Development of a supercritical fluid chromatography method ...
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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,⁎
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.