Determining Aromatic & Aliphatic Carboxylic Acids in Biomass-Derived Oils Using 2,4-Dinitrophenylhydrazine & HPLC-Tandem MSn

Samuel A. Lewis, Sr., (lewissasr@ornl.gov), R. Maggie Connatser, James R. Keiser*, Mariefel V. Olarte^, Michael Brady*, Donovan Leonard**Oak Ridge National Laboratory, Oak Ridge, TN 37831^Pacific Northwest National Laboratory, Richland, WA

Experimental

Results and Discussion

Impact

Contact information: lewissasr@ornl.gov; 865-946-1240; Oak Ridge National Laboratory, The NTRC Bldg., 2360 Cherahala Blvd., Knoxville, TN, USA, 37932Oak Ridge National Laboratory; 1 Bethel Valley Rd., Oak Ridge, TN, USA 37830

Research sponsored by the U.S. Department of Energy, Bioenergy Technologies Office

Withtheconcernssurroundingthelimitedsupplyoffossilfuelanditsadverseeffectsontheenvironment,theneedtodevelopasustainablebio-derivedfuelbecomesmorepronounced.Sourcesasdiverseasnon-foodgrasses,hardandsoftwoodtrees,shrubs,algae,andevenagriculturalandmunicipalwastestreamsarebeinginvestigatedassourcesofbiologicallyrenewablefuelsandchemicals.Byexaminingthechemicalstructuresofligninandcellulose,itiseasytoseehowcarbonylsandcarboxylicacidscanbeformedduringthepyrolysisprocess.Suchpotentiallyreactivespeciesoftencreateobstaclestoupgradingchemistriesviafouling,catalyticdeactivation,corrosion,orproductdestabilization.

• Accurateorganicacidspeciationfrombio-oilshelpsexplaincorrosionandcouldofferinsightstoavenuesofreduced-costupgradingwithhighervalueyields.

• Verifiedderivatizationoforganicacidsinbio-oilusingDNPHisachieved,eveninthepresenceofsignificantamountsofaldehydesandketones,providedadequateDNPHisavailableinexcesstoreactwithallspecies.

• Figure1affirmsthatsomeacidsarederivatizedduringthetreatmentofbio-oilevenusingtheconventionalDNPHapproach,butco-elutionwithhydroxyaldehydeisobarsskewsbothqualitative& quantitativeevaluation.

• Fragmentationofaliphaticacidsversusaromaticacidsgivesauniquemassspectrum,asnotedincomparingthecharacteristicionfragmentationpatternsshownbelowinFigures2and3.

• AcarefuladjustmentofthepHduringtheextractionprocedureandextendedheatingtimeoftheDNPHsolutionallowedforthesuccessfulderivationofaliphaticandaromaticcarboxylicacids.

• Theprocesstocreatethisbio-oilusedacatalystthattargetedthedeoxygenationofthecarbonylgroupspreferentiallytothealcohols,butthemoreconventional,rudimentaryanalyseswereunabletorevealthefactthatalargecomponentoftheconversionwasendinginbifunctionalmoleculesthatmaintainedatleastonecarbonylfunctionality.

• Fromanenergyandenvironmentalperspective,thefullerunderstandingofwhatcarboxylicacidspeciesarepresentinbiomass-derivedoilsbenefitsbio-oilupgradingresearchbyfillingininformationgapsaboutchemicalcatalystmechanismsandcorrosionpathways.

• Abilitytomeasurelargeacidsinthepresenceofothercarbonylsbenefitsabroaderaudience,includingareasofresearchspanningadiverserangefrombacterialsugarmetabolismtointernalcombustionengineemissions.

• Withagreaterawarenessofaliphaticacidspresent,separationprocessescouldcombinewithmilder,less-energy-intensivetreatmentstrategiesforhigher-valueproducts.

Waterdilute,sonicate,adjusttopH6

MeCl2 rinse,withdrawandadjustwaterphasetopH3

MixwithDNPHinMeOH;react5hrs.at80°C

MeCl2 rinse;drydown;bringupinACN

Deprotonatealllargeacids

Cross-Section SEM Microprobe Maps

STEM Cross-Section Image and Elemental Maps

• Crosssectionsof316Lstainlesssteel,above,showevidenceofreactionbetweenhotpyrolysisproductsandalloy.Thisdegradationissimilartothepreferentialgrainboundaryattackseeninother300seriesstainlesssteelcomponents.

• Bulktestscharacterizebio-oilsbasedonpropertiessuchasviscosity,overallacidity,andtotalcarbonylcontent,butcertainpossiblyimportantcompoundclassesareoverlooked.

• Morespecificchemicalanalysesfromnon-biomassresearchareas(i.e.air/exhaustsampling)canoffermoredetailedinfooncompositionbutcansufferinterferencesfrombio-oiltheyarenottailoredtoandaretherebymisleadinginsomecases.

