1. Chapter 20 # 4, 5, 12, 13, 16

2. Fig. 20-1 (p.551) Mass spectrum of ethyl benzene Fragment peaks Unit: amu or dalton Fragment peaks

3. - Sample inlet system – vaporize sample - Ion source – ionizes analyte gas molecules - Mass analyzer – separates ions according to m/z - Detector – counters ions - Vacuum system – reduces collisions between ions and gas molecules Fig. 20-11 (p.564) Components of a mass spectrometer 10 -5 -10 -8 

4. 2.1 Sample inlet 2.1.1 External (Batch) inlet systems - Liquid - Gas 2.1.2 Direct probe - Non-volatile liquid - Solid Fig. 20-12 (p.564) Sample inlet

5. 2.1.3 Chromatography/Electrophoresis -Permits separation and mass analysis - How to couple two techniques? GC/MS, Fig. 27-14 (p.799) Capillary GC-MS

6. HPLC/MS, nano flow, ESI Adapted from http://www.bris.ac.uk/nerclsmsf/techniques/hplcms.html

7. 2.2 Ion sources

8. Hard ionizationleaves excess energy in molecule – extensive fragmentation Soft ionizationlittle energy in molecule – reduced fragmentation Fig. 20-2 (p.553) Mass spectrum of 1-decanol from (a) a hard ionization source (electron impact) and (b) a soft ionization (chemical ionization)

9. 2.2 Ion sources

10. Fig. 20-3 (p.553) An electron-impact ion source 2.2.1 Gas-phase ion source (1) Electron Impact (EI) Electron bombardment of gas/vapor molecules

12. (2) Chemical ionization (CI) - EI ionization in excess (10 5 of analyte pressure) of reagent gas (methane) to generate CH 4 + and CH 3 +, then CH 4 + + CH 4  CH 5 + + CH 3 CH 3 + + CH 4  C 2 H 5 + + H 2 Ions reacts with analyte CH 5 + + A  CH 4 + AH + proton transfer C 2 H 5 + + A  C 2 H 4 + AH + proton transfer C 2 H 5 + + A  C 2 H 6 + (A-H) + hydride elimination -analyte most common ions (M+1) + and (M-1) + sometimes (M+17) + addition of CH 5 + or (M+29) + (addition of C 2 H 5 + ) Adapted from Schröder, E. Massenspektrometrie - Begriffe und Definitionen; Springer-Verlag: Heidelberg, 1991.

14. 2.2.2 Desorption/Ionization sources (For non-volatile or non-stable analytes) (1)Electrospray ionization (ESI): explosion of charged droplets containing analyte - solution of analyte pumped through charged (1-5 kV) capillary - small droplets become charged - solvent evaporates, drop shrinks, surface charge density increases - charge density reduced by explosion of charged analyte molecules (“Coulomb explosion”) Soft ionization – transfer existing ions from the solution to the gas phase, little fragmentation Easily coupled to HPLC Adapted from http://www.bris.ac.uk/theory/fab- ionisation.html

15. Fig. 20-9 (p.562) Apparatus for ESI

17. Fig. 20-10 (p.563) Typical ESI MS of proteins and peptides. - Important technique for large (10 5 Da) thermally fragile molecules, e.g., peptide, proteins - produce either cations or anions. - Analytes may accumulate multiple charges in ESI, M 2+, M 3+ … molecular mass = m/z x number of charges

18. (2)Fast atom bombardment (FAB) -Sample in glycerol matrix -Bombarded by high energy Ar or Xe atoms ( few keV) -Atoms and ions sputtered from surface (ballistic collision) -Both M + and M - produced -Applicable to small or large (>10 5 Da) unstable molecule Comparatively soft ionization – less fragmentation Adapted from http://www.bris.ac.uk/theory/fab- ionisation.html

19. (3) Matrix-assisted laser desorption/ionization (MALDI) - analyte dispersed in UV-absorbing matrix and placed on sample plate - pulsed laser struck the sample and cause desorption of a plume of ions, - energy absorption by matrix, transfer to neutral analyte desorption of matrix and neural analyte ionization via PT between protonated matrix ions and neutral analyte Fig. 20-7 (p.560) Diagram of MALDI progress.

