仪器分析CHAPTER 16

Supercriticalfluidchromatography

MaryEllenP.McNally

16.1INTRODUCTION

Whatisasupercriticalfluid?

Thediscoveryofsupercriticalfluidsoccurredin1879,whenThomasAndrewsactuallydescribedthesupercriticalstateandusedthetermcriticalpoint.Asupercriticalfluidisamaterialaboveitscriticalpoint.Itisnotagas,oraliquid,althoughitissometimesreferredtoasadensegas.Itisaseparatestateofmatterdefinedasallmatterbybothitstemperatureandpressure.Designationofcommonstatesinliquids,solidsandgases,assumestandardpressureandtemperaturecondi-tions,orSTP,whichisatmosphericpressureand01C.Supercriticalfluidsgenerallyexistatconditionsaboveatmosphericpressureandatanelevatedtemperature.Figure16.1showsthetypicalphasediagramforcarbondioxide,themostcommonlyusedsupercriticalfluid[1].

Critical point

p

Liquid

Solid

Gas

1 atm

T

Fig.16.1.Phasediagramforcarbondioxidecriticaltemperature31.31Ccriticalpressure72.9atm.

ComprehensiveAnalyticalChemistry47S.AhujaandN.Jespersen(Eds)

Volume47ISSN:0166-526XDOI:10.1016/S0166-526X(06)47016-1r2006ElsevierB.V.Allrightsreserved.

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TABLE16.1

Comparisonofphysicalpropertiesofliquids,gasesandsupercriticalfluidsPhasetypeGasat1atm,211CSupercriticalfluidLiquid

Source:VanWassenetal.[2]

Density(g/cm3)10–30.3–0.81

Diffusion(cm2/s)10À110À3–10À4o10À5Viscosity(g/cms)10À410À4–10À310À2Thecriticalpointofamaterialisthetemperatureandpressureconditionsatwhichtheliqiudstateceasestoexist.Asaliquidisheated,itbecomeslessdenseandstartstoformavaporphase.Thevaporsbeingformedbecomesmoredense,withcontinuedheatingtheliquidandvapordensitiesbecomeclosertoeachotheruntilthecriticaltem-peraturepointisreached.Atthissamepoint,theliquid-lineorphaseboundarydisappears.Thiscriticalpointwasfirstdiscoveredandre-portedin1822byBaronCharlesCagniarddelaTour.

Asafluid,thesupercriticalstategenerallyexhibitsproperitiesthatareintermediatetothepropertiesofeitheragasoraliqiud.InTable16.1,thephysicalpropertiesofliquids,gasesandsupercriticalfluidsarecompared[2].ExaminationofthevaluesinTable16.1makesthein-termediatenatureofasupercriticalfluidmoreobvious.Thedensityofasupercriticalfluidapproachesthelevelsofaliquidasdoesitsdiffusi-vity,whileitsviscosityissimilartoatypicalgas.Thesepropertiesofferrapidmovement(equilibration)asinagas,butsolvationorsolu-bilizationasfoundinaliquid.Thebestofbothworldsfromachromato-graphicviewpoint.Thisisbecauseasasolutetravelsthroughachromatographiccolumnthenumberofequilibrationpointsitreaches,asdefinedbytheVanDempterequation,defineshoweffectivetheseparationwillbe,eitherthroughthenumberoftheoreticalplates,N,theresolution,Rs,orthealphavalue,a,alsoknownasaseparationfactor.Foradetaileddescriptionofthetheoryofchromatography,thereaderisreferedtoSnyderandKirkland’stextModernPracticeofLiquidChromatography[3]orChapters12,14,15ofthistext.

Theeaseofsolubilizingtheanalyteisakeyfactoralso.Thisisbe-causetheeasieritistogetthesolutetothesepotentialequilibrationpointsinsidethechromatographiccolumnwhenthefluidismovingwithhighdiffusivity,thefastertheequilibrationsorseparationcantakeplace.Comparedtoagaswherethesolubilitiesoftheanalytesof

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interestisalmostnon-existent,theliquid-likedensityofasupercriticalfluidoffersenhancedsolubilization.

