Liquid ChromatographyCoupled to Tandem MassSpectrometryProcessesNeeded toGenerate aMassSpectrum1) Generate desolvated ions(Sources)2) Transfer ions into massspectrometer vacuum(Interface Region)3) Mass analyze ions(Quadrupoles)4) Detect ions(Detector)5) Process the data(Software)The SourceThe SourceIonization SourcesTurbo IonSpray - IonSpray (pneumaticallyassisted Electrospray) option which utilizesa heated auxiliary gas flow.Heated Nebulizer - Atmospheric PressureChemical Ionization (APCI) source whichionizes compounds in the gas phase.+-+ - -+Ion Evaporation andFormation+ + -+ - + + + -+ - - +- + + +- +-+ +IonSpray (ESI)IonSpray:- Electrospray ionization (ESI)- Gentlest ionization technique- Applicable to polar and ionic substancesIntensity,cps15+Multiply Charged Ions+Q1: from myoglobina16+etc.1060.5 1 1 3 1 .01.56e6 cps1.0e69 9 8 .09 4 2 .58 9 3 .014+1 2 1 2 .013+1 3 0 5 .05.0e58 4 8 .58 0 9 .012+1414.011+1542.510+1696.08001000120014001600m/z, amuMolecules which can be multiplyprotonated or deprotonated can exist inmore than one charge statePeaks at M nHn (n = number ofcharges)NebulizerCool ZonePrimary Flash ZoneSecondary Flash ZoneHeated Nebulizer(APCI)Heated Nebulizer: - Atmospheric Pressure Chemical Ionization (APCI)- corona discharge- polar to non-polar thermally stable compoundsAtmospheric Pressure ChemicalIonization (APCI)Corona Discharge Chemistry Positive Ions1) EI on atmosphere causes e- removal fromN2 & O2 forming N2+ & O2+ (primaryions)2) In a complex series of reactions N2+ &O2+ react with H2O & CH3OH formingH3O+ & CH3OH2+ as reagent ions for CI.3) H3O+ & CH3OH2+ donate protons toanalyte forming M+H+AnalytePolarityGC-MS vs. LC-MSIonicIonSprayAPCINon-IonicGC-MS101102103Molecular Weight104105Ion ProductionAPI ChemistryandExample SpectraIncreasingAcidity+GAS PHASE Acid-Base Scalefor Positive Ions & Neutral MoleculesNeutral MoleculesStrong acidformic acidacetic acidwatermethanolphenolformaldehydeN2Omethaneethanebenzeneacetonitrileethyl acetateammoniatriethylamineWeak acidReagent IonsWeak BaseHCOOH2+CH3COOH2+H3O+CH3OH2+C6H6OH2+HCHOH+N2OH+CH5+C2H5C6H6H+CH3CNH+C2H5OCOC2H6+NH4+C6H15NH+Strong BaseAddition of acetic acid or formic acid willincrease intensity in positive ion mode byhigher concentration of H+.Trifluoroacetic acid (TFA) works well inpositive ion mode at a concentration up to0.05%, but lingers for a long time in negativeion mode. (TFA ions are at m/z 113 and m/z227dimer)Changing from acetonitrile to methanol mayincrease signal intensity.IncreasingBacicityGAS PHASE Acid-Base Scalefor Negative Ions & Neutral MoleculesNeutral moleculesWeak AcidNH3WaterTolueneMethanolEthanolAcetonitrileAcetoneCH3SHCH3NO2HCNPhenolAcetic acidBenzoic acidHClStrong AcidReagent IonsStrong BaseNH2-OH-C6H5CH2-CH3O-C2H5O-CH2CN-CH3COCH2-CH3S-CH2NO2-CN-C6H5O-CH3COO-C6H5COO-Cl-Weak BaseFor R-COOH compounds use NH4ac buffer(pH = 6.4).Try addition of NH3 in case of neutral analytesin negative ion mode.Stay away from triethylamine, it collects in thesource!Ionization SuppressionCompounds That Cause Sensitivity Suppression Salts can interfere with ionization and can cluster tocomplicate spectrum (but also aid in identification) Strong bases or quaternary amines can interfere withpositive mode analytes, e.g. Triethylamine (TEA) Acids - Sulfuric/Sulfonic acids and TFA interfere innegative mode experiments Phosphate buffer and non-volatile ion pairing agents (e.g.SDS) can cause severe suppression and complex spectraDimerization (2M+H+) can occur at