Molecular mass spectrometry Chapter 20 The study of “molecular ions” M + e- à M.+ + 2e-Mass Spectrum of Ethyl Benzene Bar type graph. Each line represents a specific m/z M + e- à M.+ + 2e-Interpretation of SpectraIon Sources for Mass Spectrometers Unless you “create” an ion you won’t see it! Energy driven process à HARD IONIZATION, SOFT IONIZATIONMS with “Hard” and “Soft” Sources A hard ionization source (electron impact) A soft ionization source (chemical ionization)Electron Impact Source • Electrons from filament hit the sample, Path of electrons and molecules are on a right angle. • This methods is applicable to all volatile compounds (<103 Da) and gives reproducible mass spectra with fragmentation to provide structural information. Molecular mass < 103 DaWhy vacuum? • Ensure filament does not burn out • Help to vaporize samples • Reduce collision between formed ions and atmospheric gases • Remove sample from instrument after analysis !Electron Impact Source Electron Ionization (EI) M + e- (70 V) à M.+ + 2e-Electron impact source • Filament – tungsten or rhenium. Our source of 70 eV electrons. • Target – anode used in association with the filament to produce electrons. • Repeller – positively charged electrode used to “push” positive ions out of the ionization source. • Lens stack – series of increasingly more negative electrodes used to accelerate our ions to constant kinetic energy.Electron Ionization ProcessTypical Reactions during Electron Impact Energy = 70eV à 6700 kJ/mol Typical bond energies à 200 to 600 kJ/mol à EXTENSIVE FRAGMENTATIONElectron Impact Spectra • Different molecules behave differently • Good molecular ion à little fragmentation • No molecular ion à extensive fragmentation • Isotopes are extremely important! • Molecular ion isotopic cluster Isotope peaks C5H12O MW = 88From “undergraduate instrumental analysis” by James W. RobinsonChemical ionizationChemical ionization Source • A soft ionization method. • Gaseous molecules of the sample are ionized by collision reactions with ions produced from a reagent gas. • Reagent gas: methane, propane, isobutane, ammonia. They produce different spectra. • It relies on our charge being transferred from a reagent molecule to our sample. Reagent ion + molecule molecular ion + reagent ion. • This method gives molecular weight information and reduced fragmentation in comparison to EI.Chemical Ionization Source CI REAGENT GASChemical!Ioniza.on!MS!Sources!High Energy electrons è Sample Molecule MH è CH4 CH4 CH4+ CH3+ CH2+ 252433544HHCCHCHCHCHCHCH+→++→+++++625242252425HCMMHHCHCMHMHHCCHMHMHCH+→++→++→+++++++ Molecule Ions è Primary ions Secondary ions Proton transfer Hydride transferEI vs. CIEI vs. CIMass spectrum of 1-decanol from a hard ionization source and a soft ionization sourceWhy EI and CI not enough? • Sample must be in gas phase • Not for nonvolatile or thermally unstable compounds Desorption SourcesElectrospray Ionization Source Molecular mass > 100,000 DaELCTROSPRAY IONIZATION DETAILSELCTROSPRAY IONIZATION PROCESSIribarne-Thomson Model • Charge density increases • Rayleigh limit (Coulomb repulsion = surface tension) • Coulomb explosion (daughter driblets) • Evaporation of daughter droplets • Desorption (desolvation) of ions from the droplets into the ambient gas (IONS FORMED)Electrospray!Ioniza.on!MS!of!Proteins!and!Pep.des!Special Features of ESI • Little fragment • Multiple charges • Linear relationship between AVG charge and molecular weight • Coupling to HPLC and capillary electrophoresis columns directly!Matrix-Assisted Laser Desorption-Ionization (MALDI) • Matrix Assisted Laser Desorption/Ionization (MALDI) – Method where a laser is used to generate ions of high molecular weight samples, such as proteins and polymers. – Analyte is embedded in to crystal matrix – The presence of an aromatic matrix causes the large molecules to ionize instead of decomposing. – High mass range achievableMechanism of MALDI…..uncertain • Formation of a “solid solution”. • Matrix excitation • Analyte ionizationMALDI Matrix • Matrix must meet the following properties and requirements: – Be able to embed and isolate analytes (e.g. by co-crystallization) – Be soluble in solvents compatible with analyte – Be vacuum stable – Absorb the laser wavelength – Cause co-desorption of the analyte upon laser irradiation – Promote analyte ionizationMALDI MatrixMALDI!• More tolerant of salts and complex mixture analysis than ESI. • Important for huge molecules: proteins, polymers. • Spectra often contain multiple charged ions.1. First reported ~1980 Major extension in role of MS for analyses of biological materials 2. Use for compounds with MW less than <1500 with low volatility Peptides, drugs, natural products, pollutants, metalorganics 3. Samples should be easily charged in solution Acids, bases, highly polar compounds 4. Samples must be soluble in a liquid, non-volatile matrix such as glycerol 5. Use with solids probe No HPLC; No automation 6. Samples should be relatively pure and free of additional salts, buffers, etc 7. Often used to confirm MW of compounds analyzed by EI or CI May give some fragments; Suffers from high background signal No heating required; Sample directly from solution Fast Atom Bombardment Ionization (FAB) Liquid Secondary Ion Mass Spectrometry (LSIMS)FAB Ionization: Fast Atom Bombardment + SECONDARY ION MASS SPECTROMETRY àSIMSFAB Ionization Xe+ Xe0 Primary Ion Beam Atom Gun Secondary ion Beam to MSFast Atom Bombardment Source • Soft ionization technique. • Samples in condensed state (glycerol solution matrix) are ionized by bombardment with energetic xenon or argon atoms. • Rapid sample heating, little loss of compound. • Matrix absorbs part of energy. • Suitable for the analysis of low volatility species, typically producing large peaks for the pseudo-molecule ion species [M+H]+ and [M-H]-, along with structurally significant fragment ions and some higher mass cluster ions and dimers. • Choosing correct matrix is