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SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingPracticalTechniquesforLC/MSAnalysisRobertClassonShimadzuScientificInstrumentsrjclasson@shimadzu.comRevised3/15/2005SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingLC/MSseminaroverviewDevelopingmethodsforLC/MSGettingthemostoutofeachionizationtechniquePracticalconsiderationswithLC/MSSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingTermsandDefinitionsAPI-AtmosphericPressureIonizationVAtmosphericPressureIonization:Allowscreationofionsatatmosphericpressure.Usesnebulizergasat1atmospheretoassistindryingliquidaerosol.UsuallyreferstothecollectionoftechniquesusedtoionizesolutesincludingAPCIandESI.Source(calledinterfaceorprobebychromatographers)VESI-ElectrosprayIonizationsourceESI:ElectrosprayIonization.Themostcommontechniqueforionizingmolecules.Usestheionsformedinsolution,evaporatesthemobilephaseusingnebulizergaspressure,leaveionsbehind.Canproducesinglyormultiplychargedionsdependingonmolecularstructure.VAPCI-AtmosphericPressureChemicalIonizationsourceAPCI:AtmosphericPressureChemicalIonization.OneoftheothercommonionizationtechniquesusedtointerfaceHPLCtoMS.Typicallyusesheatandgaspressuretodrydroplets,andusesaelectricaldischargetoionizemobilephaseandgasthatisusedtotransferachargetothesolutemolecule.Onlycreatessinglychargedions.CID:Useofvoltageandgaspressureinthefirstvacuumregiontobreakionsintosmallerfragments.AlsocalledIn-sourcefragmentation.SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingHPLCversusMSHPLCPrefersneutralspeciesRequiresdissolvedmoleculeSeparatesbasedonsolubility(ie,retentiontime)MassSpectrometerPrefersionizedspeciesRequiresdryordesolvatedmoleculeSeparatesbasedonmassSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingHPLCversusMSProblemHPLCprefersneutralcompoundsinordertoachieveagoodseparationLCMSrequiresionicspeciesinordertoobtaingoodsignallevelsSolutionsPickacompromisepHthatgivessomeseparationandsomeionizationChangepHpostcolumnUseHILIC(hydrophilicinteractionchrom)(normalphasecolumnwithreversephasesolvents)UseAPCIanddoionizationingasphaseConsidertheuseofvolatileion-pairingreagentstocreateneutralspeciesforseparationandionicspeciesfordetectionSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingTerms&DefinitionsMass:Aquantitativemeasureofmatterresistancetobeingaccelerated.Nominal(orunit)mass:Themassbasedonnominalatomicweights:I.e.Carbon=12;Hydrogen=1;Oxygen=16,etc.MonoisotopicMass:Themassofanionorcompoundbasedonusingthemostabundantisotopeofeachelement.ForCarbon,themonoisotopicmass=12.0000.ForHydrogen,monoisotopicmass=1.00782VMassSpectrometristsonlyuseMonoisotopicmass.AverageMass:Theaverageweightedmassbasedonalltheisotopesnaturallyoccurring.Averagemassofcarbon=12.011.Hydrogen=1.00794.Chlorine35=34.968852[itisamixtureoftheisotopeat35(76%ofthetotal)and37(24%ofthetotal)].MolecularWeight:Thesumofalltheaveragemassesofeachoftheelementspresentinacompound.SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingMassversusMolecularWeight3-trichloromethylhydrochlorothiazide(diuretic,hypertenside)Formulaweight=415.10Nominalmass=413.00Monoisotopicmass=412.86Whythedifferences?DifferentmeasurementtechniquesElementswithmultipleisotopesSNHSNHOOOONH2ClClClClSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingIsotopesoftheElementsRnAtPoBiPbTlHgAuPtIrOsReWTaHfLaBaCs6XeITeSbSnInCdAgPdRhRuTcMoNbZrYSrRb5KrBrSeAsGeGaZnCuNiCoFeMnCrVTiScCaK4ArClSPSiAlMgNa3NeFONCBBeLi2HeH1VIIIVIIVIVIVIIIIII12345678+#ofnaturalisotope:SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingSampleIsotopicAbundanceSpectraCisplatinTributyltinbenzoateHexachloro-pheneBromsulphaleinClomethacillinTrichlordiurideMercurichromeSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingImprovingQuantitationReducenumberofisotopicelementstoreducenumberofionsdilutingthesignallevelsVEg,forCisplatin,hydrolyzetwochlorineatomstohydroxylstogain~70moresignalintensity.CisplatinafterhydrolysisCisplatinSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingLysineIonizationMolecularWeight=146.1m/z=148/2=74NH2OHONH2m/z=145m/z=147SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingHowtoDevelopaLCMSMethodusepHtocreatetypeofionrequiredLysine00.20.40.60.8102468101214pHRelativeionization[M+2H]+2[M+H]+1[M-H]-1NH2ONH2O-NH2ONH3+OHNH3+ONH3+OHNote:atanypH,thiscompoundisanion!!SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingAminoAcidsHowdoyouanalyzeanaminoacidifitisionicatallpHvalues?IfeachaminoacidhasauniquepKa,howtoanalyzeamixtureofthemwithouthavingtochangechemistryforeachone?Howtoavoiddoingderivitization?Howtoseparatethemwithouthavingtodoionexchangeseparations?CantheystillbedetectedbyLC/MS?Aspositiveornegativeions?SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingAnalysisofAminoAcidsusingaVolatileIonPairReagent2.55.07.510.012.515.017.520.022.5min250e3500e3750e31000e31250e31500e31750e32000e32250e32500e32750e33000e3Int.156.00(1.00)175.10(1.00)147.00(1.00)148.00(3.08)134.00(8.59)116.00(2.35)182.
本文标题:液质LCMS教程
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