Insupercriticalfluidchromatography,fluidsabovetheircriticalpointareusedasmobilephases.Thischapterdiscussestheprinciplesofoperation,mobilephaseconsiderations,parametersthatcanbead-justedinmethoddevelopmentaswellasanoverviewofinstrumenta-tionrequiredandafewpertinentexamplesfromcurrentliterature.Noteverythingcanbeillustrated,buttheadvantagesofthisdiversetechno-logywillbehighlighted.

16.2HISTORYOFSFC

Intermsofchromatography,thefirstindividualcreditedwiththeuseofsupercriticalfluidsasthemobilephaseisErnstKlesperwhenin1962hereportedontheseparationofmetalporphyrinsusingdense-gaschromatography(GC)orSFC[3].Butitwasnotuntilthe1980sthattheanalyticalcommunitytookholdoftheabilitiesandadvantagesofthetechniquewiththeadventofseveralcommercialinstrumentationventures.

Twoapproachesweretakenduringthistime,onebygaschromato-graphersandtheotherbyliquidchromatographers.Practicinggaschromatographerswhoexperimentedwithsupercriticalfluidchromato-graphyforitsenhancedsolvatingpowerspursuedthefirstapproach.Theycoupledsupercriticalfluids,ingeneralpurecarbondioxide;withsmallnarrowborecapillarycolumns,similartoleadingGCcolumnsofthetime.WiththatcouplingtheywereabletogetenhancedresolutionofcompoundsthataretoodifficulttoanalyzebyGC,becausetheywerenotsolubleinthenitrogenandheliummobilephasescommonlyusedinGC.

Thesecondapproachwastakenbypracticingliquidchromato-graphers.Theyroutinelydealtwiththermallylabile,highlypolarmole-culesandfrequentlysacrificedresolution,andspeedintheirsepara-tionsbecauseoftheaqueousmobilephasesthatwererequired.Withtheenhanceddiffusionanddecreasedviscosityofsupercriticalfluidsoverliquids,chromatographicrun-timeandresolutioncouldbeim-provedwhensupercriticalfluidswereused.Butsolubilityinpurecar-bondioxidemobilephases,whichhasthesolvatingpowersfromhexanetomethylenechlorideundernormaldensityranges,wasaproblemforthesepolarmolecules.Tocompensateforthis,experimentalistsstartedworkingwithmixedmobilephases.Thesemixedphaseswerebasedon

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theadditionofpolarmodifiersoradditivestothecarbondioxidemobilephase.Similartothefirstapproach,thissecondgroupofscientistscontinuedtousethecolumnswithsimilardimensionstoliquidchroma-tography(LC)columnsprevalentinthe1980sthattheywerealreadyusing,i.e.,25cmby4.6mm;thecolumnswerealsofunctionalizedwiththecommonC-18,C-8andphenylphasesasfoundinLC.

Withboththeseapproachesrapidlybeingpursuedbyawidevarietyofacademic,industrialandinstrumentcompanylaboratories,thegrowthandpublicationrateinsupercriticalfluidchromatographydur-ingthemid-late1980sandtheearly1990swasexponential.Awidevarietyofapplicationsareavailablefromthattimeperiodaswellasseveralreferencesthatexplaintheadvantagesofoptimizingasuper-criticalfluidseparationusingalltheparametersavailableinSFCcom-paredtoGCorLC[4–8].

16.3BASICPRINCIPLESINSFC

ThemostcommonandwidelyusedsupercriticalfluidinSFCiscarbondioxide.Itisinert,inthatitisnon-toxicandnon-flammable,italsohasmildcriticalparameters,alowcriticaltemperatureof31.31Candacriticalpressureof72.8atm[1].Usingpure,supercriticalcarbondi-oxideeliminatesorganicsolventwasteandwithitwastedisposalcostsandconcerns.Thisisextremelypracticaladvantageintheindustrialenvironmentwherethegenerationofwasterequiresspecialhandlingandsignificantcost.

Beyondthispracticalconsideration,theadvantagesofSFCfromatechnicalperspectiveareinthewiderrangeofparametersthatcanbeoptimizedtoachievethebestseparation[4].Takenfromthesametwoapproachesdescribedabove,theparameterthatismostcommonlyad-justedtochangeresolutionandretentiontimeinGCisthetemper-ature,afterthattheonlychoiceanexperimentalistmighthaveistochangethecolumnusedforseparation.However,fromtheliquidchromatographersperspective,themobilephasecompositionistheprincipalcomponentthatcanbechangedtoeffectabetterseparation,themobilephasecomponentsaregenerallythesecondchoiceandthenfinallythechoiceofchromatographiccolumn.UsingsupercriticalfluidchromatographygivesalltheparametersoptimizedinLCaswellastheabilitytoseedrasticretentionchangesduetoatemperaturegradient,asinGC.Inaddition,SFChastheaddedparametersofpressureand/ordensitythatcanbeselectedtoachievethebestseparationconditions.

