile/Water(98/2;v/v)( ).

alt-text:Fig.5

Fig.6Increaseinthenumberofpeaksdetectedat210nmasafunctionof(a)theinitialcomposition(reference5%)forisocraticpart( )andgradientpart( )and(b)BPRpressurefor3columntemperatures:20°C( ),30°C( )and60°C( )(Reference30°Cand150bar).Other

conditions:AcquityBEH2-EP(3×100mm;1.7μm);A:CO2;B:ACN/H2O(98/2v/v);1.4mL/min.

alt-text:Fig.6

Table2RSDofretentiontimesforreferencepeaksdependingontheinitialcompositionofthegradientelution(30°Cand150bar).

alt-text:Table2

Initialcomposition(%) Peak1 Peak2 Peak3 Peak4 Peak5

RSD(%) Tr(min) RSD(%) Tr(min) RSD(%) Tr(min) RSD(%) Tr(min) RSD(%) Tr(min)

5 0.27 0.70 0.16 0.92 0.48 3.08 0.41 5.61 0.53 9.47

1 0.29 1.08 0.52 2.57 0.19 5.52 0.27 7.89 0.15 12.48

As regards thegradientpart, asdiscussed in the first section (seeSection3.1), a lineargradientwasexpected tobe suitable for the separationofbio-oils (small variationofSwith log (k0)).Withaview tooptimize the

normalizedgradientslope,wecomparedthenumberofpeaksandthepeakintensityofareferencepeakbothasafunctionofthegradienttime.AsshowninFig.7,thenumberofpeaksincreaseswiththegradienttime.Howeverthisis

correlatedtoadecreaseinsensitivity.Agoodtrade-offbetweenthesetwooppositeeffectsconsistedinselectingagradienttimeintherange10–20minsothattheslopeofthecurvegivingthevariationofthenumberofpeakswiththe

gradienttimewasnottoolow.Accordingly,anormalizedgradientslopeof0.01(i.e.agradienttimeof14min)wasselected.

3.4OptimizationofthecolumntemperatureandBPRpressureBothpressureandtemperaturehaveapotentialeffectonmobilephasedensityandhenceonsoluteretention.Forthisreason,weoptimizedBPRpressure(intherange100–150bar)andthecolumntemperature(intherange

30°Cto60°C)simultaneously.Usualconditions(30°Cand150bar)weredefinedasreferenceinFig.6b,showingtheincreaseinthenumberofpeaksdetectedasafunctionofBPRpressurefor3columntemperatures.

ResultsreportedinFig.6bshowthat,withinthestudiedrange,theeffectoftemperatureandBPRpressureonthenumberofpeaksdetectedisnotassignificantastheeffectoftheinitialcomposition(Fig.6a).Theincreasein

thenumberofpeaksdoesnotexceed10%exceptfor150barat60°C.However60°Cisthemaximumrecommendedtemperatureforthisstationaryphase.Inordertopreventanythermaldegradation,thisconfigurationwasnot

selected.Consideringthesmallincreaseinthenumberofpeaksunderalternativeconditions,wepreferredmaintaintheoriginalconditions(i.e.150barand30°C).Thesmallvariationofpeakcapacitywithtemperatureandpressure

canbeexplainedbythecomplexityofthesample.Severalstudies[21–23]reportedtheeffectoftemperatureand/orpressureonretention,buttherelatedsamplescontainedafewcomponentsonly,whichdidnotgiverisetomany

coelutions.Incaseofcomplexmixtures,DavisandGiddings[24]demonstratedthatonly18%ofthetheoreticalpeakcapacitycouldcorrespondtoasingle-componentpeak.Inourcase,itisobviousthatevenunderthebestconditions,

numerousco-elutionsoccur.Althoughtheimpactofpressureandtemperatureisdifferentdependingonthecomponent,thenumberofpeaksdetectedshouldremainnearlythesame.Toconclude,usualSFCconditions(i.e.30°Cand

150bar)seemtobesuitablefortheanalysisofthefastpyrolysisbio-oil.

3.5FinalSFC-UVmethodByfollowingthedifferentstepspresentedinFig.3,weselectedtheconditionsallowingtodetectalargenumberofpeaks(i.e.122)andhencetoprovidealargesamplepeakcapacityforabio-oilwithinareasonableanalysis

timeof22min.TherelevanceofthisapproachcanbeassessedbycomparinginFig.8twoseparationsofbio-oil.Thefirstone(Fig.8a)wasnotoptimized.Inparticular,bothstationaryandmobilephaseswerenotsuitableforbio-oil

compounds.Intheseinappropriateconditions,only15peaksweredetected.Thesecondseparation(Fig.8b)wasobtainedundertheaboveoptimizedconditions.Usingourmethodology,wedeterminedthatthebestsetofconditions

Fig.7Numberofpeaks(blue)andpeakintensityofareferencepeak(red)observedat210nmasafunctionofthegradienttime.Conditions:AcquityBEH2-EP(3×100mm;1.7μm),A:CO2;B:ACN/H2O(98/2v/v);from99:1(A:B)to60:40(A:B);30°C;BPRpressure:150bar;

1.4mL/min.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle).

alt-text:Fig.7

involvespolarstationaryphases(BEH-2EP)associatedtoamixofacetonitrileandwateraspolaritymodifier.Withafurtheradjustmentoftheconditions,namelyalowinitialcomposition(1%),anormalizedgradientslopeof1%,a

temperatureof30°CandBPRpressureof150bar,thenumberofpeaksdetectedinUV(210nm)wasincreasedtoabout120.

