2. Mass Spectrometry A key Tool for the chemist’s toolbox. The logic is, we always want the molecular weight. Second, we can smash out fragments that are intact structurally These are easier to solve and relate back to the starting structure Implication is, we don’t get the sample back; a destructive method

3. Mass Spectrometry A primary tool for chemists from almost every discipline Molecular Weights are fundamental to almost every structural question. Molecular weight is not ambiguous. A compound has a unique MW. Our ability to analyze compounds on this basis, depends completely on being able to generate ions from the compound. Specifically molecular ions*, whose weight is equal to the MW of the compound, are critical. Once produced, our analysis according to MW depends on differential mobility or acceleration of ions proportionate to the MW. *We can usefully broaden this definition from [M  ] + to embrace [M+H] +, [M-H] -, Chemical Ionization adducts, etc.

4. What’s in a Mass Spectrum? Ion Abundance (as a %of Base peak) Mass, as m/z. Z is the charge, and for doubly charged ions (often seen in macromolecules), masses show up at half their proper value High mass Not usually scanned below m/z=32 (Why?) [M+H] + (CI) Or M + (EI) “molecular ion” Unit mass spacing Fragment Ions Derived from molecular ion or higher weight fragments In CI, adduct ions, [M+reagent gas] +

5. Molecular Ions give us the molecular mass Chemical Ionization M H+H+ H+H+ H+H+ H+H+ H+H+ [M+ H ] + Weighs one more than MW Dislodges an electron Electron Impact M -e-e -e-e -e-e -e-e -e-e -e-e 2e - M +

6. Identifying Molecular Ions Potential question; Is the largest m/z the molecular ion or is it a prominent fragment from an even heavier molecule? Increase sample loading In EI, can lower the beam voltage (make the M + less energetic, perhaps more long-lived.) Logical interval between significant peaks and suspected M +. i.e. the loss of 3-14 mass units is unusual, as is loss of 19-25 (except F). Loss of 33, 35, 38 also unusual. However a loss of 15, 18, 31 is good evidence for a molecular ion. Switch to CI, vary reagent gas. Positive, negative probes. Check for CI adduct ions. e.g. C 2 H 5 +, CH 5 +, C 3 H 5 + Find MW by other method Prepare derivative Other compounds present may give ions that deceive us. May be more detectable. MS intensities are problematic

7. The “Nitrogen Rule” Molecules containing atoms limited to C,H,O,N,S,X,P of even-numbered molecular weight contain either NO nitrogen or an even number of N This is true as well for radicals as well. Not true for pre-charged, e.g. quats, (rule inverts) or radical cations. In the case of Chemical Ionization, where [M+H] + is observed, need to subtract 1, then apply nitrogen rule. Example, if we know a compound is free of nitrogen and gives an ion at m/z=201, then that peak cannot be the molecular ion.

8. E lectron I mpact and C hemical I onization EI Sometimes too energetic for molecular ion to survive Rich harvest of fragment ions “fingerprint” nature of fragment patterns lends itself to database library searches CI Stronger, more reliable molecular ions Fewer fragments Can choose different reagent gasses and exploit chemistry, giving different fragmentation. e.g. NH 3 /ND 3 Adduct ions give support to identities Nitrogen rule works but inverted Can do negative ion Mass Spec EI CI

9. When would you use CI, EI? EI When “fingerprint” is needed for Identification by comparison screening in databases Trace analysis Forensics Environmental Total unknowns, e.g natural products Fragment homology within a series, e.g. of natural products CI When rapid, reliable identification of molecular ion is needed. LC-MS Following a synthetic chemistry route, tentative impurity ID Biological samples, other fragile or sensitive to decomposition; Drug or other metabolite ID When reagent gas chemistry is key, e.g. exchange D in for H Minimize fragmentation, get most intensity in molecular ion.