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Fromanoverallperspective,boththetemperatureandpressureofthemobilephasecontrolthedensityorelutionpower.Achangeineithertemperatureorpressurechangesthemobilephasedensityandwillalterthechromatographicelution.ItshouldbenotedthatincreasingthetemperatureinSFCincreasestheretentiontime,generallyanun-desirableeffectandtheoppositeeffectthatisexhibitedinGC.Becauseofthis,reversetemperaturegradients,fromahightemperaturetoalowtemperature,areutilizedinSFCtodecreaseretention.Withde-creasingtemperature,densityofthefluidincreases;thesehigherden-sitymobilephasessolubilizetheanalytesofinterestandresultsinearlierelutionofthecompoundsofinterest.

ThesolvatingabilityofcarbondioxideinSFCissignificantwhencomparedtogasesusedinGC.Thisisbecauseasapurecomponent,achangeindensityofcarbondioxidecausesachangeinthematerial’sHildebrandsolubilityparameter.Incarbondioxide,thisrangeofsolu-bilitiescanbeconsideredequivalenttothesolubilitiesseenfromhexanetomethylenechloride.However,intermsofsolvatingmoder-atelypolarorhighlypolarmolecules,thissolubilityparameterrangeisnotsufficientandmodifiersoradditivestocarbondioxidemustbeincluded.Onceamodifierhasbeenaddedtothemobilephase,thecriticalparametersoftheoriginalsolventarenolongervalidandatwo-phasesystemcanexist.Asanexample,a10%methanolincarbondioxidesolutionhasacriticaltemperatureof51.51Cwithacriticalpressureof74.2atm.Thecriticalparametersofpuremethanolare240.51C,temperature,and78.9atm,pressure.Fromthesevaluesitcaneasilybedeterminedthatthereisnolinearrelationshipforcriticalpointchangewhenthecomponentsofatwo-phasesystemaremixedtogether.Whatshouldbenotedthoughisthatabovethecriticalpointofamixture,themobilephaseisonephase.Thisisextremelyimpor-tantinchromatography,anequilibrium-basedprocess,wherebydefi-nitionthemovementoftheanalyteisbetweentwophases.Ifoneofthephasesisanotapurecomponent,equilibriumisdifficulttoreproduceprecisely,theanalyteisnottransferringbackandforthbetweenthestationaryphaseandthemobilephase,butinsteadbetweenthreephases,thestationaryphaseandatwo-componentmobilephase.Thisdiscussionemphasizesthat,fromapracticalstandpoint,oneofthekeyconsiderations,oftenneglectedbyinitialpractitioners,istheneedtomaintainthemobilephaseaboveitscriticalpointatalltimesduringthechromatographicseparation.Thismaybedifficultwiththewiderangeofparametersthatareadjustedtooptimizeaseparation.

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16.3.1

Parameteroptimization

Beingabletochangethedensity,viaeitherchangesinpressureortemperature,isthekeydifferenceinSFCoverGCandLCseparations.Typicaldensityrangesarefrom0.3to0.8g/mlforpurecarbondioxide.Table16.2showsdataobtainedfromISCO’sSF-SolverProgramforthecalculationofdensity(g/ml),HildebrandSolubilityParameterandarelativeequivalentsolventforpurecarbondioxideataconstantpres-sureof6000psi,approximately408atm.

ButtherealadvantageinSFCisnotjusttheabilitytoadjustthedensityofthemobilephasebuttheabilitytoadjustitfromtwodif-ferentdirections.Densitychangesachievedbyachangeinpressurecanyielddifferentseparationfactorsthendensitychangesachievedbyad-justingthetemperature.Thisoffersanadvantageinmethoddevelop-mentbySFCnotavailableinGCorLC.

