液质LCMS教程

SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingPracticalTechniquesforLC/[e-mail]3/15/2005SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingLC/MSseminaroverview„DevelopingmethodsforLC/MS„Gettingthemostoutofeachionizationtechnique„PracticalconsiderationswithLC/MSSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingTermsandDefinitions„API-AtmosphericPressureIonizationVAtmosphericPressureIonization:Allowscreationofionsatatmosphericpressure.Usesnebulizergasat1atmospheretoassistindryingliquidaerosol.UsuallyreferstothecollectionoftechniquesusedtoionizesolutesincludingAPCIandESI.„Source(calledinterfaceorprobebychromatographers)VESI-ElectrosprayIonizationsourceŒESI:ElectrosprayIonization.Themostcommontechniqueforionizingmolecules.Usestheionsformedinsolution,evaporatesthemobilephaseusingnebulizergaspressure,leaveionsbehind.Canproducesinglyormultiplychargedionsdependingonmolecularstructure.VAPCI-AtmosphericPressureChemicalIonizationsourceŒAPCI:AtmosphericPressureChemicalIonization.OneoftheothercommonionizationtechniquesusedtointerfaceHPLCtoMS.Typicallyusesheatandgaspressuretodrydroplets,andusesaelectricaldischargetoionizemobilephaseandgasthatisusedtotransferachargetothesolutemolecule.Onlycreatessinglychargedions.„CID:Useofvoltageandgaspressureinthefirstvacuumregiontobreakionsintosmallerfragments.AlsocalledIn-sourcefragmentation.SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingHPLCversusMSHPLC„Prefersneutralspecies„Requiresdissolvedmolecule„Separatesbasedonsolubility(ie,retentiontime)MassSpectrometer„Prefersionizedspecies„Requiresdryordesolvatedmolecule„SeparatesbasedonmassSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingHPLCversusMS„ProblemƒHPLCprefersneutralcompoundsinordertoachieveagoodseparationƒLCMSrequiresionicspeciesinordertoobtaingoodsignallevels„SolutionsƒPickacompromisepHthatgivessomeseparationandsomeionizationƒChangepHpostcolumnƒUseHILIC(hydrophilicinteractionchrom)(normalphasecolumnwithreversephasesolvents)ƒUseAPCIanddoionizationingasphaseƒConsidertheuseofvolatileion-pairingreagentstocreateneutralspeciesforseparationandionicspeciesfordetectionSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingTerms&Definitions„Mass: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-pheneBromsulphaleinClomethacillinTrichlordiurideMercurichromeSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingImprovingQuantitation„ReducenumberofisotopicelementstoreducenumberofionsdilutingthesignallevelsVEg,forCisplatin,hydrolyzetwochlorineatomstohydroxylstogain~70moresignalintensity.CisplatinafterhydrolysisCisplatinSHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingLysineIonization„MolecularWeight=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!!SHIMADZULCMSTrainingSHIMADZULCMSTrainingSHIMADZULCMSTrainingAminoAcids„HowdoyouanalyzeanaminoacidifitisionicatallpHvalues?„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.