• HPLC-ESI-MS/MSisusedtodiscernderivatizedcarboxylicacid-containingmoleculespresentinbio-oilsfromthealdehydesandketonestraditionallymeasuredinairmonitoringusinganoptimizeddinitrophenylhydrazine todinitrophenylhydrazone reaction.

• Bio-oiltestedforthisworkwasproducedfromamixofpine,forestresidueandindustrialconstructionwasteattheNRELinGolden,CO,andupgradedatPNNLinRichland,WA.

Sample Preparation &Derivatization

Highperformanceliquidchromatography

Negativeionmodeelectrospray

ionization&tandemmassspectrometry

Identificationofdistinctivefragmentsby

comparisonwithstandards,literature

Separation &Structural

Determination

Reducechar,sugar

backgroundspecies

CreateDNP-hydrazone**derivativesofacidsbyaddingenoughenergy(heating,time)tothesystem&guaranteeingprotonremainswith-COOH

group(pH3)todiscouragetheresonance

stabilizationthatusuallydefeatsthehydrazoneformationreaction

MassSpec:BrukerEsquire6000IonTrap

OperatedinNegESImode;H2O,EtOH,&10%

NH4OHaddedtoLCeffluentviasyringepumptoinsureionization

oftheDNP-hydrazones**

Massscanrange:50-320amuSecondaryionization

voltage:1volt

HPLC:HP1100Column:WatersX-BridgePhenyl

3x100mm,3.5umparticlesColumnTemp:

50°CMobilePhase:ACN/water

Reaction*ofDNPHwithcarboxylicacid,followedbyketo-enoltautomerization**.

*FromUchiyama,etal.AnalyticalChemistry,Vol.76,(19),2004:5849-5854.

formaldehydepropionic acidorhydroxypropanaldehyde

propionaldehyde

propionaldehyde withbutyricacidorhydroxybutyraldehyde

Figure1

TheDNPHreactionproceedsbyanucleophilicaddition-elimination.

ResonancestabilizationofthecarbonylCbythealcoholOinthecarboxylicacidrendersthatCmoreelectronegativeand,thereby,lesssubjecttonucleophilicattack.Largeracids,akinlargeraldehydesandketones,alsosuffersterichindranceandareslower,

moreenergy-intensivetoderivatize.

DNPhydrazide at wavelength 360 nm. Acetic acid, propionic acid,and butyric acid exhibit similar behavior with longer reaction timein order of increasing carbon number.It is postulated that carboxylic acids react with DNPH to

initially form corresponding hydrazone derivatives, which thenisomerize to hydrazides by keto-enol tautomerization.

The derivatives eluted from the DNPH cartridges were identi-fied by comparison of the HPLC retention times with authentic

samples prepared by direct reaction of the carboxylic acids withDNPH (Experimental Section). Two HPLC columns with differingselectivities (C18 and RP-Amide C16) were used to confirm theretention time assignments.Characterization. The structures of the synthesized hy-

drazides are consistent with the NMR data. 1H NMR data of thederivatives were as follows: formic DNPhydrazide 1H NMR (CD3-CN, TMS), δ (ppm): 7.25 (d 1H, J) 9.6, a), 8.28 (dd 1H, J) 2.5,9.4, b), 8.94 (d 1H, J) 2.8, c), 9.36 (s 1H, d), 8.49 (s 1H, e), 8.23(s 1H, f). Acetic DNPhydrazide 1H NMR (CD3CN, TMS), δ(ppm): 7.25(d 1H, J) 9.2, a), 8.25 (dd 1H, J) 2.75, 9.6, b), 8.92(d 1H, J) 2.8, c), 9.37 (s 1H, d), 8.49 (s 1H, e), 2.02 (s, 3H, CH3).Propionic DNPhydrazide 1H NMR (CD3CN, TMS), δ (ppm): 7.22(d 1H, J) 9.6, a), 8.23 (dd 1H, J) 2.52, 9.39, b), 8.91 (d 1H, J)2.29, c), 9.37 (s 1H, d), 8.48 (s 1H, e), 2.30 (t, 2H, CH2), 1.13 (t,3H, CH3). Butyric DNPhydrazide 1H NMR (CD3CN, TMS), δ(ppm): 7.21 (d 1H, J ) 9.15, a), 8.24 (dd 1H, J ) 2.75, 9.62, b),8.91 (d 1H, J ) 2.75, c), 9.39 (s 1H, d), 8.49 (s 1H, e), 2.27 (m,2H, J) 7.56, CH2), 1.66 (t, 2H, J) 7.42, CH2), 0.964 (t, 3H, J)7.327, CH3).Carboxylic DNPhydrazide derivatives demonstrated excellent

thermal stability on DSC analysis. Melting points are listed inTable 1 and are higher than those of corresponding hydrazonesby 32-50 °C.UV-visible absorption spectra of the carboxylic DNPhydrazide

derivatives (20 µmol/ L acetonitrile solution) are presented inFigure 2. For reference, the absorption spectrum of DNPH is alsoshown.The spectral profiles of carboxylic DNPhydrazide derivatives

exhibited similar curves. Specifically, acetic, propionic, and butyricDNPhydrazide showed much the same maximum absorptionwavelengths (333-334 nm) and molar absorption coefficients (1.42× 104 to 1.44 × 104 L/ mol/ cm) (see Table 1).Separation of Carboxylic DNPhydrazide Derivatives . Ana-

lytical conditions for formic, acetic, propionic, and butyric DN-Phydrazide derivatives were examined using a C18 and an RP-Amide C16 column. Figure 3 shows a chromatogram of C1-C4

(30) Budavari, S.; O’Neil, M.; Smith, A.; Heckelman, P.; Kinneary, J. The MerckIndex. An Encyclopedia of Chemicals, Drugs and Biologicals; Chapman andHall: London, UK, 1996; p 2576.

