SPE-通过固相萃取进行样品富集和纯化

PoorSamplePreparation[Yellowextract]PoorSamplePreparation[Yellowextract]SPE――通过固相萃取进行样品富集和纯化为何使用固相萃取（SPE）技术您需要从样品中去除特定干扰物，以免它们在目标分析物的检测和定量过程中影响实验结果。在此处所示的示例中，不适当的样品制备方案未能去除干扰物，导致提取物呈现出残留的黄色干扰物，色谱图中目标分析物与多个干扰峰发生了重叠。OptimizedSPE[Colorlessextract]您需要提高初始样品中目标分析物的浓度，以便所用的分析技术能够更轻松地对其进行检测和准确定量。如果目标分析物可被较强地保留，那么可能需要在 SPE色谱柱上加载较大的样品量，随后仅以极小体积的洗脱液将此分析物洗脱下来，由此提高样品中分析物的浓度。fraceEnrichment|AfterSPEAnalyteConcentrationOriginalSample您需要去除样品中的干扰物（即使不可见），这些干扰物会在质谱检测中抑制目标分析物的信号在此处的示例中，蛋白沉淀法无法去除血浆提取物中的磷脂，从而造成严重的离子抑制。优化的复合模式SPE方案可获取最纯净的提取物，并可在最大程度上降低离子抑制效应。100%ProteinPrecipitation:80%tonsuppression1100%-I- 一SPE:1 1 V 1 1MinimalionsuppressionI_WhatisSolid-PhaseExtraction(SPE)?Don'tbeconfusedbythetermsolid-phaseextraction[SPE].AtypicalSPEdevicehas50timesmoreseparationpowerthanasimple,singleliquid-liquidextraction.SPEisactuallycolumnliquid-solidchromatography.SinceSPE isliquidchromatography[LC],itspracticeis

governedbyLCprinciples.Asampleisintroducedintoacolumnoracartridgedevicecontainingabedofappropriateparticles,orotherform,ofachromatographicpackingmaterial[stationaryphase].Solvent[mobilephase]flowsthroughthebed.Bychoosinganappropriatecombinationofmobileandstationaryphases,samplecomponentsmaypassdirectlythroughthecolumnbed,ortheymaybeselectivelyretained.Individualcompoundsinthesampleeachtypicallyappeartotravelatdifferentspeedsthroughthedevice.Usingaweakersolventcausesthemtomoveslowlyand/orbestronglyretained.Astrongersolventspeedsuptheirpassagethroughthebedandelutestheanalyte(s)inamoreconcentratedvolume.ElutionfromanSPEdeviceisusuallydonebyincreasingthestrengthofthemobilephaseinaseriesofdiscrete,ratherthancontinuous,stepsduringwhichselectedanalytesorinterferencesareeitherfullyretainedorrapidlyeluted-thisvariationofgradientelutioncalleda stepgradient.Mostcommonly,SPEispracticedusingminiaturecolumnorcartridgedevices.Anexampleisshownhere.Amixtureofthreedyesisloadedontothecartridgeinaweaksolvent,causingstrongsampleretentioninanarrowbandthatappears blackatthecolumninlet.Subsequentgradientsteps,eachwithasuccessivelystrongersolvent,areusedtoelutethedyesindividually[yellow,red,thenblue].LoadSample EhtionElutionElutionStep3NOTE]DiHerentstrengthsolventscanbeusedtoisparot^rheLoadSample EhtionElutionElutionStep3NOTE]DiHerentstrengthsolventscanbeusedtoisparot^rheOneSPEco/tridgecanseporoteallthreedyesTypicalSPEcartridgesarelow-pressuredevices-constructedofsolvent-resistantplasticorglass-filledwithparticles>30卩mindiameter.Suitableflowratesmaybeachievedbygravityglass-filledwithparticlesorwiththeassistanceofvacuumorlowpositivepressure.