20. Fig. 20-8 (p.561) MALDI-TOF spectrum from nicotinic acid matrix irradiated with a 266-nm laser beam. MALDI spectrum contains: dimmer, trimmer, multiply charged molecules no fragmentation, Soft ionization

21. Matrix: small MW absorb UV able to crystallize

22. 2.3 Mass analyzer (separate ions to measure m/z and intensity) Resolution: -ability to differentiate peaks of similar mass R = mean mass two peaks / separation between peaks = (m 1 +m 2 )/2(m 1 -m 2 ) - Resolution depends on mass R=1000, able to separate 1000 & 1001, or 100.0 & 100.1, or 10000& 10010 - High resolution necessary for exact MW determination - Nominal MW =2 8 - Actual MW C 2 H 4 + = 28.0313 - CH 2 N + = 28.017 - N 2 + = 28.0061, R > 2570

23. 2.3.1 magnetic sector analyzers Fig. 20-13 (p.567) Schematic of a magnetic sector spectrometer. Kinetic energy of ion: KE = z  e  V = 1/2  m  2 Magnetic force: F B = B  z  e  Centripetal force: F c = m 2 /r Only for ions with F B = F C can exit the slit m/z = B 2 r 2 e/2V For fixed radius & charge -use permanent magnet, and vary A and B potential V -Fixed V, vary B of electromagnet

24. 2.3.2 quadrupole analyzer Fig. 11-6 (p.283) A quadrupole mass spectrometer V RF cos(2  ft) U DC Ions travel parallel to four rods Opposite pairs of rods have oppositive VRFcos(2  ft) and U DC Ions try to follow alternating field in helical trajectory

25. Fig. 11-7 (p.288) operation of a quadrupole in xz plane V RF cos(2  ft) + U DC

26. - Stable path only for one m/z value for each field frequency U DC = 1.212mf 2 r 0 2 V RF = 7.219mf 2 r 0 2 U DC /V RF = 1.212/7.219 = 0.1679 R=0.126/(0.16784-U DC /V RF ) - Harder to push heavy molecule – m/z max < 2000 - R max ~ 500 Fig. 11-7 (p.288) Change of U DC and V RF during mass scan

27. Fig. 11-10 (p.290) A TOF mass spectrometer 2.3.3 Time-of-flight (TOF) analyzer

28. Unlimited mass range m/z max > 100 kDa Poor resolution R max < 1000 Poor sensitivity

29. 2.4 Detectors 2.4.1 Electron Multipliers Fig. 11-2 (p.284) Electron multiplier

30. 2.4.2 Microchannel Plates (MCP) Fig. 11-4 (p.286) MCP

31. Identification of Pure compounds (a) Nominal M + peak (one m/z resolution) (or (M+1) + or (M-1) + ) give MW (not EI) (b) Exact m/z (fractional m/z resolution) can give stoichiometry but not structure (double-focusing instrument) (c) Fragment peaks give evidence for functional groups (M-15) + peak  methyl (M-18) +  OH or water (M-45) +  COOH series (M-14) +, (M-28) +, (M-42) + …  sequential CH 2 loss in alkanes (d) Isotopic peaks can indicate presence of certain atoms Cl, Br, S, Si (e) Isotopic ratios can suggest plausible molecules from M +, (M+1) + and (M+2) + peaks 13 C/ 12 C = 1.08%, 2 H/ 1 H = 0.015% (M+1) peak for ethane C 2 H 6 should be (2x1.08) + (6x0.015)=2.25% M + peak (f)Comparison with library spectra

32. Fig. 20-4 (p.556) EI mass spectra of methylene chloride and 1-pentanol What about peaks at greater m/z than M + ? Two sources -Isotope peaks –same chemical formula but different masses - 12 C 1 H 2 35 Cl 2 m=84 - 13 C 1 H 2 35 Cl 2 m=85 - 12 C 1 H 2 35 Cl 37 Cl m=86 - 13 C 1 H 2 35 Cl 37 Cl m=87 - 13 C 1 H 2 37 Cl 2 m=88 - Heights vary with isotope abundance 13 C 1.08% 12 C, 2 H is 0.015% 1 H, 37 C is 32.5% 35 C CH 2 Cl 2, 13 C, 1 x 1.08 = 1.08 37 Cl, 2 x 32.5% 2 H, 2 x 0.015 = 0.030% (M+1) + /M + = 1.21%(M+2) + /M + =65%

34. One of the most powerful analytical tools Sensitive (10 -6 -10 -13 g) Range of ion sources for different situation Element comparison for small and large MW –biomolecules Limited structural information Qualitative and quantitative analysis of mixtures Composition of solid surfaces Isotopic information in compounds But Complex instrumentation Expensive: high resolution Structure obtained indirectly Complex spectra/fragmentation for hard ionization sources Simple spectra for soft ionization sources