LC-MS液质联用资料系列1.ppt

,Liquid Chromatography Coupled to Tandem Mass Spectrometry,Processes Needed to Generate a Mass Spectrum,1) Generate desolvated ions (Sources) 2) Transfer ions into mass spectrometer vacuum (Interface Region) 3) Mass analyze ions (Quadrupoles) 4) Detect ions (Detector) 5) Process the data (Software),The Source,The Source,Ionization Sources,Turbo IonSpray - IonSpray (pneumatically assisted Electrospray) option which utilizes a heated auxiliary gas flow.,Heated Nebulizer - Atmospheric Pressure Chemical Ionization (APCI) source which ionizes compounds in the gas phase.,+,-,+ - -,+,Ion Evaporation and Formation,+ + - + - + + + + - + - - + +,+,- + + + + +,+ - +- + + +,+ + +,IonSpray (ESI),IonSpray:,- Electrospray ionization (ESI) - Gentlest ionization technique - Applicable to polar and ionic substances,Intensity,cps,15+,Multiply Charged Ions,+Q1: from myoglobina,16+ etc.1060.5 1 1 3 1 .0,1.56e6 cps,1.0e6,9 9 8 .0 9 4 2 .5 8 9 3 .0,14+ 1 2 1 2 .0,13+ 1 3 0 5 .0,5.0e5,8 4 8 .5 8 0 9 .0,12+ 1414.0,11+ 1542.5,10+ 1696.0,800,1000,1200,1400,1600,m/z, amu Molecules which can be multiply protonated or deprotonated can exist in more than one charge state Peaks at M nHn (n = number of charges),Nebulizer,Cool Zone,Primary Flash Zone Secondary Flash Zone,Heated Nebulizer,(APCI),Heated Nebulizer: - Atmospheric Pressure Chemical Ionization (APCI),- corona discharge,- polar to non-polar thermally stable compounds,Atmospheric Pressure Chemical,Ionization (APCI),Corona Discharge Chemistry Positive Ions 1) EI on atmosphere causes e- removal from,N2 & O2 forming N2+ & O2+ (primary,ions),2) In a complex series of reactions N2+ &,O2+ react with H2O & CH3OH forming H3O+ & CH3OH2+ as reagent ions for CI. 3) H3O+ & CH3OH2+ donate protons to,analyte forming M+H+,AnalytePolarity,GC-MS vs. LC-MS Ionic IonSpray APCI,Non-Ionic,GC-MS,101,102,103 Molecular Weight,104,105,Ion Production,API Chemistry,and,Example Spectra,IncreasingAcidity,+,GAS PHASE Acid-Base Scale for Positive Ions & Neutral Molecules,Neutral Molecules Strong acid formic acid acetic acid water methanol phenol formaldehyde N2O methane ethane benzene acetonitrile ethyl acetate ammonia triethylamine Weak acid,Reagent Ions Weak Base HCOOH2+ CH3COOH2+ H3O+ CH3OH2+ C6H6OH2+ HCHOH+ N2OH+ CH5+ C2H5 C6H6H+ CH3CNH+ C2H5OCOC2H6+ NH4+ C6H15NH+ Strong Base,Addition of acetic acid or formic acid will increase intensity in positive ion mode by higher concentration of H+. Trifluoroacetic acid (TFA) works well in positive ion mode at a concentration up to 0.05%, but lingers for a long time in negative ion mode. (TFA ions are at m/z 113 and m/z 227dimer) Changing from acetonitrile to methanol may increase signal intensity.,IncreasingBacicity,GAS PHASE Acid-Base Scale for Negative Ions & Neutral Molecules,Neutral molecules Weak Acid NH3 Water Toluene Methanol Ethanol Acetonitrile Acetone CH3SH CH3NO2 HCN Phenol Acetic acid Benzoic acid HCl Strong Acid,Reagent Ions Strong Base NH2- OH- C6H5CH2- CH3O- C2H5O- CH2CN- CH3COCH2- CH3S- CH2NO2- CN- C6H5O- CH3COO- C6H5COO- Cl- Weak Base,For R-COOH compounds use NH4ac buffer (pH = 6.4). Try addition of NH3 in case of neutral analytes in negative ion mode. Stay away from triethylamine, it collects in the source!,Ionization Suppression,Compounds That Cause Sensitivity Suppression, Salts can