highconcentration, leading to non-linearity duringquantitation Dimer Signal at m/z = (MW*2)+1 Can cause non-linearity at high concentrationsProgesterone in Positive Ion ESI-MSM+ Na+Intensity,cpsIntensity,cps6e7Effect of Phosphate BufferQ1 MCA (10 scans): from cck q1 -ve nano254.43424.538.78e7 cps8e77e7CCK dissolved in Phosphate B uffer566.445e74e73e7733.932e7835.931e7898.221000.421 1 4 21164.6130040050060070080090010001100m/z, amuQ1 MCA (10 scans): from CCK PURIFIED Q1 SACN 16.74.0e73.5e7570.444.44e7 cps3.0e72.5e72.0e71.5e7Phosphate B uffer removed from CCK sample1142.031.0e7292.82377.02439.03497.03700.63798.63879.235.0e630040050060070080090010001100m/z, amu+-+-Adducts & Clusters formed in LC/MSAdduct*/Adduct Ion FormedCauseIonicAPCIESI+/-m/z of IonLithiumM+LiAmmoniaM + NH4WaterM + H3OSodiumM + NaLithium SaltsAmmonia/NH4OHWater/AcidsSodium saltsIonicAdductAdductIonicM + H + 6M + H + 17M + H + 18M + H + 22MethanolM + H + CH3OHChlorideM + ClPotassiumM + KAcetonitrileM + H + CH3CN)Formic AcidM - H + HCO2HAcetic AcidM - H + CH3CO2HTFAM - H + CF3CO2HMethanol inSolventChlorinated solventPotassium saltsAcetonitrile insolventFormic AcidAcetic AcidTFAAdductIonicIonicAdductAdductAdductAdduct+/-+/-M + H + 32M - H + 32M + 35 (& 37)M + H + 38M + H + 41M - H + 41M - H + 46M - H + 60M- H + 114* Adducts can often be broken up by using a higher DP voltage.Ion TransferIon Path“Electronic Ion Ski Slope”ORSKIQ1Q0STRO1ST2RO2 (LINAC)ST3RO3EXIT CEMDFOR =45V0V, SK at groundMoves with C.E.Q0= -10VRO1 =-11VRO3, moves withRO2 (-2-3V)Lens (IQn) and rod (ROn) voltages are linkedKeep ions moving at correct speed through system For bestsensitivity, proper mass filteringMS &MS/MS (tandem)Quadrupole TheoryQuadrupole acts as a mass filter Separates ions based on m/z ratioQuad. made of 4 rods “A” pole - vertical rods; “B” pole -horizontal rods (by convention)DC & RF voltages are imposed: U = (DC)A - (DC)B (FDC) V = RF voltage (RFp-p) V = Const.Mr022Note: M V = RF frequency r0 = distance between rodsU(DC)Operating LineStability DiagramDC/RF(through the apices) HighUnitLowV (RF)MS OperationScan ModesQ1 MS ScansStart/Stop ModeQ3 Single-Stage MS ScansStart/Stop ModeMS/MS Scan ModesAvailable Scan Modes Product IonProvides structural information Precursor IonProvides structural information complementary toProduct Ion Neutral LossProvides compound class specificity MRMUsed for QuantitationIn all MS/MS modes, Q3 is a mass filter Q3 may scan or be fixed at a given m/z Q2 is “source” of product ions entering Q3MS/MS Product Ion Scanm1+ fixedm3+ scannedMS/MS Precursor Ion Scanm1+ scannedm3+ fixedMS/MS Neutral Loss Scanm1+ scannedmm3+ scannedMS/MS MRM(Multiple Reaction Monitoring)Precursor ionfixedFragmentation(CAD)Product ionfixedFragmentationWhen an ion fragments the following formulas apply: Positive mode: Negative mode:Precursor+Precursor- Product+ Product-+ Neutral+ NeutralTwo modes of fragmentation on Sciex Instruments: CAD = Collisionally Activated DissociationEnergy controlled by CE; occurs in Q2 (collision cell)Ions collide with N2 molecules (CAD gas), collision energy isconverted into vibrational energy, and bonds break CID = Collision-Induced DissociationEnergy controlled by DP; occurs between orifice and skimmerIons collide with N2 (from curtain gas) and solvent molecules,collision energy is convertedIon DetectionDetector & Signal HandlingDeflectorIon PathSignal OutputCEM1 M1000 pF10 kVSignal HandlingBoardCEM HornVoltage (-)CEM BiasVoltage (+)System Controller(in SystemElectronics Box)