SPE-通過固相萃取進(jìn)行樣品富集和純化-waters

1、SPE通過固相萃取進(jìn)行樣品富集和純化PoorSamplePreparationYellowextract為何使用固相萃?。⊿PE）技術(shù)1 .您需要從樣品中去除特定干擾物，以免它們?cè)谀繕?biāo)分析物的檢測和定量過程中影響實(shí)驗(yàn)結(jié)果。在此處所示的示例中，不適當(dāng)?shù)臉悠分苽浞桨肝茨苋コ蓴_物，導(dǎo)致提取物呈現(xiàn)出殘留的黃色干擾物，色譜圖中目標(biāo)分析物與多個(gè)干擾峰發(fā)生了重疊。OptimizedSPEColorlessextract2 .您需要提高初始樣品中目標(biāo)分析物的濃度，以便所用的分析技術(shù)能夠更輕松地對(duì)其進(jìn)行檢測和準(zhǔn)確定量。如果目標(biāo)分析物可被較強(qiáng)地保留，那么可能需要在SPE色譜柱上加載較大的樣品量，隨后僅以極小體積

2、的洗脫液將此分析物洗脫下來，由此提高樣品中分析物的濃度。TraceEnrichmentni1JI_-F-"->-B-|AfterSPEAnalyteConcentrationOriginalSample3 .您需要去除樣品中的干擾物（即使不可見），這些干擾物會(huì)在質(zhì)譜檢測中抑制目標(biāo)分析物的信號(hào)。在此處的示例中，蛋白沉淀法無法去除血漿提取物中的磷脂，從而造成嚴(yán)重的離子抑制。優(yōu)化的復(fù)合模式SPE方案可獲取最純凈的提取物，并可在最大程度上降低離子抑制效應(yīng)。100%ProteinPrecipitation:80%EonsuppressionIB*J_IIlilIII100%產(chǎn)1ISPE:

3、MinimalionsuppressionI.1lf1WhatisSolid-PhaseExtraction(SPE)?Don'tbeconfusedbythetermsolid-phaseextractionSPE.AtypicalSPEdevicehas50timesmoreseparationpowerthanasimple,singleliquid-liquidextraction.SPEisactuallycolumnliquid-solidchromatography.SinceSPEisliquidchromatographyLC,itspracticeisgoverne

4、dbyLCprinciples.Asampleisintroducedintoacolumnoracartridgedevicecontainingabedofappropriateparticles,orotherform,ofachromatographicpackingmaterialstationaryphase.Solventmobilephaseflowsthroughthebed.Bychoosinganappropriatecombinationofmobileandstationaryphases,samplecomponentsmaypassdirectlythrought

5、hecolumnbed,ortheymaybeselectivelyretained.Individualcompoundsinthesampleeachtypicallyappeartotravelatdifferentspeedsthroughthedevice.Usingaweakersolventcausesthemtomoveslowlyand/orbestronglyretained.Astrongersolventspeedsuptheirpassagethroughthebedandelutestheanalyte(s)inamoreconcentratedvolume.Elu

6、tionfromanSPEdeviceisusuallydonebyincreasingthestrengthofthemobilephaseinaseriesofdiscrete,ratherthancontinuous,stepsduringwhichselectedanalytesorinterferencesareeitherfullyretainedorrapidlyeluted-thisvariationofgradientelutioncalledastepgradient.Mostcommonly,SPEispracticedusingminiaturecolumnorcart

7、ridgedevices.Anexampleisshownhere.Amixtureofthreedyesisloadedontothecartridgeinaweaksolvent,causingstrongsampleretentioninanarrowbandthatappearsblackatthecolumninlet.Subsequentgradientsteps,eachwithasuccessivelystrongersolvent,areusedtoelutethedyesindividuallyyellow,red,thenblue.LoadSample(Bfcrd)Elu

8、tionSlep1ElutionStep2日uhonStep3SlahanoryPhoie一ftitleiNOTt:Differentstrengthsolventscanbeusedtoieparot由ady6sOneSPEco/tridgecanseporoteallthreedyesTypicalSPEcartridgesarelow-pressuredevices-constructedofsolvent-resistantplasticorglass-filledwithparticles>30mmindiameter.Suitableflowratesmaybeachieve