interfere with ionization and can cluster to complicate spectrum (but also aid in identification), Strong bases or quaternary amines can interfere with,positive mode analytes, e.g. Triethylamine (TEA) Acids - Sulfuric/Sulfonic acids and TFA interfere in,negative mode experiments, Phosphate buffer and non-volatile ion pairing agents (e.g. SDS) can cause severe suppression and complex spectra,Dimerization (2M+H+) can occur at high concentration, leading to non-linearity during quantitation, Dimer Signal at m/z = (MW*2)+1, Can cause non-linearity at high concentrations,Progesterone in Positive Ion ESI-MS,M+ Na+,Intensity,cps,Intensity,cps,6e7,Effect of Phosphate Buffer,Q1 MCA (10 scans): from cck q1 -ve nano 254.43,424.53,8.78e7 cps,8e7 7e7 CCK dissolved in Phosphate B uffer 566.44 5e7 4e7 3e7 733.93 2e7 835.93,1e7,898.22,1000.42,1 1 4 21164.61,300,400,500,600,700,800,900,1000,1100,m/z, amu,Q1 MCA (10 scans): from CCK PURIFIED Q1 SACN 16.7 4.0e7 3.5e7,570.44,4.44e7 cps,3.0e7 2.5e7 2.0e7 1.5e7,Phosphate B uffer removed from CCK sample,1142.03,1.0e7,292.82,377.02,439.03,497.03,700.63,798.63,879.23,5.0e6,300,400,500,600,700,800,900,1000,1100,m/z, amu,+,+,+,+,+,+,+,+,+,+,+,+,+,+,-,-,-,-,+,+,+,+,+,-,-,-,-,-,-,-,-,-,-,Adducts & Clusters formed in LC/MS Adduct*/,Adduct Ion Formed,Cause,Ionic,APCI,ESI,+/-,m/z of Ion,Lithium M+Li Ammonia M + NH4 Water M + H3O Sodium M + Na,Lithium Salts Ammonia/ NH4OH Water/ Acids Sodium salts,Ionic Adduct Adduct Ionic,M + H + 6 M + H + 17 M + H + 18 M + H + 22,Methanol M + H + CH3OH Chloride M + Cl Potassium M + K Acetonitrile M + H + CH3CN) Formic Acid M - H + HCO2H Acetic Acid M - H + CH3CO2H TFA M - H + CF3CO2H,Methanol in Solvent Chlorinated solvent Potassium salts Acetonitrile in solvent Formic Acid Acetic Acid TFA,Adduct Ionic Ionic Adduct Adduct Adduct Adduct,+/- +/-,M + H + 32 M - H + 32 M + 35 (& 37) M + H + 38 M + H + 41 M - H + 41 M - H + 46 M - H + 60 M- H + 114,* Adducts can often be broken up by using a higher DP voltage.,Ion Transfer,Ion Path “Electronic Ion Ski Slope”,OR,SK,IQ1,Q0,ST,RO1,ST2,RO2 (LINAC),ST3,RO3,EXIT CEM,DF,OR = 45V,0V, SK at ground,Moves with C.E.,Q0= -10V,RO1 = -11V,RO3, moves with RO2 (-2-3V),Lens (IQn) and rod (ROn) voltages are linked Keep ions moving at correct speed through system For best sensitivity, proper mass filtering,MS &,MS/MS (tandem),Quadrupole Theory,Quadrupole acts as a mass filter Separates ions based on m/z ratio,Quad. 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.Mr022,Note: M V, = RF frequency, r0 = distance between rods,U(DC),Operating Line,Stability Diagram,DC/RF,(through the apices) ,High,Unit Low,V (RF),MS Operation,Scan Modes,Q1 MS Scans,Start/Stop Mode,Q3 Single-Stage MS Scans,Start/Stop Mode,MS/MS Scan Modes,Available Scan Modes, Product Ion,Provides structural information, Precursor Ion,Provides structural information complementary to Product Ion, Neutral Loss,Provides compound class specificity, MRM,Used for Quantitation,In 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 Q3,MS/MS Product Ion Scan,m1+ fixed,m3+ scanned,MS/MS Precursor Ion Scan,m1+ scanned,m3+ fixed,MS/MS Neutral Loss Scan,m1+ scanned,m,m3+ scanned,MS/MS MRM (Multiple Reaction Monitoring),Precursor ion fixed,Fragmentation (CAD),Product ion fixed,Fragmentation When an ion fragments the following formulas