BeyondthedensitychangesthatcanbeusedtocontrolmethodmodificationsinSFC,themobilephasecompositioncanalsobead-justed.TypicalLCsolventsarethefirstchoice,mostlikelybecauseoftheiravailability,butalsobecauseoftheircompatibilitywithanalyticaldetectors.Themostcommonmobilephasemodifiers,whichhavebeenused,aremethanol,acetonitrileandtetrahydrofuran(THF).Additives,definedassolutesaddedtothemobilephaseinadditiontothemodifiertocounteractanyspecificanalyte–columninteractions,arefrequentlyincludedalsotoovercomethelowpolarityofthecarbondioxidemobilephase.Aminesareamongthemostcommonadditives.

TABLE16.2

Calculationofdensity,HildebrandsolubilityparameterusingISCO’sSFsolverprogramatconstantpressureof6000psi(408atm)Temperature(1C)404955647073798594

Density(g/mL)0.9670.9370.9170.8860.8650.8550.8340.8150.785

Hildebrandsolubility8.2487.9027.8147.5547.3757.2877.1136.9446.694

EquivalentsolventC6H12C2H4CF4C8H16C6H16O2C4H10C8H18F2

CyclohexaneEthylene

Carbontetrafluoriden-Octanen-HexaneOxygenn-Butane

2-TrimethylpentaneFluorine

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ModifiersandgradientcompositionsinLCareusedbroadly.

Agradientthatrunswith30–80%methanoloracetonitrileisnotuncommon.Thisamountofmodifierisgenerallynotneededinsuper-criticalfluidchromatographytoaffectthesameseparation.TypicalmodifiercompositioninSFCis1.0–10%andwouldachievehigherHildebrandSolubilityParameteradjustmentoverallthanthebroadergradientsfoundinLC.

16.3.2

Instrumentrequirements

Themajordifferenceinsupercriticalfluidchromatographyandcon-ventionalLCequipmentisthepumpingsystemsaswellasthesafetyfeaturesinstalledtomaintainhigherpressure.UniqueSFCequipmentdifferencesare:1.2.3.4.5.6.

Carbondioxidetankformobilephasesupply

a.EquippedwithapressurereliefvalueandrupturediskHigh-pressurepump

a.ChillertomaintainmobilephaseinliquidstateHigh-speedinjectorPressurerestrictor

a.High-pressuretubing

High-pressureflowcellforUVdetection

Solventcollectiondevicewithabilitytoventtoalaboratoryhoodorelephanttrunk.

Carbondioxideisusuallypurchasedinatank,insidethetankthemobilephaseexistsasaliquid.Typically,thetankdoesnotcomewithapressuregaugebutishookeduptoapressurereliefvalveandrupturedisk,whicharesetabovethetankpressureshouldatankleakoccur.High-pressurepumpsusedinSFCcancomeinavarietyoftypes;mostofthemaremodificationsofpumpsusedinLC.Piston,diaphragmandsyringepumpsareused.

Thepumpsdeliverthemostaccurateflowofthecarbondioxideifitispumpedintheliquidstate.Sincethatisthecase,ifthereisagreatdistancebetweenthetankandthepumpthenachillerisusuallyplacedsothatthetubingcontainingthecarbondioxidefromthetankcanbecooledmaintainingthecarbondioxideintheliquidstate.Mostcom-merciallyavailablepumpsoperateathighenoughpressuresthatthepumpheadsorpumpbodiesdonotneedancillarycooling,butoldermodelsfrequentlyrequiredachillerthatprovidedacirculationofcold

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fluidstothepartsofthepumpwherethecarbondioxidehasapotentialtovaporizeoutoftheliquidstate.

Ahigh-speedinjectorisrequiredinsupercriticalfluidchromato-graphy.Thisistopreventlossofpressureduringtheinjectionprocess.Thereareavarietyofrestrictortypesthatcontrolthepressureofthefluidduringthechromatographicprocess.Eachmanufacturerhaspatentedtheirindividualrestrictiondevices.Itisbeyondthescopeofthistexttogointothedetailaboutalloftheserestrictors.However,bywayofexplanation,mostofthemechanicalrestrictorsthatareusedincommercialequipmentsoperateundertheprincipleofdecreasingavolumeoraspacethatthemobilephasemustpassthroughbysomemechanicalmeans.Thisdecreaseinvolume,ifmeteredaccurately,in-creasesthepressureinthesysteminacontrolledmanner.Asasafetyprecaution,foranyequipmentwhereelevatedpressuresareused,tub-ingshouldberatedwithasafetyfactorofatleast1.5timesthemaxi-mumpressuretheSFCsystemcanachieve.