9、dbygravityorwiththeassistanceofvacuumorlowpositivepressure.Thelatterrequiresputtingacapontheopeninletofacolumnorusingasealeddevicewithinletandoutletfittings.ImportanceofSamplePreparationInthelasttwodecades,dramaticadvancesinanalyticalinstrumentationandlaboratoryinformationmanagementsystemsshiftedthe

10、analyst'spredominanttasksfromassaymeasurementstosamplepreparationanddataprocessing.Asthestringencyofrequirementsforhighersensitivity,selectivity,accuracy,precision,andnumberofsamplestobeprocessedhasescalated,thecorrespondingincreasesinspeedandsophisticationofanalysisanddatacollectionhaveoutpaced

11、improvementsinthemanytraditionaltechniquesofsamplecollectionandpreparation.Bysomeestimates,75to80%oftheworkactivityandoperatingcostinacontemporaryanalyticallabisspentprocessingandpreparingsamplesforintroductionorinjectionintoananalyticalseparationand/ormeasurementdevice.Clearly,effortsdirectedandpro

12、ductsdesignedtostreamlinesamplepreparationprotocolsareessentialtofutureprogressinanalyticalscience.GoalsofSamplePreparationSuccessfulsamplepreparationformostanalyticaltechniquesHPLC,GC,spectrophotometry,RIA,etc.hasathreefoldobjective:namely,toprovidethesamplecomponentofinterestinsolutionfreefrominte

13、rferingmatrixelementsataconcentrationappropriatefordetectionormeasurement.Toaccomplishthesegoals,asample,orarepresentativeportionthereofnotalwayseasytoobtain,ispreparedviatraditionalmethodsofdissolution,homogenization,extractionliquid-orsolid-phase,filtration,concentration,evaporation,separation,che

14、micalderivatization,standardizationinternalorexternal,etc.Usuallysuchmethodsareusedincombinationsofmultiplesteps,whichformasampleprepprotocol.Thefewerstepsandmethodsusedinanygivenprotocol,thesimpler,moreconvenient,costeffective,andlesstimeconsumingitis.Simplerprotocolslendthemselvesmorereadilytoauto

15、mationandalsoleadtoincreasedaccuracy,reliability,reproducibility,andsafety.InnovationinSamplePreparationMethodsTherearemanywaystocombinestandardtoolsandtechniquestoaccomplishthegoalsofsampleprep.However,itisbesttoseekinnovativemeanstostreamlinesampleprepprotocols:tocombinethefunctionsofseveralsteps,

16、ifpossible,intooneoperation;toeliminateneedlesssampletransfersandmanipulations;toreducethescaleasmuchaspracticablegainingeconomiesoftime,labor,andcost;tousenewtoolsincreativeways.BenefitsofSolid-PhaseExtractionSPECartridgesWhencomparedtoothersamplepreparationprocesses,solid-phaseextractionusingSPEca

17、rtridgesoffers:LowerCostGreaterRecoverieslowersolventconsumptionlowerreagentconsumptionlessapparatusFasterProtocolGreaterSafetyGreaterAccuracyNoEmulsionProblemsNoTransportingofSamplestoLabReducedHarmtoLabileSamplesMinimalGlassBreakage?fewersteps?lessexposuretotoxicagents?nocrosscontamination?lesssam

18、plehandling?fewersteps?directfieldsampling?minimalevaporation?lessglasswareused,lesstowashminimalsampletransferAchievingSamplePreparationObjectiveswithSolid-PhaseExtractionSPEToremovesampleconstituentsthateluteaftertheanalytesofinterestorarestronglyadsorbed:usesolid-phaseextractionwithsorbentsurface

19、chemistrythatisthesameasthatintheanalyticalHPLCcolumn.tailorthegradientstepstoeluteanalytesselectively.Toremovesampleconstituentsthatcoelutewithananalyteofinterest:usesolid-phaseextractionwithsorbentsurfacechemistryand/orseparationmodedifferentfromthatintheanalyticalcolumn.tailorthegradientstepstoel

20、uteanalytesselectively.Toenrichsamplecomponentspresentinlowconcentration:tailorthegradientstepstoeluteanalytesselectively.use"large"samplevolumesinadsorption-promotingsolvent.use"small"collectionvolumeindesorption-promotingsolvent.usesorbentchemistrytailoredtotheanalyte,independe

21、ntofthatinanalyticalcolumn.carefullychoosechemistryofsolid-phaseextractioncolumnsofurthersampleprepwillbeunnecessary.Todesaltsamples:first,adsorbanalytesonreversed-phasesorbentwhilesaltbreaksthroughunretained.then,afterusingwatertowashawayresidualsalt,desorbanalytesusingwater-miscibleorganicsolvent.