[Thelatterrequiresputtingacapontheopeninletofacolumnorusingasealeddevicewithinletandoutletfittings.]ImportanceofSamplePreparationInthelasttwodecades,dramaticadvancesinanalyticalinstrumentationandlaboratoryinformationmanagementsystemsshiftedtheanalyst'spredominanttasksfromassaymeasurementstosamplepreparationanddataprocessing.Asthestringencyofrequirementsforhighersensitivity,selectivity,accuracy,precision,andnumberofsamplestobeprocessedhasescalated,thecorrespondingincreasesinspeedandsophisticationofanalysisanddatacollectionhaveoutpacedimprovementsinthemanytraditionaltechniquesofsamplecollectionandpreparation.Bysomeestimates,75to80%oftheworkactivityandoperatingcostinacontemporaryanalyticallabisspentprocessingandpreparingsamplesforintroductionorinjectionintoananalyticalseparationand/ormeasurementdevice.Clearly,effortsdirectedandproductsdesignedtostreamlinesamplepreparationprotocolsareessentialtofutureprogressinanalyticalscience.GoalsofSamplePreparationSuccessfulsamplepreparationformostanalyticaltechniques[HPLC,GC,spectrophotometry,RIA,etc.]hasathreefoldobjective:namely,toprovidethesamplecomponentofinterestinsolutionfreefrominterferingmatrixelementsataconcentrationappropriatefordetectionormeasurement.Toaccomplishthesegoals,asample,orarepresentativeportionthereof[notalwayseasytoobtain],isprepared viatraditionalmethodsofdissolution,homogenization,extraction[liquid-orsolid-phase],filtration,concentration,evaporation,separation,chemicalderivatization,standardization[internalorexternal],etc.Usuallysuchmethodsareusedincombinationsofmultiplesteps,whichformasampleprepprotocol.Thefewerstepsandmethodsusedinanygivenprotocol,thesimpler,moreconvenient,costeffective,andlesstimeconsumingitis.Simplerprotocolslendthemselvesmorereadilytoautomationandalsoleadtoincreasedaccuracy,reliability,reproducibility,andsafety.InnovationinSamplePreparationMethodsTherearemanywaystocombinestandardtoolsandtechniquestoaccomplishthegoalsofsampleprep.However,itisbesttoseekinnovativemeanstostreamlinesampleprepprotocols:tocombinethefunctionsofseveralsteps,ifpossible,intooneoperation;toeliminateneedlesssampletransfersandmanipulations;toreducethescaleasmuchaspracticable[gainingeconomiesoftime,labor,andcost];tousenewtoolsincreativeways.BenefitsofSolid-PhaseExtraction[SPE]CartridgesWhencomparedtoothersamplepreparationprocesses,solid-phaseextractionusingSPEcartridgesoffers:

LowerCostGreaterRecoveriesFasterProtocolLowerCostGreaterRecoveriesFasterProtocolGreaterSafetyGreaterAccuracyNoEmulsionProblemsNoTransportingofSamplestoLabminimalsampletransferfewerstepslessexposuretotoxicagentsnocrosscontaminationlesssamplehandlingfewerstepsReducedHarmtoLabileSamplesMinimalGlassBreakageminimalevaporationReducedHarmtoLabileSamplesMinimalGlassBreakageminimalevaporationlessglasswareused,lesstowashAchievingSamplePreparationObjectiveswithSolid-PhaseExtraction[SPE]Toremovesampleconstituentsthateluteaftertheanalytesofinterestorarestronglyadsorbed:usesolid-phaseextractionwithsorbentsurfacechemistrythatisthesameasthatintheanalyticalHPLCcolumn.tailorthegradientstepstoeluteanalytesselectively.Toremovesampleconstituentsthatcoelutewithananalyteofinterest:usesolid-phaseextractionwithsorbentsurfacechemistryand/orseparationmodedifferentfromthatintheanalyticalcolumn.tailorthegradientstepstoeluteanalytesselectively.Toenrichsamplecomponentspresentinlowconcentration:tailorthegradientstepstoeluteanalytesselectively.use"large"samplevolumesinadsorption-promotingsolvent.use"small"collectionvolumeindesorption-promotingsolvent.usesorbentchemistrytailoredtotheanalyte,independentofthatinanalyticalcolumn.carefullychoosechemistryofsolid-phaseextractioncolumnsofurthersampleprepwillbeunnecessary.Todesaltsamples:first,adsorbanalytesonreversed-phasesorbentwhilesaltbreaksthroughunretained.then,afterusingwatertowashawayresidualsalt,desorbanalytesusingwater-miscibleorganicsolvent.Toexchangesolvents:adsorbthesamplecompletelyontoastronglyretentivesorbentandflushawaytheoriginalsolventwithaweakereluent.elutetheanalytewiththedesiredsolvent.Tofractionateclassesofcompounds:useastep-gradientsequencetodivideasample-onthebasisofhydrophobicity,polarity,orcharge-intofractionscontaininggroupsofanalytesthatsharecommonproperties.Toderivatizeanalytesusingsolid-phasereagents:adsorbaderivatizationreagentonthesurfaceofthesorbent;then,collectthesample(usuallyagas)underconditionsthatfavorcompleteadsorptionoftheanalyte;waitforthereactiontooccurandthenselectivelyelutethederivative.SPEisChromatographyKeepinmindthatsolid-phaseextractionhasthesamefundamentalbasisasHPLC.Anyknowledgeofthechromatographicbehavioroftheanalytesofinterest,andofothermatrixcomponents,canhelpinchoosingthepropersorbentandeluents.If,forexample,youknowthatcertainchromatographicconditionsprovideexcellentseparationofyouranalytefrominterferences,thenyoumaychooseasimilarSPEsorbentandsolventcombination.Similarly,ifyouaretryingtoremoveaninterferencethatcoelutesinHPLC,thenyouknow apriorithatsimilarSPEconditionswillnotbesuccessful.GeneralElutionProtocolsTherearetwogeneralstrategiesforisolatingandcleaningupsamplecomponentsofinterest:adsorbmatrixinterferenceswhilecomponentsofinterestpassthroughthecartridgeunretained.adsorbcomponentsofinterestwhilematrixinterferencespassthroughthecartridgeunretained.Thefirststrategyisusuallychosenwhenthedesiredsamplecomponentispresentinhighconcentration.Whencomponentsofinterestarepresentatlowlevels,ormultiplecomponentsofwidelydifferingpolaritiesneedtobeisolated,thenthesecondstrategyisgenerallyemployed.Traceenrichmentofcompoundspresentatextremelylowlevelsandconcentrationofdilutesamplesarealsoachievedbythesecondstrategy.StepsofaSolid-PhaseExtractionProcedureThefollowingsectiondescribesthestepsinvolvedinacompletesolid-phaseextractionprocedure.Inmanyapplications,oneormoreofthesteps,listedbelowandsubsequentlydescribedbygeneralexamples,canbeomitted,therebysimplifyingtheprocedure.Theproceduresillustratedhereusesamplescontainingdyessothatseparationsmaybeeasilyvisualized.Keepinmindthatmostsamplescontaincolorlesscomponentsthatrequiresometypeofdetectorortesttolocatetheminthecollectedfractions.Usethefollowinginformationasaguidelineinthedevelopmentofyourownprocedureorwhenmodifyingprocedurespublishedintheliterature.PretreatmentofthesampleConditioningofthecartridge(Silica,Florisil,Alumina,Diol,CN,NH (Silica,Florisil,Alumina,Diol,CN,NH 2)LoadingthesampleElutionofthefractionsPrincipalSeparationModesinSolid-PhaseExtraction[SPE]Normal-PhaseChromatographyThismodeisclassicallyusedtoseparateneutralorganiccompoundswhosechemicalnaturerangesfromhydrophobictomoderatelypolar.Toperformnormal-phasechromatographywithSPEcartridges,useastepgradientofnonpolarsolventswithapolarpackingmaterial.Conditionthecartridgewithsixtotenhold-upvolumesofnon-polarsolvent,usuallythesamesolventinwhichthesampleisdissolved.Loadthesamplesolutionontothecartridgebed.Eluteunwantedcomponentswithanon-polarsolvent.Elutethefirstcomponentofinterestwithamorepolarsolvent.Eluteremainingcomponentsofinterestwithprogressivelymorepolar[stronger]solvents.Whenyourecoverallofyourcomponents,discardtheusedcartridgeinasafeandappropriatemanner.Thisprocedureisillustratedinthefigurebelowforasamplecontainingamixtureofthreeneutral,relativelynon-polarorganicdyes[yellow,red,andblue]thatappearsblackwheninitiallyloadedontothecartridgebed.IllustrationofaGeneralElutionProtocolforNormal-PhaseChromatographyonSPECartridgesLoodsompls 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sonipl*residueReversed-PhaseChromatographyBecauseofthemultiplicityofaqueoussamplesspanningabreadthofapplicationsfromenvironmentalwatertofruitsandvegetables,frombeveragestobiologicalfluids,reversed-phasechromatographyhasbecomethepredominantmodeofSPE.Toperformreversed-phasechromatographywithSPEcartridges,useagradientofstronglytoweaklypolarsolvents[fromweaktostrongsolventelutionstrength]withanon-polarpackingmaterial.Solvatethesilica-bondedphaseorpolymerpackingwithsixtotenhold-upvolumesofmethanoloracetonitrile.Flushthecartridgewithsixtotenhold-upvolumesofwaterorbuffer.Donotallowthecartridgetodryout[unlessusingHLB].Loadthesampledissolvedinastronglypolar[weak]solvent[typicallywater].Eluteunwantedcomponentswithastronglypolarsolvent.Eluteweaklyretainedcomponentsofinterestwithalesspolarsolvent.Elutemoretightlyboundcomponentswithprogressivelymorenon-polar[stronger]solvents.Whenyourecoverallthecomponentsofinterest,discardtheusedcartridgeinasafeandappropriatemanner.Thisprocedureisillustratedinthefigurebelowforasampleofanaqueousgrapedrinkcontainingtwopolarfooddyes[redandblue],aswellassugarandartificialflavor[butnorealgrapejuice!].Asprepared,thisdrinkappearslightpurpleinaglass,sincethedyeconcentrationisdilute.WhenaportionisloadedontoapreparedSPEcartridge,thestronglyretaineddyesbecomeconcentratedneartheinletinadarkpurpleband.