10. How can I tell which (EI or CI) was run? Adduct ions higher m/z than MH +,,[M+C 2 H 5 ] +,[M+C 3 H 5 ] + [M+NH 4 ] + Large molecular ion Relatively few fragment ions Chemical Ionization No ions higher m/z than M + Smaller M + intensity Rich family of fragment ions Electron Impact

11. The “Rule of 13” as an aid to guessing a molecular Formula Take the Weight of ion, divide by 13 This answer is N, for (CH) N and any numerical remainder is added as H e.g.; 92 92/13 = 7 with remainder = 1; C 7 H 8 weighs 92. This is our candidate formula Can evaluate other alternative candidate formulas possessing heteroatoms. For each member of the list below, replace the indicated number of CHs in the above answer Hetero substitution CH replacement Hetero substitution CH replacement OCH 4 PC2H7C2H7 NCH 2 SC2H8C2H8 O+NC2H6C2H6 O+SC4C4 FCH 7 IC 10 H 7 SiC2H4C2H4 Cl,Br(use isotopes)

12. Analyzing Ion Clusters: a way to rule candidate structures in or out Mass spectrometry “sees” all the isotopomers as distinct ions An ion with all 12 C is one mass unit different from an ion with one 13 C and the rest 12 C Since the isotope distribution in nature is known* for all the elements ( 13 C is 1.1%), the anticipated range and ratios of ions for a given formula can be predicted and calculated Follows a binomial expansion: e.g.; for N carbon atoms (% 12 C + % 13 C) N

13. Clusters of Ions Spaced by unit mass Each peak is for the same molecular formula Different peaks because there are some molecules with 13 C, 2 H etc. Especially significant for Cl, Br m/z The Nominal mass is m/z of the lowest member of the cluster. This is the isotopomer that has all the C’s as 12 C, all protons as 1 H, all N’s as 14 N, etc.

14. Isotope Patterns in Ion Clusters Here are two molecular ions of nearly the same m/z. One of them is “carbon-rich”, and has a larger number of 13 C’s The other, presumably has proportionately, more heteroatoms C 24 H 50 C 12 H 22 O 11

15. Why is this Important? All 12 C 1 13 C C 10 C 100 1 13 C 2 13 C From this, it is clear that for large or macromolecules, there will be practically no population having all 12 C or even only 1 13 C A rule of thumb, made possible by knowing the isotopic abundance is that the number of C in a formula is given by: N=

16. Fragmentation The “Even Electron Rule” dictates that even (non-radical) ions will not fragment to give two radicals (pos + neutral) (CI) Better carbocation wins and predominates (“Stevenson’s Rule”) [M · ] + A + +B · (neutral) or B + +A · EI CI [M+H] + PH + +N (neutral)

17. Reading a Mass Spec from the M + Down (EI) FragmentDue to loss of…Interpretation M + -1-HAldehydes, tert. Alcohols, cyclic amines M + -2Multiple -HSecondary alcohols M + -3Multiple -HPrimary alcohols M + -4 to -13(doubtful)Consider contaminants M + -14(doubtful)CH 2, N not good losses M + -15CH 3 Available methyl groups, methylesters M + -16OPeroxides M + -17OHAlcohols, phenols, RCO 2 H M + -18H2OH2Oalcohols M + -19-F M + -20-HF M + -21 to -25No peaks expected M + -26 HC  CH M + -27HC=CH2 or HCNHCN from pyridine, anilines M + -28 C  O or CH 2 =CH 2 Check for McLafferty R&R

18. How Do I go about using Mass Spec Data for Unknowns? First, get the molecular weight Identify prime, smaller mass losses like water, etc. Now stop. Don’t worry about the fragments till you have some candidate structures Based on NMR, IR get some notions of structure candidates or partial structures, functional groups Now go back to MS, predict some fragments your structure will give, calculate the molecular weights and check MS Back and forth with other data, to corroborate or refute a possible structure.