22、Toexchangesolvents:adsorbthesamplecompletelyontoastronglyretentivesorbentandflushawaytheoriginalsolventwithaweakereluent.elutetheanalytewiththedesiredsolvent.Tofractionateclassesofcompounds:useastep-gradientsequencetodivideasample-onthebasisofhydrophobicity,polarity,orcharge-intofractionscontainingg

23、roupsofanalytesthatsharecommonproperties.Toderivatizeanalytesusingsolid-phasereagents:adsorbaderivatizationreagentonthesurfaceofthesorbent;then,collectthesample(usuallyagas)underconditionsthatfavorcompleteadsorptionoftheanalyte;waitforthereactiontooccurandthenselectivelyelutethederivative.SPEisChrom

24、atographyKeepinmindthatsolid-phaseextractionhasthesamefundamentalbasisasHPLC.Anyknowledgeofthechromatographicbehavioroftheanalytesofinterest,andofothermatrixcomponents,canhelpinchoosingthepropersorbentandeluents.If,forexample,youknowthatcertainchromatographicconditionsprovideexcellentseparationofyou

25、ranalytefrominterferences,thenyoumaychooseasimilarSPEsorbentandsolventcombination.Similarly,ifyouaretryingtoremoveaninterferencethatcoelutesinHPLC,thenyouknowapriorithatsimilarSPEconditionswillnotbesuccessful.GeneralElutionProtocolsTherearetwogeneralstrategiesforisolatingandcleaningupsamplecomponent

26、sofinterest:adsorbmatrixinterferenceswhilecomponentsofinterestpassthroughthecartridgeunretained.adsorbcomponentsofinterestwhilematrixinterferencespassthroughthecartridgeunretained.Thefirststrategyisusuallychosenwhenthedesiredsamplecomponentispresentinhighconcentration.Whencomponentsofinterestarepres

27、entatlowlevels,ormultiplecomponentsofwidelydifferingpolaritiesneedtobeisolated,thenthesecondstrategyisgenerallyemployed.Traceenrichmentofcompoundspresentatextremelylowlevelsandconcentrationofdilutesamplesarealsoachievedbythesecondstrategy.StepsofaSolid-PhaseExtractionProcedureThefollowingsectiondesc

28、ribesthestepsinvolvedinacompletesolid-phaseextractionprocedure.Inmanyapplications,oneormoreofthesteps,listedbelowandsubsequentlydescribedbygeneralexamples,canbeomitted,therebysimplifyingtheprocedure.Theproceduresillustratedhereusesamplescontainingdyessothatseparationsmaybeeasilyvisualized.Keepinmind

29、thatmostsamplescontaincolorlesscomponentsthatrequiresometypeofdetectorortesttolocatetheminthecollectedfractions.Usethefollowinginformationasaguidelineinthedevelopmentofyourownprocedureorwhenmodifyingprocedurespublishedintheliterature.1. Pretreatmentofthesample2. Conditioningofthecartridge3. Loadingt

30、hesample4. ElutionofthefractionsPrincipalSeparationModesinSolid-PhaseExtractionSPENormal-PhaseChromatographyThismodeisclassicallyusedtoseparateneutralorganiccompoundswhosechemicalnaturerangesfromhydrophobictomoderatelypolar.Toperformnormal-phasechromatographywithSPEcartridges,useastepgradientofnonpo

31、larsolventswithapolarpackingmaterial.1. Conditionthecartridgewithsixtotenhold-upvolumesofnon-polarsolvent,usuallythesamesolventinwhichthesampleisdissolved.2. Loadthesamplesolutionontothecartridgebed.3. Eluteunwantedcomponentswithanon-polarsolvent.4. Elutethefirstcomponentofinterestwithamorepolarsolv