Becausethedensityisacontrollingfactorinachievingthesepara-tion,maintainingtheelevatedpressureusedintheseparationisneces-saryuptothepointofdetection.ForUVmeasurements,thisrequiresahigh-pressurecellalsoratedtothemaximumpressuretheSFCsystemcanachievewithadesiredsafetyfactorof1.5times.Forotherdetectionmethods,i.e.,flameionizationdetectionandmassspectrometry,wherethesampleisnebulizedintothedetector,theoutputoftherestrictorisgenerallyrightatthenebulizationpoint.Thispositioningofthecolumnoutleteliminatesanypeakmergingthatcouldoccurunderlow-orno-pressuremovement.16.4

CURRENTEXAMPLES

Theexamplesillustratedhereinarenotall-inclusivebutshouldgivearepresentationoftheadvantagesofthetechniqueanditsmostcommonuses.

16.4.1

Chiralseparations

Theuseofsupercriticalfluidstoseparateenantiomersisoneofthemostimportanttasksinseveralareasofresearch,especiallypharma-ceuticalsandagrochemicals.Thisisbecauseitiswellknownthatthetwoenantiomericformsofamoleculecandisplaydramaticallydif-ferentbiologicalactivity.Theuseofsupercriticalfluidstoseparate,withhigherefficienciesandshorterretentiontimes,enantiomersis

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extremelyadvantageousinthisdifficultwork.Intheirarticleentitled‘‘Chiralseparationofsometriazolepesticidesbysupercriticalfluidchromatography,Toribioandcoworkers,separatedsixtriazolepesti-cidesandshowedtheeffectsofdifferentorganicmodifiersontheres-olutionandretentionviak0value[9].Themodifierstheyexaminedweremethanol,ethanoland2-propanol.Theadditionofadditiveswasalsoexamined.Table16.3showssomeoftheirmodifierresults.Ingeneral,separationscouldbeconductedinlessthan10minandthebestmodifierfortheindividualseparationswascompounddependent.This

TABLE16.3

Valuesofcapacityfactorsandresolutionsobtainedfordiniconazole,

tetraconazole,hexaconazoleandtebuconazoleusingdifferentmodifiers[9]CompoundHexaconazole

Methanol(%,v/v)51015

Ethanol(%,v/v)51015

2-Propanol(%,v/v)51015

TetraconazoleMethanol(%,v/v)51015

Ethanol(%,v/v)51015

2-Propanol(%,v/v)51015

k10

k20

RsCompound

k10

k20

Rs5.355.680.672.332.591.021.431.610.875.146.232.412.012.281.141.191.320.654.385.010.542.712.900.481.451.450

Tebuconazole

Methanol(%,v/v)513.2314.741.28105.265.821.17153.113.421.03Ethanol(%,v/v)57.167.160105.045.040152.692.6902-Propanol(%,v/v)520.3622.530.98107.618.351.2153.543.911.15Diniconazole

Methanol(%,v/v)57.2912.446.08102.815.696.90151.633.435.87Ethanol(%,v/v)56.9710.634.55102.533.873.77151.372.083.112-Propanol(%,v/v)512.4621.061.23104.094.090152.042.040

4.305.231.55

2.052.411.271.061.831.024.265.293.052.242.842.491.681.982.065.868.754.983.285.464.012.403.693.43

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isconsistentwiththeunderstandingofmodifierinteractionswiththesolutesandtheireffectontheoverallequilibriumbetweenthestation-aryandmobilephasesinSFCaspreviouslydescribed.Recalling,ac-ceptableresolution,Rs,valuesforquantitationpurposesaregreaterthan1.5,valuesthataregreaterthan1.5forresolutionarehighlightedinTable16.3.Figure16.2showsanexamplechromatogramofatypicalseparation.TheseseparationswereconductedonaHewlett-Packard1205Asupercriticalfluidchromatographequippedwithaphoto-diodearraydetector;detectionwasat220nm.AChiralpakAD25cmÂ4.6mmcolumnpackedwiththe3,5-dimethylphenylcarbamatederivativeofamylose,coatedona10mmsilicasupportwasused.ThemobilephasewascarbondioxidemodifiedasoutlinedinTable16.3foreachoftheindividualseparations.Otherchromatographicconditionswere351C,2ml/minflowrateandapressureof200bar(2960atm)(Fig.16.3).(Note:Becauseofthelowtemperatureoftheseparations,themobilephasewasnotlikelyinthesupercriticalstateforallofthemodifierconcentrationsexaminedinthisreport.)