19. Nominal Mass Here for example is a list of the compounds in the Merck Index (9th ed) that weigh nominally, 200 Exact mass measurements can easily distinguish These instruments (and other) can generate exhaustive lists of possible structure formulas near the exact mass value.

20. molar mass: 157 Formula M+1 M+2 MM e/o dbr HN2O8 1.05 1.60 156.9732 e 1.5 HN10O 3.75 0.20 157.0337 e 5.5 H3N3O7 1.41 1.40 156.9971 o 1 H3N11 4.12 0.00 157.0576 o 5 H5N4O6 1.78 1.20 157.021 e 0.5 H7N5O5 2.14 1.00 157.0448 o 0 CHO9 1.45 1.80 156.9619 e 1.5 CHN8O2 4.15 0.43 157.0224 e 5.5 CH3NO8 1.81 1.60 156.9858 o 1 CH3N9O 4.51 0.24 157.0463 o 5 CH5N2O7 2.17 1.41 157.0096 e 0.5 CH5N10 4.88 0.04 157.0702 e 4.5 CH7N3O6 2.54 1.21 157.0335 o 0 C2HN6O3 4.55 0.66 157.0111 e 5.5 C2H3N7O2 4.91 0.47 157.0350 o 5 C2H5O8 2.57 1.61 156.9983 e 0.5 C2H5N8O 5.27 0.28 157.0589 e 4.5 C2H7NO7 2.93 1.42 157.0222 o 0 C2H7N9 5.64 0.09 157.0827 o 4 C3HN4O4 4.94 0.89 156.9998 e 5.5 C3H3N5O3 5.31 0.70 157.0237 o 5 C3H5N6O2 5.67 0.51 157.0476 e 4.5 C3H7N7O 6.03 0.33 157.0714 o 4 C3H9N8 6.4 0.14 157.0953 e 3.5 C4HN2O5 5.34 1.11 156.9885 e 5.5 C4H3N3O4 5.70 0.93 157.0124 o 5 C4H5N4O3 6.07 0.74 157.0362 e 4.5 C4H7N5O2 6.43 0.56 157.0601 o 4 C4H9N6O 6.79 0.38 157.0840 e 3.5 C4H11N7 7.16 0.20 157.1078 o 3 C5HO6 5.74 1.32 156.9772 e 5.5 C5H3NO5 6.10 1.15 157.0011 o 5 C5H5N2O4 6.46 0.97 157.025 e 4.5 C5H7N3O3 6.83 0.79 157.0488 o 4 C5H9N4O2 7.19 0.61 157.0727 e 3.5 C5H11N5O 7.55 0.44 157.0965 o 3 C5H13N6 7.92 0.26 157.1204 e 2.5 C6HN6 8.84 0.33 157.0264 e 9.5 C6H5O5 6.86 1.19 157.0136 e 4.5 C6H7NO4 7.22 1.02 157.0375 o 4 C6H9N2O3 7.59 0.84 157.0614 e 3.5 C6H11N3O2 7.95 0.67 157.0852 o 3 C6H13N4O 8.31 0.50 157.1091 e 2.5 C6H15N5 8.68 0.32 157.1329 o 2 C7HN4O 9.23 0.58 157.0151 e 9.5 C7H3N5 9.60 0.41 157.0390 o 9 C7H9O4 7.98 1.07 157.0501 e 3.5 C7H11NO3 8.35 0.90 157.0739 o 3 C7H13N2O2 8.71 0.73 157.0978 e 2.5 C7H15N3O 9.07 0.56 157.1217 o 2 C7H17N4 9.44 0.40 157.1455 e 1.5 C8HN2O2 9.63 0.81 157.0038 e 9.5 C8H3N3O 9.99 0.65 157.0277 o 9 C8H5N4 10.36 0.49 157.0516 e 8.5 C8H13O3 9.11 0.96 157.0865 e 2.5 C8H15NO2 9.47 0.80 157.1104 o 2 C8H17N2O 9.83 0.64 157.1342 e 1.5 C8H19N3 10.20 0.47 157.1581 o 1 C9HO3 10.03 1.05 156.9925 e 9.5 C9H3NO2 10.39 0.89 157.0164 o 9 C9H5N2O 10.75 0.73 157.0403 e 8.5 C9H7N3 11.12 0.57 157.0641 o 8 C9H17O2 10.23 0.87 157.1229 e 1.5 C9H19NO 10.59 0.71 157.1468 o 1 C9H21N2 10.96 0.55 157.1706 e 0.5 C10H5O2 11.15 0.97 157.029 e 8.5 C10H7NO 11.51 0.81 157.0528 o 8 C10H9N2 11.88 0.66 157.0767 e 7.5 C10H21O 11.35 0.79 157.1593 e 0.5 C10H23N 11.72 0.64 157.1832 o 0 C11H9O 12.27 0.90 157.0654 e 7.5 C11H11N 12.64 0.75 157.0892 o 7 C12H13 13.40 0.84 157.1018 e 6.5 C13H 14.32 0.97 157.0078 e 13.5 Example, m/z’s for 157 Clearly, some are not realistic!

