1. Molecular mass spectrometry Chapter 20 The study of “molecular ions” M + e -  M. + + 2e -

2. Ionization energy?

3. EI Ionization??

4. MS Terms Fragmentation Fragmentation Molecular ions Molecular ions Daughter ions Daughter ions Base peak Base peak Isotopic peaks Isotopic peaks Collision product peaks (MH + ……) Collision product peaks (MH + ……) Hard and soft ionization sources Hard and soft ionization sources Gas-phase sources Gas-phase sources Desorption sources Desorption sources

5. Mass Spectrum of Ethyl Benzene

6. Ion Sources for Mass Spectrometers Unless you “create” an ion you won’t see it! Energy driven process  HARD IONIZATION, SOFT IONIZATION

7. MS with “Hard” and “Soft” Sources

8. Electron Impact Source Electron Ionization (EI) M + e -  M.+ + 2e -

9. Why vacuum? Ensure filament does not burn out Ensure filament does not burn out Help to vaporize samples Help to vaporize samples Reduce collision between formed ions and atmospheric gases Reduce collision between formed ions and atmospheric gases Remove sample from instrument after analysis Remove sample from instrument after analysis

11. Typical Reactions during Electron Impact Energy = 70eV  6700 kJ/mol Typical bond energies  200 to 600 kJ/mol  EXTENSIVE FRAGMENTATION 2(ABCD). +

12. Electron Impact Spectra Different molecules behave differently Good molecular ion  little fragmentation No molecular ion  extensive fragmentation Isotopes are extremely important! Molecular ion isotopic cluster

15. CI REAGENT GAS

16. Chemical Ionization MS Sources High Energy electrons  Sample Molecule MH  CH 4 CH 4 CH 4 + CH 3 + CH 2 + Molecule Ions 

17. EI vs. CI

19. Why EI and CI not enough? Sample must be in gas phase Sample must be in gas phase Not for nonvolatile or thermally unstable compounds Not for nonvolatile or thermally unstable compounds

21. Electrospray Ionization Source Several KV

22. ELCTROSPRAY IONIZATION DETAILS

23. Iribarne-Thomson Model Charge density increases Charge density increases Rayleigh limit (Coulomb repulsion = surface tension) Rayleigh limit (Coulomb repulsion = surface tension) Coulomb explosion (daughter driblets) Coulomb explosion (daughter driblets) Evaporation of daughter droplets Evaporation of daughter droplets Desorption (desolvation) of ions from the droplets into the ambient gas (IONS FORMED) Desorption (desolvation) of ions from the droplets into the ambient gas (IONS FORMED)

24. Electrospray Ionization MS of Proteins and Peptides

25. Special Features of ESI Little fragment Little fragment Multiple charges Multiple charges Linear relationship between AVG charge and molecular weight Linear relationship between AVG charge and molecular weight Coupling to HPLC directly! Coupling to HPLC directly!

26. An example Applications: Determination of MW and charges for each peak (Smith et al. Anal. Chem., 1990, 62, 882-899): Assumptions Assumptions The adjacent peaks of a series differ by only one charge The adjacent peaks of a series differ by only one charge For proteins, the charging is due to proton attachment to the molecular ion. For proteins, the charging is due to proton attachment to the molecular ion. This has been an excellent (but not crucial) assumption of nearly all proteins studied to data where alkali attachment contributions are small. This has been an excellent (but not crucial) assumption of nearly all proteins studied to data where alkali attachment contributions are small. Ionization of only the intact molecule. Ionization of only the intact molecule.

27. Advanced Analytical Chemistry – CHM 6157® Y. CAIFlorida International University Updated on 10/9/2006Chapter 7Chromatogr./Mass Spec. Coupling Given these assumptions, eq 1 describes the relationship between a multiply charged ion at m/z P 1 with charge z 1 and molecular weight M. Z1Z1 Z2Z2 M/Z P1P2 P 1 Z 1 = M + MaZ 1 = M + 1.0079Z 1 [1] Assume that the charge carrying species (Ma) is a proton. The molecular weight of a second multiply protonated ion at m/z P 2 (where P 2 > P 1 ) that is j peaks away from P 1 (e.g. j = 1 for two adjacent peaks) is given by P 2 (Z 1 -j) = M + 1.0079(Z 1 -j)[2] Equations 1 and 2 can be solved for the charge of P 1. Z 1 = j(P 2 -1.0079)/(P 2 -P 1 )[3] The molecular weight is obtained by taking Z 1 as the nearest integer valve.

28. FAB Ionization: Fast Atom Bombardment + SECONDARY ION MASS SPECTROMETRY  SIMS

29. FAB Ionization Xe + Xe 0 Primary Ion Beam Atom Gun Secondary ion Beam to MS

30. MS Instrument Components

31. Gas/Liquid Inlet System

32. Solid/Matrix Inlet Systems Sample

33. More MS Analyzers Resolution? Resolution? The capability of a MS to differentiate between masses R = m/∆m ∆m: mass different between two adjacent peaksthat are just resolved (height of the valley < 10% of the peak). M: the norminal mass of the first or the mean of the two peaks

34. Resolution Estimate the accuracy of the measurement if resolution is known Estimate the accuracy of the measurement if resolution is known If R = 5000, at mass 500 range 5000 = 500/ ∆m ∆m = 0.1 Determine R required Determine R required C 2 H 4 + and CH 2 N + have masses of 28.0313 and 28.0187 R = 28.025/0.0126 = 2220

35. Magnetic Sector Analyzer

36. Magnetic Sector Physics

38. Single Focusing Magnetic Sector  Ions at source with same mass-to-charge ratio  Ions with diverging directional distribution will be acted upon in the same way  Brings ions with different directional orientations to focus  Limits the resolution

39. Double Focusing Mass Spectrometers  Passes ions through an electrostatic analyzer (ESA) which limits the range of the kinetic energy of ions reaching the magnetic sector  Only ions with the same average kinetic energy pass through the ESA slits into the magnetic sector  Two focal planes at the ion collector  Energy focal plane  Directional focal plane  Increases resolution

40. Double Focusing Mass Spectrometer

41. MS-MS

42. Daughter ion scan The parent ion mass is fixed and the masses of all daughter ion formed from the parent ion are measured. The first stage of analysis selects ions of particular m/z. This parent ion is passed onto the reaction region. The daughter ions formed in the reaction region are then analyzed by mass. These daughter ions are generally characteristic of the structure of the parent ions and thus provide a means of determining that structure. Instrument design

44. Daughter ion scan The parent ion mass is fixed and the masses of all daughter ion formed from the parent ion are measured. The first stage of analysis selects ions of particular m/z. This parent ion is passed onto the reaction region. The daughter ions formed in the reaction region are then analyzed by mass. These daughter ions are generally characteristic of the structure of the parent ions and thus provide a means of determining that structure. Instrument design