16.4.2

Polymerseparations

Theadvantagesofsupercriticalfluidchromatographyforpolymersep-arationshavebeenillustratedintheliteratureformanyyears.Arecentexampleistheseparationoflong-chainpolyprenolsusingSFCwithmatrix-assistedlaser-desorptionionizationTOFmassspectrometry[10].Thegenericnamefor1,4-polyprenylalcoholsispolyprenol;thesecompoundsgenerallyhavesmallerpolymerizationchainsofless

200150100500

0(A)mAU246Min.810200150100500

0mAU(B)246Min.

810Fig.16.2.Enatiomericseparationofhexaconazoleat200bar,351C,2ml/minand10v/v2-propanol.(A)Withoutusingadditives;(B)using0.1%(v/v)tri-ethylamineand0.1%(v/v)trifluoroaceticacid[9].570

Supercriticalfluidchromatography

Fig.16.3.Enatiomericseparationat200bar,351C,2ml/min.(A)Tetraconazolewith4%(v/v)ethanol;(B)diniconazole15%(v/v)ethanol[9].

than30monomers.ChromatographicseparationofthesechainswasconductedusingaJascoSuper-201chromatograph.Thesystemhastwoseparatepumps,onedeliversthecarbondioxide,andtheotherdeliveredtheTHFmodifier.Thecolumntemperaturewas801C,thepressure,controlledbyabackpressureregulatorwascontrolledat19.6MPa.AnInertsilPh-3(25cmÂ4.6mm),5mmcolumnwasused.AflowgradientwasusedtointroducetheTHFmodifier,thisisnottyp-ical,butitispossiblewiththetwopumpsonthisinstrument.Theconsistentflowrateofthecarbondioxideis3.0ml/min;theTHFflowratestartedat0.8ml/minandwasadjustedover30min-to2.0mL/minandthenheldconstant.Figure16.4illustratestheresultantchromato-gramobtainedforEucimmiaulmoidesleaves,aplantproducingfibrousrubber;over100-mer(MW:6818)componentswerecompletelysepa-ratedusingtheconditionsdescribedabove.

16.4.3Highthroughputscreeningofpharmaceuticals

AtAbbottLaboratories,HochlowskiandcoworkersusedpreparatoryscaleSFCtoscreentheoutputofhighthroughputorganicsynthesis(HTOS)forpuritylevelsandclassifychemicalstructuresintolibrariesofsimilaranalogs[11].ThesescientistsusedaBergerInstrumentsSFCself-modifiedforthisparticularuse.Figure16.5showstheschematicofthismodifiedsystem.Inthisapplication,theauthorsreportedaddingSFCcapabilitytotheirrepertoireofinstrumentationtosolvetheiranalyticalchallenges.Figure.16.6showsthecomparisonofatypicalreactionmixtureanalyzedbybothhighperformanceliquidchromato-graphy(HPLC)andSFC.

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38

100

20

01020Min

304050Fig.16.4.SFCchromatogramoflong-chainpolyprenolsinE.ulmoidesleaves.ThesamplehadMn3.99Â103(calibratedagainstcis-1,4-polyisoprenestand-ards)andMw/Mn1.41.Thenumbersinthechromatogramrepresentdegreesofpolymerizationforpolyprenolhomologues[10].

Autosampler

CO2 deliverysystem

Customsoftware

Methanol wash

system

Dual armfraction collector

Fig.16.5.Preparativesupercriticalfluidchromatographicsystem,customizedforhighthroughputorganicsynthesis(HTOS)screening[11].