21. Calculated mass distributions Isotopic Element Massesand Atomic Weights:Lide, D.R., Ed., CRC Handbook of Chemistry and Physics,74th Ed., CRC Press, Boca Raton FL,(1993) Isotope Distribution:Rockwood, A. L., Van Orden, S. L.,Smith, R. D.,Anal. Chem., 67, 2699, (1995) iMass is freeware. Contact: ursr@mac.comursr@mac.com IMASS for Mac OSX Version 1.0 (v2A15) © 2000 - 2002, Urs Roethlisberger,

22. Fragment Ions The Game is, to rationalize these in terms of the structure Identify as many as possible, in terms of the parent structure Generally, simply derived from the molecular ion Or, in a simple fashion from a significant higher mw fragment. Simply, here means, ions don’t fly apart, split out neutrals and then recombine. Fragments will make chemical sense A good approach is the “rule of 13” to write down a molecular formula for an ion of interest. Especially in EI, we only identify major fragments

23. Chemical Ionization Fragmentation Loss of neutral molecules, small stable, from MH + Loss of neutrals from protonated fragments Subsequent reprotonation after a loss Typically there is no ring cleavage (needs radical) or two bond scissions. Depends highly on ion chemistry specifically acid- base (proton affinities)

24. Some popular cleavages Cleave at a branch point. Loss of radical or other neutral to provide a more stable cation Cleave  to a heteroatom (capable of supporting positive charge) Note the use of “half arrow” for one-electron movements. e.g homolytic cleavage CH 3 CH 3 CH 3 CH 3 CH 3 C + CH 3 CH 3. Obs. in Mass Spec + neutral + RO RO RO : : Obs. in Mass Spec Resonance stabilized neutral + +

25. Some examples Primary alcohols, m/z=31 CH 2 =OH + Primary amines, m/z=30 CH 2 =NH 2 +

26. Commonly encountered Electron-Impact fragments N CH 2 + 92 + + 91 H H H H H + 77 CH 2 + 43 H O + 29

27. McLafferty Rearrangements Radical cations localized on keto-type oxygen give  cleavage The mechanism limits this to EI fragmentation Needs a H atom on a  sp 3 carbon Ketones, esters, carboxylic acids all give McLafferty products O + H R2 R1 O + H OH R1 R2 + Loss of neutral alkene The new radical cation is stabilized by resonance Note the use here, of the “half arrow” to represent “1-electron flow”

28. Important example of McLafferty R&R m/z = 60 + Seen for primary carboxylic acids

29. Non-Sequential Losses MW=152 M + -CH 3 CO M + -CH 3

30. Hydrocarbons Weak [M]+ Intense C n H 2n+1 Good 43 m/z = C 3 H 7 protonated cyclopropane 57 m/z = C 4 H 9 + 71 m/z = C 5 H 11 Hydrocarbon chains characterized by successive losses of m/z=14 (clusters)