3.6HyphenationwithMSdetectionToenhance informationabout fast pyrolysisbio-oil composition, hyphenationof ourSFC-UVdevicewithmass spectrometry (MS)was carriedout.As themobilephase comingout fromSFCcolumnwaspressurized (i.e.

150bar),adecompressioncouldoccurattheMS-sourceinlet,therebypreventingsufficientvacuumtobeensured.ThatmeansthatonlyMSsourcesworkingatatmosphericpressurecanbeused.Inthiswork,electrosprayionization

(ESI)andatmosphericpressurechemicalionization(APCI)sourceswerechosentocoveralargerangeofpolarityandmolecularweight.

To take intoaccount the specificityof aCO2-basedmobilephase,MSparametershad tobeoptimized.The source temperatureneeded tobehigher than that inusual conditions toprevent fromany freezingduring the

decompressionofCO2andtolimittheformationofclustersduetothecoldspray.ThedecompressionofCO2requirestheuseofanadditionalsolvent(make-upflow)beforetheentryintheionizationsource.Withoutthat,solutesmay

precipitateinthetransfercapillary.Theadditionofmethanol(proticsolvent)asmake-upsolvent,ensuredasuitableinletflowandenhancedtheionizationprocessbychargeexchangewithanalytes.CO decompressionha dalsoan

impactonthenebulizingflow.Thislatterhadtobeaslowaspossibletoavoidatoowidesprayandhencealossofinformation.TheFig.9illustratestheinterfaceusedinbothESIandAPCImodes.

In optimized conditions, positive-ion and negative-ion ESI and APCI chromatograms of the bio-oil are presented in Fig. 10. The different ionization modes enabled a successful ionization and the detection of compounds

containedinthebio-oil.Itisinterestingtonoticethatthefourobtainedchromatogramsarequitedifferent.Asexpected,ourresultsdemonstratethatthesedifferentmodesofdetectionarecomplementaryandthuscanprovidean

extensiveinformationaboutthechemicalcompositionofbio-oils.36peaks,46peaks,61peaksand59peaksweredetectedinpositive-ionandnegative-ionESIandpositive-ionandnegative-ionAPCIchromatogramsrespectively.The

numberofpeaksdetectedislowerthanwithUVdetection(i.e.122)whichisconsistentwiththefactthat(i)MSdetectionismorespecificand(ii)additionalpeaktailingduetodispersionphenomenainMSreducespeakseparationand

sensitivity(iii)the210nmselectedasdetectionwavelengthintheUVallowsamuch‘broader'detectionofcompounds.

Fig.8SFC-UVchromatogramofthefastpyrolysisoilusing(a)badconditions(stationaryphase:AcquityFluorophenyl,modifier:MeOH,temperature:30°C,BPRpressure:150bar)and(b)optimizedconditions(stationaryphase:AcquityBEH2-EP,modifier:ACN/H2O(98/2),

temperature:30°C,BPRpressure:150bar).

alt-text:Fig.8

2 s

Fig.9SchemaofinterfacebetweenSFC-UVandIonizationsourceofMS.

alt-text:Fig.9

Theuseofpositive-ionmodes(APCIandESI)seemstoberelevanttoenhanceinformationonlessretainedcompounds.ESInegativemodeseemstobethebestonetoanalyzethelastpartofthechromatogram.Itcanalsobe

expectedthatnegative-ionmodewillbemoreappropriateforacids,phenoliccompounds,whereaspositive-ionmodewillbepreferredforcarbohydrates,ketonesandfurans.Asuitablecombinationofthesedifferentdetectionmodes

willberequiredtogetanexhaustiveinformationaboutalargenumberof compoundspresentinbio-oils.

4ConclusionDespiteSFCanalysishasgainedincreasinginterestforseveralyearsnow,abetterknowledgeisrequiredonhowkeyexperimentalparameterssuchasstationaryphase,temperature,pressure,mobilephasecompositionaffect

the retention of oxygenated polyfunctional compounds present in complex products. In this work, an approach for the selection of suitable SFC conditions for separating a biomass fast pyrolysis oil is proposed based on three

quantitative,objectiveandeasy-to-usecriteria.Thenoveltyofthismethodologystemsfromthefactthattherewasnoneedformodelcompoundssincemethoddevelopmentwasdirectlyperformedwiththebio -oil.Stationaryand

mobilephases,initialmobilephasecomposition,andgradientslopewereoptimizedinordertoachieveamaximumseparationpower.

Wefoundthatapolarstationaryphase(i.e.ethylpyridinebondedsilica)associatedwithamixof98%acetonitrileand2%water,asstrongsolvent ledtothebestchromatographicsystem.Underoptimizedconditions, the

numberofpeaksdetectedbyUVspectroscopycouldreachmorethan120in22min.Somefirstpreliminaryresultsusingasimplequadripolemassspectrometerwereshowntoillustratethepotentialinterestofsuchahyphenated

system.Togofurtheron,weanalyzedbiomassfastpyrolysisoilsusingahighresolutionmassspectrometer(SFC-IT-Tof/MS).Resultsshouldbesoonsubmittedinadedicatedpaper.

AppendixA.SupplementarydataSupplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,athttp://dx.doi.org/10.1016/j.chroma.2017.06.003.

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AppendixA.SupplementarydataThefollowingareSupplementarydatatothisarticle:

MultimediaComponent1

Highlights

• AnovelSFC-UV/MSmethodfortheanalysisofabiomassfastpyrolysisoil.

• Optimizationbasedonacomplexindustrialsampleusingthreespecificcriteria.

• Separationofmorethan120peaksbySFC-UV.

• ComplementaryinformationobtainedwithAPCI+/−andESI+/−modes.