API3000LCMSMS操作培训手册

API3000OperatorTrainingCourseAnalyst-NTFosterCityCAPALLabWhatisLC/MS•LC:LiquidChromatography•MS:MassSpectrometeraninstrumentthatproducesionsandseparatestheminthegasphaseaccordingtotheirmass-to-chargeratio(m/z)ElementMass•Averagemassofallnaturalisotopes•CarbonAbundanceIntegerMass12C=12.0000Da98.91%1213C=13.00341.09%13(12.0000)(0.9891)+(13.0034)(.011)=12.011NaturalisotopesAbundanceElementAbundanceExactMassX+1FactorX+2Factor1H99.991.00782H0.012.014110B49.910.012954.03NB11B50.111.009312C98.9112.000013C1.0913.00341.1NC0.0060(NC*NC)14N99.6314.003115N0.3715.00010.37NN35Cl75.7734.968937Cl24.2336.965932.5NCl79Br50.778.918381Br49.380.916398.0NBr127I100126.9045NaturalIsotopesAbundance•ElementAbundanceExactMassX+1FactorX+2Factor•16O99.7615.9949•17O0.0416.99910.04NO•18O0.2017.99920.02NO•19F10018.9984•28Si92.227.9769•29Si4.728.97655.1Nsi•30Si2.229.97383.4NSi•31P10030.9738•32S95.031.9721•33S0.7532.97150.8NS•34S4.2233.96794.4NsDefiningthemasses•IntegerMass-SumofProtonsandNeutronsofanelement•NominalMass-Sumoftheintegermassesofthemostabundantisotopesoftheelementsinacompound•AverageMass-Sumoftheatomicweightsoftheelementsinacompound•Exactmass-Sumofthemostabundantisotopesofelementsinacompound-Theoreticalcalculationonly•Accuratemass-experimentallydeterminedmass•Monoisotopicmass-sumoftheexactmassesofthemostabundantisotopesofelementsinacompoundExamplesofMassesHaloperidol:C21H23NO2FClNominalMass:375AverageMass:375.8737ExactMass:375.1401Monoisotopicmass375.1401m/z365376386378100755025%IntensityC21H23NO2FClMultiplyChargedIonsMultiplyChargedIons:Signal=(MW+N)/N(N=#ofcharges)SincetheQuadseparatesionsbasedonm/zratioandionscanhavemultiplechargesinIonSpray.Aseriesofmultiplychargedionsisusuallyproducedforlargermolecules.CommonadductsPOSITIVEMODENa22Da.higherthanM+HK38Da.higherthanM+HLi9Da.higherthanM+HNH418Da.higherthanM+HACN41Da.higherthanM+HNEGATIVEMODETFA114Da.higherthenM-H(113and227background)Acetate60Da.higherthenM-HFormic46Da.higherthenM-HGASPHASEAcid-BaseScaleforPositiveIon&NeutralMoleculesGASPHASEAcid-BaseScaleforNegativeIons&NeutralMoleculesIonizationSuppression•CompoundsThatCauseSensitivitySuppressionSaltscaninterferewithionizationandcanclustertocomplicatespectrum(butalsoaidinidentification)Strongbasesorquaternaryaminescaninterferewithpositivemodeanalytes,e.g.Triethylamine(TFA)Acid-Sulfuric/SulfonicacidsandTFAinterfereinnegativemodePhosphatebufferandnon-volatileionpairingagents(e.g.SDS)cancauseseveresuppressionandcomplexspectrum•Dimerization([2M+H]+)canoccurathighconcentration,leadingtonon-linearityduringquantitationDimerSignalatm/z=(MW*2)+1Cancausenon-linearityathighconcentrationsBasicstepsofLC/MSanalysis•SampleIntroduction：inliquidstatesbysyringepumporLCpump•Ionization：IonSpray，APCI•Vacuumsystem:preventcollisionsofionswithresidualgasmoleculesintheanalyzerduringtheflightfromionsourcetothedetector•Interface:preventexcessivegasload•Massanalysis:QuadrupoleMassfilter,TimeofFlight•Dataprocesssystem:computerbasedAPIAnalyticalDomainsNeutral101102103104105MolecularWeightIonicAnalytePolarityIonSprayAPCIGC/MSQuadrupoleTheory•ThreetypesofvoltagespresentonaQuadrupole:RFp-p–Fixedfrequency(1MHz)oscillatingvoltage(VACMass);establishesharmoniciontrajectories.FilteringDC(FDC)–DCvoltagedifferencebetweenpoles(lineartoRFp-p)thatdefinestheresolution.RodOffsetVoltage–LowDCvoltagethatdefinesaxialionenergy(velocitythroughquadrupole).•“RFonly”modemeans:NoFDCbetweenpoles;onlyRFp-p,RodOffsetpresent.Quad.isin“TotalIonMode”(transmittingallions,notfiltering).QuadrupoleTheory(cont.)•Quadrupolesanalyzersoperateinconstantpeakwidthmode(SameResolutionoverMassrange)APIResolutionSpecificationUnit=0.7(±0.1amuFWHH)High=.5(±0.1amuFWHH)Low=~1.1(±0.1amuFWHH)Usercansethigherresolutiondependingonproblemtobesolved(i.e.,settingsingly-chargedresolutionto0.5–0.6amuFWHHaidsindetectingdoubly-chargedions)ResolutionoffsetssettheDC/RFratioatvariouspointsalongmassaxisResolutionAtUnit,HighandLowautomaticallytunedItisalwaysrecommendedtocalibratemassaxisafterresolutionisadjustedforaquadrupoleResolution=0.7amuFWHHLC2Tune1.4b11.pictLC2Tune1.4b11.pictResolution=0.6amuFWHHResolution=0.5amuFWHHQuadrupoleTheory&IonEnergy•IonenergyEffectsResolutionandPeakShapeLowerenergy(slower)=morecycles,betterresolutionHigherenergy(faster)=lesscycles,lowerresolution(typically0.5–2eV)isgivenbythevoltagedifferencebetweentheions“startingpotential”(Q0)andtheQ1rodoffset(RO1)(timesthenumberofcharges)Inc.IEalsoinc.Eofionstodistortpeakshape.AlwaysatradeoffbetweenresolutionandsensitivityinallquadrupoleanalyzersQuadrupoleTheory&IonEnergy•Q1Scan=n(Q0-RO1);Q1IE=0.5-2eV•Q3Scan=n(RO2-RO3);Q3IE=(1-6eV)•CollisionEnergyforMS/MS:CADenergy=n(Q0-RO2);n=#chargesProductionenergy=n(RO2-RO3);n=#chargesthisaffectsresolutionofproductions(lowerenergyyieldsbetterresolution)IonEnergiesExamplesofanIonEnergyof.5eVinQ1LowerVelocitythroughQuadsallowsforgreaterfilteringandbetterresolutionSensitivityisofcourselostasresolutionincreasesIonEnergiesExamplesofanIonEnergyof1.5eVinQ1Higherveloci