31. Cleavage  to C=O groups O O O :. + + + : + : : Obs. in mass spec. Acylium ions are resonance-stabilized neutral Prominent for ketones CH 3 C=O + m/z=43

32. Example M+ -45, loss of ethoxy radical

33. Example M + -43; also tropylium ion

34. Cleave  to Heteroatoms like O, N O R :. + Heterolytic cleavage R O : :. neutral + Observed in Mass Spec provided that a good stabilized carbocation can form +

35. Rearrangements and fragmentations to give good Carbocations CH 2 + C H + CH 2 H C CH 2 CH+ + Benzylic cation (stabilized including “tropylium” ion m/z =91 Good cleavage  to aromatic rings

36. Example Bromine pattern Tropylium ion

37. Carboxylic acids  H present?; can give McLafferty R&R to alkene plus CH2=C(OH)(OH) + at m/z=60 Loss of water, especially in CI Loss of 44 is loss of CO 2 m/z=45 suggests O  C–OH +

38. Amines N + - R R Cyclic amines will lose adjacent H, form iminium ion In CI,  NH+ can eliminate adjacent alkene, reprotonate

39. Silyl Ethers Si O + Loss of CH 3 from Si Loss of R in  cleavage Loss of CR 3 then CH 3 to (CH 3 ) 2 Si=OH + m/z=75 Total loss of carbinol to (CH 3 ) 3 Si + m/z=73

40. H transfer in heterosubstituted Anisoles Loss of CH 2 O Extra H transfer mediated by adjacent heteroatom H O + R H H +

41. Nitroaromatics N + O O Loss of N=O Loss of C  O m/z= 93 (this can form from lots of different origins) Aromatic! m/z=65 Good test for aryloxy

42. Sulfur Compounds Fortunately there is an [M+2]+ of 4% for the natural abundance of 34 S. This is diagnostic for S vs 2x 16 O Aliphatic thiols can split out H 2 S, [M-34] Alpha cleavage at carbon bearing the sulfur in thiols, thioethers, similar to ethers, etc. R -R

43. The “retro Diels-Alder” Cleavage + + + + + Observed! Typically you see both. More stable cation will predominate Also works for hetero-substituted (e.g. make enol) Both EI (shown) and in Chemical Ionization. (protonated molecular ion, cleave, then reprotonation Cyclohexenes, with favorable 6-membered transition state. Can include heteroatoms (N,O, driven by keto-enol like stability.

44. An Example from Terpenoid Chemistry + + +  12 -Oleanene m/z 204

45. A good example for Retro Diels Alder fragmentation 4-terpineol MW 154 mz 86 + + mz 68 EI Mass Spectrum +

46. Double bonds can isomerize MW=396 m/z136 C 9 H 12 O + m/z118 136-water  -cleavage following double bond migration

47. Mass Spectral “shifts” Note highly conserved regions; series of related compounds Losses down to ions common in series. Variation can not influence the fragmentation or introduce new fragmentation, e.g. internal fission not possible for homologs

48. Using the Information in Ion Clusters--Halogens CH 3 Cl One chlorine CHCl 3 Three chlorines 35 Cl 37 Cl CH 3 Br One bromine CHBr 3 Three bromines 79 Br 81 Br 81 Br 2 81 Br 1 The paired appearance flags the ions as to the number of halogens Fragment ions with the same halogen count preserve the pattern

49. Great Websites http://medlib.med.utah.edu/masspec/elcomp.htm Calculate potential molecular formulas from m/z (neutrals only) http://www.colby.edu/chemistry/PChem/Fragment.html Wizard calculates both odd, even electron species based on m/z The same folks provide a online wizard for calculating ion clusters (isotope patterns) from a suggested formula. http://www.colby.edu/chemistry/NMR/IsoClus.html http://webbook.nist.gov/chemistry/ Free search of name, formulas