32、ent.5. Eluteremainingcomponentsofinterestwithprogressivelymorepolarstrongersolvents.6. Whenyourecoverallofyourcomponents,discardtheusedcartridgeinasafeandappropriatemanner.Thisprocedureisillustratedinthefigurebelowforasamplecontainingamixtureofthreeneutral,relativelynon-polarorganicdyesyellow,red,an

33、dbluethatappearsblackwheninitiallyloadedontothecartridgebed.2)IllustrationofaGeneralElutionProtocolforNormal-PhaseChromatographyonSPECartridges(Silica,Florisil,Alumina,Diol,CN,NHLoodsompl?mixtureYoumayconditioncartridgewithnonpolarsolventWaslitoeluteweaklyretain&dyMMwdyeElut電ElulebluedyereddyeDi

34、scoidtheusedcorhidgeconloiningwkeni&sampleresidueU$eprogressivelystrongermorepolar國口1請(qǐng)色nt&IoetutethreecomponentofmterealReversed-PhaseChromatographyBecauseofthemultiplicityofaqueoussamplesspanningabreadthofapplicationsfromenvironmentalwatertofruitsandvegetables,frombeveragestobiologicalfluid

35、s,reversed-phasechromatographyhasbecomethepredominantmodeofSPE.Toperformreversed-phasechromatographywithSPEcartridges,useagradientofstronglytoweaklypolarsolventsfromweaktostrongsolventelutionstrengthwithanon-polarpackingmaterial.1. Solvatethesilica-bondedphaseorpolymerpackingwithsixtotenhold-upvolum

36、esofmethanoloracetonitrile.Flushthecartridgewithsixtotenhold-upvolumesofwaterorbuffer.DonotallowthecartridgetodryoutunlessusingHLB.2. Loadthesampledissolvedinastronglypolarweaksolventtypicallywater.3. Eluteunwantedcomponentswithastronglypolarsolvent.4. Eluteweaklyretainedcomponentsofinterestwithales

37、spolarsolvent.5. Elutemoretightlyboundcomponentswithprogressivelymorenon-polarstrongersolvents.6. Whenyourecoverallthecomponentsofinterest,discardtheusedcartridgeinasafeandappropriatemanner.Thisprocedureisillustratedinthefigurebelowforasampleofanaqueousgrapedrinkcontainingtwopolarfooddyesredandblue,

38、aswellassugarandartificialflavorbutnorealgrapejuice!.Asprepared,thisdrinkappearslightpurpleinaglass,sincethedyeconcentrationisdilute.WhenaportionisloadedontoapreparedSPEcartridge,thestronglyretaineddyesbecomeconcentratedneartheinletinadarkpurpleband.IllustrationofaGeneralElutionProtocolforReversed-P

39、haseChromatographyonSPECartridges(C18,tC18,C8,CN,Diol,HLB,PorapakRDX,NH2)LoadaqueoussamplegrapedrinkmixtureSolvo杷ihesinrionoryphasewatileppr卓qwithqlehd,tfienHushwithwaterand/orbuHerWashgpah?,un-untsd8mp產(chǎn)?帥sugarrwlhCompor#nt1ReddyeC。筋N字卜2Bluedyediscard“轉(zhuǎn)dcartridgeq口川口也由9solvent&samplewidueflavoro

40、ils.UseprogreisivelystrongerTejspolartoelutefirstondsecondcomponenfsc/imerestIon-ExchangeChromatographyAnionandCationExchangeCompoundsthatareionicorionizableareoftenbestisolatedusingsomeformofion-exchangechromatography.Thisseparationmodeisorthogonaltothemorewidelyusednormal-phaseandreversed-phasemod

41、esandprovidesapowerful,selectiveseconddimensiontosamplepreparationprotocols.IllustrationoftheTwoMajorTypesofPhasesandHowTheySelectivelyAttractandRetainMoleculesofOppositeChargeNegativelyChargedAnalyteAnionAttractedto/PositiveSurfoce(+Anion+ExchangerStationary-phase5歲ParticlePosilivelyChargedAnclyteC

42、ationAttractedtoNegafivaceYpICation_ExchangerStationary-phaseToperformion-exchangechromatographywithSPEcartridges,useagradientofpHorionicstrengthwithanionexchangepackingmaterial.1. Conditionthecartridgewithsixtotenhold-upvolumesofdeionizedwaterorweakbuffer.2. Loadthesampledissolvedinasolutionofdeion