Actualoperatingconditionscanbefoundinthefigurecaption.AgivenlimitationofSFC,relativetoHPLC,asdescribedistheabilitytodissolvesamplesinasolventsystemcompatiblewiththemethanol/carbondioxidemobilephase.Forthisparticularmobilephase,othercompatiblesamplediluentsthatworkedeffectivelyarepuremethanol,

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1000750mAU50025000

(a)11008250mAU65002750

0

(b)

0

1

2

31

3

4

1

2

3

54

Minutes2

6

7

8

9

10

1

23

4

45Minutes

678

Fig.16.6.(a)HPLCchromatogramsforreactionmixtureA.HPLCconditions:C18(20Â100mmNova–PakTM),gradient5–95%acetonitrile/water,with0.1%TFA;(b)SFCchromatogramforreactionmixtureA.SFCconditions:Diolcolumn(21.2Â150mm,BergerInstruments),gradient5–60%Methanolwith0.5%dimethylethylamineincarbondioxide[11].

ormixturesofeithermethanolandacetonitrileormethanolanddi-chloromethane.Dimethylsulfoxide,DMSO,ontheotherhandwasnotcompatiblewiththecarbondioxidemobilephaseused.

16.5CONCLUSIONS

Supercriticalfluidsofferpropertiesintermediatetogasandliquids.Assuch,supercriticalfluidchromatographyisanalternativetechniquetobothLCnotliquidandorGC.Thedistinctadvantagesofsupercriticalfluids,becauseofgaslikedensitiesyieldsfasterchromatographicelu-tionandthereforeshorteroverallruntimesthaninLC.Greatersol-vatingpowerthangases,makesitmoreapplicabletoawidervarietyofanalytesthatcantypicallybeanalyzedbyGC.AbilitytooptimizeawidervarietyofparametersthanGCorLCisalsoadvantageous;den-sity,temperatureandpressure,mobilephasecomposition,gradientel-utionandtheadditionofadditivestothemobilephaseareallavailable.But,thechallengetotheanalyticalchemistortechnicaloperatoristhatthereismoretounderstandtoutilizethetechniquecorrectly,workin

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theoptimizedrangeofaphasediagramanddealwiththehigher-pressureequipmentandsafetyprecautionsthatarenecessary.Becauseofthiscomplication,SFChasnotreplacedLCorGCinthemainstreamanalyticallaboratoryalthoughithasthecapabilitytodoso.

REFERENCES

SupercriticalFluidExtraction:PrinciplesandPractice,M.A.McHughandV.Krukonis,2ndEdition,Butterworths-Heinemann,1994,608.ISBN:0750692448

2U.VanWassen,I.SwaidandG.M.Schneider,Angew.ChemieInt.Ed.

Engl.,19(1980)575.

3IntroductiontoModernLiquidChromatography,L.R.Snyderand

J.J.Kirkland,2ndEdition,WileyInterscience,1979

4SupercriticalFluidsinChromatographyandExtraction,R.M.Smithand

S.B.Hawthorne,Elsevier,1997,414,ISBN:0-444-82869-95J.A.CrosandJ.P.Foley,Anal.Chem.,62(1990)378–386.

6M.E.McNallyandJ.R.Wheeler,J.ofChromatogr,477(1988)53–63.7M.E.McNallyandJ.R.Wheeler,LC/GCMagazine,6(9)(1988).8M.E.McNally,Anal.Chem.,67(9)(1995)308A–314A.

9L.Toribio,M.J.delNozal,J.L.Bernal,J.J.JimenezandC.Alonso,

J.ChromatogrA.,1046(2004)249–253.

10T.Bamba,E.Fukusaki,Y.Nakazawa,H.Sato,K.UteT.Kitayamaand

A.Kobayashi,J.Chromatogr.A.,995(2003)203–207.

11J.Hoxhlowski,J.Olson,J.Pan,D.Sauer,P.SearleandT.Sowin,

J.Liq.Chromatgr.&Rel.Tech.,26(3)(2003)3333–3354.1

REVIEWQUESTIONS1.

DescribetheparametersthatareavailabletothepracticingchromatographerinLC,GCandSFCtoconductmethoddevelop-mentandobtainthebestresolutionofthecomponentsofamixture.

WhatistheprincipalrequirementinSFCinstrumentationtocon-trolthepressure?

Definethecriticalpointofasubstance.

Describethechangethatoccursinthecriticalpointofasubstanceifasecondcomponentisadded.

WhatisthemainadvantageofSFCoverLCandGC?Howisthisobtained?

2.3.4.5.

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