(31) The 2003-2004 Aldrich Handbook of Fine Chemicals; Sigma-Aldrich: St.Louis, MO.

Figure 1. The reaction of adsorbed formic acid and DNPH withtime. (λ ) 360 nm).

Table 1. Maximum Absorpt ion Wavelengths (λMax) andMolar Absorpt ion Coeffic ients (E) of C1-C4 CarboxylicAcid and Aldehyde DNPH Derivat ivesa

λmax(nm)

ϵ(L/ mol/ cm)

mp(°C)

formic DNPhydrazide 331 1.36 × 104 185.7acetic DNPhydrazide 333 1.42 × 104 201.5propionic DNPhydrazide 334 1.44 × 104 192.0butyric DNPhydrazide 334 1.44 × 104 167.1DNPhydrazine 351 1.55 × 104 ∼20030formaldehyde DNPhydrazone 349 1.91 × 104 153-15631acetaldehyde DNPhydrazone 359 2.10 × 104 165-16831propionaldehyde DNPhydrazone 360 2.10 × 104 152-15531butyraldehyde DNPhydrazone 361 2.18 × 104 117-11931

a Melting points of aldehyde-2,4-DNPhydrazones are the literaturevalues.

Analytical Chemistry, Vol. 76, No. 19, October 1, 2004 5851

Principles of Drug Action 1, Spring 2005, Carboxylic Acids Part 1

7

R' NR

O..

..R'

O

NR

..R'

O

O

H

R'

O

O....

..

..R' O

O

Carboxylic Acid Resonance Stabilization of the Conjugate Base

R'

O

NH

R....

Amide Resonance Stabilization of the Conjugate Base

Sulfonamides, can be regarded as "isosteres" of the amides in which the carbonyl grouphas been replaced with a SO2 moiety. Sulfonamides typically have pKas in the range of8-10 and thus are "stronger acids" than amides (pKas > 14), but "weaker acids" thatcarboxylic acids (pKas 3-5). The enhanced acidity relative to amides results because thenegative charge formed in the conjugate base can be stabilized over more electronegativeatoms as shown by the following resonance structures. Yet the charge forms on a lesselectronegtive nitrogen, and thus sulfonamides are less acidic than acids:

SO

ON

H

HSO

ON

HSO

NHO

SO

NHO

Base

It is also important to understand that the acidity of carboxylic acids may vary widely,depending on the overall structure of the acids. It was noted above that carboxylic acidstypically have pKas in the range of 3 to 5. Such is the case for the simplest acids, formicacid and acetic acid as shown below. Note the difference in pKa values for thesecompounds. Formic acid is nearly ten-times more acidic than acetic acid! In formic acidthe carboxyl carbon is attached to a hydrogen atom, while in acetic acid this atom islinked to a "methyl" group. Hydrogen atoms may be regarded as "electroneutral" atoms,while alkyl groups such as methyl as capable of electron donation by induction (+I)through sp3 hybridized bonds. Since the ionization associated with acidity generates anegative charge in the conjugate base (an anion), functional groups donating electrondensity toward the site of ionization have a "destabilizing effect" on the conjugate base.Thus the "additional methyl" group present in acetic acid impedes ionization and thusreduces basicity relative to formic acid:

HO

O H

Formic acid: pKa 3.75 Acetic acid: pKa 4.76

O

O HCH3

Objective:Developmentananalyticalmethodthatgivesinsightintocarboxylicacidsandotheroxygenatedspeciescontainedinbio-oils.

Background

TheMSMSofbio-oilconstituentofmass239,likelyaceticacid,showingtheformationofion167,indicativeofanaliphaticcarboxylicacid.Otherlikelyaliphaticcarboxylicacidswerefoundinthebio-oil,asidentifiedbythepresenceofthecharacteristic2° ionat167amu,andarelistedinthechart,inset.

TheMSMSofbenzoicacid(anaromaticacid),withitsmolecularionpeakofmass301amu,lacksthestrong167amu signalindicativeofaliphaticacids.Standardswerecreatedandanalyzedviatheexactsequenceofderivatization,chromatography,andspectrometryofbenzoicandseveralotherlargeracidscommontobio-oilsinordertovalidatestructuralidentificationbytandemMS.

Figure2

Figure3

1° Ion Mass 2° Ion Mass223 167237 167239 167267 167