43、izedwaterorbuffer.3. Eluteunwanted,weaklyboundcomponentswithaweakbuffer.4. Elutethefirstcomponentofinterestwithastrongerbuffer(changethepHorionicstrength).5. Eluteothercomponentswithprogressivelystrongerbuffers.6. Whenyourecoverallofyourcomponents,discardtheusedcartridgeinanappropriatemanner.Thispro

44、cedureisillustratedinthefigurebelowforasampleofanaqueousmixtureoftwoionicdyeswithdifferentpKavalues.Whenloadedontothecartridge,botharestronglyretained,andthecombinationofblueandyellowcomponentsappearsasagreenbandneartheinlet.IllustrationofGeneralElutionProtocolforIon-ExchangeChromatographyonSPECartr

45、idges(NH2,Accell?PlusQMA,AccellPlusCM,SCX,SAX,WCX,WAX)LcodsamplemixiureUseprogres$ivelysirargerbufferscontaining2dyesctiangingpHorionicstrength,orbothCondifionccidridgswifhMaferorbufferDiscardlhensedcartridgecontainingsolvent&mpieresiduefoelutefirstandsecondcomponentsafinfereslWnshtoduleComponen

46、t1Comporienr2unwontedYellowdyeBluedyacompdnenJ|ilwiih而口徑water口w電曰kbuHer1*Inmixed-modeionexchange,onintermediatewo&hwithorganicsolventTncrybeusedioeluteneutralcompoundscrthosewithsamechcrgdossorbenhlboundviahydrophobicinteraction.Cationandanionexchangersarefurthercategorizedaseitherweakorstrongex

47、changers,dependinguponthetypeofionicgroupontheirsurface.StrongcationexchangerspossessanacidicsurfacemoietysuchasasulfonicacidthatisalwaysionizednegativelychargedoverthewholepHrange.WeakcationexchangerspossessanacidicsurfacemoietysuchasacarboxylicacidthatisnegativelychargedathighpHbutneutralatlowpH.S

48、imilarly,stronganionexchangerstypicallybearquaternaryammoniumgroupsthatarealwayspositivelycharged,whileweakanionexchangerspossessprimary,secondary,ortertiaryaminegroupsthatmaybepositivelychargedatlowpHbutneutralathighpH.UsethefollowingtableasaguidelinetochoosetheappropriateSPEion-exchangecartridgety

49、peforyourparticularanalyte.Ion-ExchangeGuidelinesFirsi,del<s-rmineondl«typeIheriiWlowcorrespondingarrowsdownforreconmendadparticleondmobilepha5epH.AnalyteTypeWentACID，日.*3-5StrongACIDW&akBASE8”pKti-ID5gngBASEChargeStatevs.pH*Mdth口也小pH<anno州，7anion|AlwciyaChargedcoliGWl|npH<SNoChang

50、s口八12+1|&dlfon)Alwa/iChargedStationaryPhaseParticleAnionhanger科AnionExchangert.y-r眼LIQSlrofigCationExchongerCaticmExchanger,s.g-i$ChargeStatevspH1+Alwci亡hbg&J1alpH48Noa?PH>12Alwciya仁）口+火Na-cdpH<3pH>7MobilePhasepHRangeloReloinanolytecapfurepHaFPHapHtSpH?7ioReteoseanaSyteeloie州七字rH>

51、;2pH下12pH<3NoteRcmg«areftppraxinnafisTl注y皿盟tfepwidupon$pacificanalyk*andpaFtidecharacfHfistki.Mixed-modeionexchangechromatographycombinestheuseofreversed-phaseandion-exchangemodesintoasingleprotocolonasingleSPEcartridge.Itcanbeusedtoisolateandseparateneutral,acidic,andbasiccompoundsfromasing

52、lecomplexmatrix.Anidealmixed-modeSPEsorbentsubstrateremainswater-wettablewhileexhibitingstrongreversed-phaseretentionofhydrophobiccompounds.Onitssurfaceareion-exchangefunctionalitiesofoneofthefourgeneraltypesjustdescribedabove.Intermediatewasheswithorganicsolventmixturesofappropriateelutionstrengthmaybeusedtoisolateneutralcompoundsincludingionizableanalytesintheirneutralstate.Selectiveelutionofionicallyboundanalytesmaybeattainedbymanipulatingthechargeofeithe