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[flavoroils.]Ion-ExchangeChromatographyCompoundsthatareionicorionizableareoftenbestisolatedusingsomeformofion-exchangechromatography.Thisseparationmodeisorthogonaltothemorewidelyusednormal-phaseandreversed-phasemodesandprovidesapowerful,selectiveseconddimensiontosamplepreparationprotocols.—AnionandCationExchange—AnionandCationExchangeandHowTheySelectivelyAttractandRetainMoleculesofOppositeCharge

+++++Anion++十Exchanger-+++++Anion++十Exchanger-+Stationary-phaseApTide+〃NegaJivelyChargedAnalyte[Anion]Attractedto 斗PositiveSurfoce(LCation-Exchanger__Stationary-phaseKrParticle——.PosilivelyChargedAnclyte[CaHon]AttractedtoNegafiv^^^^e/^pToperformion-exchangechromatographywithSPEcartridges,useagradientofpHorionicstrengthwithanionexchangepackingmaterial.Conditionthecartridgewithsixtotenhold-upvolumesofdeionizedwaterorweakbuffer.Loadthesampledissolvedinasolutionofdeionizedwaterorbuffer.Eluteunwanted,weaklyboundcomponentswithaweakbuffer.Elutethefirstcomponentofinterestwithastrongerbuffer(changethepHorionicstrength).Eluteothercomponentswithprogressivelystrongerbuffers.Whenyourecoverallofyourcomponents,discardtheusedcartridgeinanappropriatemanner.ThisprocedureisillustratedinthefigurebelowforasampleofanaqueousmixtureoftwoionicdyeswithdifferentpKavalues.Whenloadedontothecartridge,botharestronglyretained,andthecombinationofblueandyellowcomponentsappearsasagreenbandneartheinlet.IllustrationofGeneralElutionProtocolforIon-ExchangeChromatographyonSPECartridges(NH2,Accell?PlusQMA,AccellPlusCM,SCX,SAX,WCX,WAX)Loodsamplenriixiur^

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占口mpieresidueInmixed-modeionexchange,onintermediatewo&hwithorganicsolventTnaybeusedioeluteneutralcompounds[orthosewithsamechcrg^ossorbenhlboundviahydrophobicinteraction.Cationandanionexchangersarefurthercategorizedaseitherweakorstrongexchangers,dependinguponthetypeofionicgroupontheirsurface.Strongcationexchangerspossessanacidicsurfacemoietysuchasasulfonicacidthatisalwaysionized[negativelycharged]overthewholepHrange.WeakcationexchangerspossessanacidicsurfacemoietysuchasacarboxylicacidthatisnegativelychargedathighpHbutneutralatlowpH.Similarly,stronganionexchangerstypicallybearquaternaryammoniumgroupsthatarealwayspositivelycharged,whileweakanionexchangerspossessprimary,secondary,ortertiaryaminegroupsthatmaybepositivelychargedatlowpHbutneutralathighpH.UsethefollowingtableasaguidelinetochoosetheappropriateSPEion-exchangecartridgetypeforyourparticularanalyte.

Ion-ExchangeGuidelinesFirst,determineanal^4«typeThen」Mowcorrespondingorrowsdownforreconmendadporticloondmobilepha^epH.AnalyteTypeWentACID*■0 =5S叱zigACIDW&okBASEw声廿-io5切ngBASEChargeStategpH'MachargehpH<[anno]>0i[anion|山阳口餌Charged-p[asliGWl|ntpH<SHaChargepH^IZ+1|&dN«n)AJw甲iChargedStationaryPhaseParticleStrongAnionhangerAniqnExchangert.y-r呎1=1砂SlrongCationExchongerCaticmExchanger■s.g-ip和-$ChorgeStatsvspH1+Alw-c^iChailed(llpH48NomPH>12AlwciyaNa-ElpH<30pH>7MobilePhasepHRangeloReloinanolyte[capfure]pHA孑PHapHtSpHt-7ioReleaseanaSyle[eloie]p*1«3州>}2pH^>\2pH<3*MotepHRcmg«口净仃卩praxinn口怕Tl^ytfepwidupon$pacificanalyk*andpdFticlecha(acfHfisti<$.Mixed-modeionexchangechromatographycombinestheuseofreversed-phaseandion-exchangemodesintoasingleprotocolonasingleSPEcartridge.Itcanbeusedtoisolateandseparateneutral,acidic,andbasiccompoundsfromasinglecomplexmatrix.Anidealmixed-modeSPEsorbentsubstrateremainswater-wettablewhileexhibitingstrongreversed-phaseretentionofhydrophobiccompounds.Onitssurfaceareion-exchangefunctionalitiesofoneofthefourgeneraltypesjustdescribedabove.Intermediatewasheswithorganicsolventmixturesofappropriateelutionstrengthmaybeusedtoisolateneutralcompounds[includingionizableanalytesintheirneutralstate].Selectiveelutionofionicallyboundanalytesmaybeattainedbymanipulatingthechargeofeitherthe