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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,*,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 nH,n,(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,N,2,&O,2,forming N,2,+,&O,2,+,(primary,ions),2)In a complex series of reactions N,2,+,&,O,2,+,react with H,2,O&CH,3,OH forming,H,3,O,+,&CH,3,OH,2,+,as,reagent ions,for CI.,3)H,3,O,+,&CH,3,OH,2,+,donate protons to,analyte forming M+H,+,AnalytePolarity,GC-MS vs.LC-MS,Ionic,IonSpray,APCI,Non-Ionic,GC-MS,10,1,10,2,10,3,Molecular Weight,10,4,10,5,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,N,2,O,methane,ethane,benzene,acetonitrile,ethyl acetate,ammonia,triethylamine,Weak acid,Reagent Ions,Weak Base,HCOOH,2+,CH,3,COOH,2+,H,3,O,+,CH,3,OH,2+,C,6,H,6,OH,2+,HCHOH,+,N,2,OH,+,CH,5+,C,2,H,5,C,6,H,6,H,+,CH,3,CNH,+,C,2,H,5,OCOC,2,H,6+,NH,4+,C,6,H,15,NH,+,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,NH,3,Water,Toluene,Methanol,Ethanol,Acetonitrile,Acetone,CH,3,SH,CH,3,NO,2,HCN,Phenol,Acetic acid,Benzoic acid,HCl,Strong Acid,Reagent Ions,Strong Base,NH,2-,OH,-,C,6,H,5,CH,2-,CH,3,O,-,C,2,H,5,O,-,CH,2,CN,-,CH,3,COCH,2-,CH,3,S,-,CH,2,NO,2-,CN,-,C,6,H,5,O,-,CH,3,COO,-,C,6,H,5,COO,-,Cl,-,Weak Base,For R-COOH compounds use NH,4,ac buffer,(pH=6.4).,Try addition of NH,3,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+NH,4,Water,M+H,3,O,Sodium,M+Na,Lithium Salts,Ammonia/,NH,4,OH,Water/,Acids,Sodium salts,Ionic,Adduct,Adduct,Ionic,M+H+6,M+H+17,M+H+18,M+H+22,Methanol,M+H+CH,3,OH,Chloride,M+Cl,Potassium,M+K,Acetonitrile,M+H+CH,3,CN),Formic Acid,M-H+HCO,2,H,Acetic Acid,M-H+CH
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