1. Syed Ghulam Musharraf Assistant Professor H.E.J. Research Institute of Chemistry International Centre for Chemical and Biological Sciences (ICCBS) University of Karachi, Karachi-75270 E mail: musharraf1977@yahoo.com Interpretation of Mass Spectrometric Data

2. Course Outline Introductory lectures on gas phase ion reactions using Electron Impact (E.I) source. E.I fragmentation patterns of different classes of compounds and their spectral interpretations. Interpretation of Fast Atom Bombardment (FAB) and Chemical Ionization (CI)-MS spectra. Gas chromatography-mass spectrometry (GC-MS) data analysis and its spectral interpretation. Analysis of polar compounds by Electrospray ionization mass spectrometry (ESI-MS). ESI-fragmentation patterns of different classes of compounds and their interpretations. ESI-MS analysis of proteins/peptides and their spectra interpretations. MALDI-MS analysis of polar compounds and their spectral interpretation. Use of modern software for MS spectral interpretation.

3. Lecture 1: Introductory lecture on gas phase ion reactions using Electron Impact (EI) source

4. Mass Spectra EI-MS CI-MS FAB-MS ESI-MS MALDI-MS Which Mass Spectrum You are Going to Interpretate?

5. E.I. Mass Spectrometric Data The Mass Spectrum: A.Presentation of data 1.The mass spectrum is presented in terms of ion abundance vs. m/e ratio (mass) 2.The most abundant ion formed in ionization gives rise to the tallest peak on the mass spectrum – this is the base peak 3.All other peak intensities are relative to the base peak as a percentage. 4.If a molecule loses only one electron in the ionization process, a molecular ion is observed that gives its molecular weight – this is designated as M +. on the spectrum M +. Base peak Region A Region B

6. Interpretation of E.I. Mass Spectrometric Data A- Find out the molecular ion peak: B- Structural information extracted from the molecular ion peak: 1 st Step for Mass Spectral Interpretation

7. Interpretation of E.I. Mass Spectrometric Data 1-The molecular ion must be the highest mass ion in the spectra, discounting isotope peaks. 3-The ion must be an odd-electron (OE) ion. 2-The compound represented by the molecular ion must be capable of producing the important and logical fragment ions. A-Find out the molecular ion peak: “Some molecules are highly fragile and M +. peaks are not observed” Three facts must be fulfilled by molecular ion peaks:

8. Interpretation of E.I. Mass Spectrometric Data How we can know that ion must be odd-electron (OE)? By the calculation of saturation index : saturation index: (R + DB) R = number of rings DB = number of double bonds The total number of rings + double bonds = x - 1/2y + 1/2z + 1 For the general formula C x H y N z O n : Si is treated as C P is treated as N S is treated as O F, Cl, Br and I are treated as H

9. Interpretation of E.I. Mass Spectrometric Data Some Calculations: possible molecular ions? CH 4 C 3 H 3 F C 6 H 6 C 7 H 6 O 2 C 7 H 5 O “This is an important characteristic of even-electron ions-they will never have whole number values for their saturation index” For an even electron ion RDB = must end with ½ For an odd electron ion RDB = must end with whole number “Words of Caution” “It is true that all molecular ions will be odd-electron ions, not all odd-electron ions are molecular ions”. Many compounds can form odd-electron ions by breaking two chemical bonds, like in McLafferty rearrangement.

10. B- Structural informations extracted from the molecular ion peak (Low resolution analysis) 1-Generate molecular formula tentatively? Generate base formula by the rule of Thirteen 1 M/13 = n + r/13 M = molecular weight n = number of C and H atoms R = reminder C n H n+r Example: 1 = Bright, J. W., and Chen C. M., Journal of Chemical Education, 60 (1983): 557 M = 94, molecular formula = ? 94/ 13 ) 9413 91 7 3 Possible molecular formula = C 7 H 10 Other possible molecular formulas = C 6 H 6 O, C 5 H 2 O 2, C 6 H 8 N, C 5 H 2 S, CH 3 Br, Interpretation of E.I. Mass Spectrometric Data When a molecular mass, M +., is known, a base formula can be generated from the following equation:

11. Lung Cancer: Biological Samples 94 95 96 M +. + 1 M +. + 2 M +. What are the isotopic peaks: Peak (s) generated due to their naturally occurring heavier isotopes 2-Isotopic peaks B- Structural in formations extracted from the molecular ion peak (Low resolution analysis) Interpretation of E.I. Mass Spectrometric Data 1-Monoisotopic:  A or X elements  19 F, 23 Na, 31 P, 127 I  Others are 27 Al, 45 Sc, 55 Mg, 59 Co, 103 Rh, 133 Cs 2-Di-isotopic element: a-X+1 Element  12 C, 13 C; 14 N, 15 N; 1 H, 2 H b-X+2 Element  35 Cl, 37 Cl; 79 Br, 81 Br; 63 Cu, 65 Cu; 69 Ga, 71 Ga;  107 Ag, 109 Ag; 113 In, 115 In; 121 Sb, 123 Sb. c-X-1 Elements  6 Li, 7 Li; 10 B, 11 B; 50 V, 51 V 3-Polyisotopic element: Isotopic Classification of the Element:

12. Elements containing only one important isotopic form Element Mass F(A) 19 P(A) 31 I(A) 127 Elements containing two important isotopic forms Element Mass % Abundance Mass % Abundance H(A + 1) 1 100 2 0.01 C(A + 1) 12 100 13 1.1 N(A + 1) 14 100 15 0.37 Cl(A + 2) 35 100 37 32.5 Br(A + 2) 79 100 81 98.0 O(A + 2) 16 100 18 0.20a Elements containing three important isotopic forms Element Mass %AbundanceMass %AbundanceMass % Abn. Si(A + 2) 28 100 29 5.1 30 3.4 S(A + 2) 32 100 33 0.80 34 4.4 Mass and relative abundance of common organic elements Interpretation of E.I. Mass Spectrometric Data

13. 1- Nominal Mass: “integer mass of the most abundant naturally occurring stable isotope of an element” SnCl 2 (120 + 35 x 2) = 190 u 3- Relative Mass: “Sum of the average weight of the naturally occurring isotopes of an element” 100 + 31.96 Mr = 35.4528 u 2- Monoisotopic Mass: “The Exact mass of the most abundant isotope of an element” Different masses used in MS Mr =100 x 34.968853 u + 31.96 x 36.965903 u Cl 2 =

14. Interpretation of E.I. Mass Spectrometric Data B- Structural informations extracted from the molecular ion peak (Low resolution analysis) “Number of carbon atoms can be estimated” 1-Information from M +1 Peak: C = 100 Y/1.1 X C= numbers of carbon X = amplitude of the M ion Y = amplitude of the M+1 ion Peak m/zIntensityC = 100 Y/1.1 X 72 M+73.0 (X) = 100. 3.3/1.1. 73 = 4 73 M+13.3 (Y) 74 M+20.2 0.3% = Absence of S (4.4%), Cl (33%), Br (98%) So the probable molecular formula is C 4 H 8 O For a molecular formula composed of C and H = C 4 H 24 An example: 2-Information from M +2 Peak: Presence of S or Si Presences of Br and Cl (A characteristics peak intensity pattern observe)

15. Interpretation of E.I. Mass Spectrometric Data B- Structural informations extracted from the molecular ion peak (Low resolution analysis) 1-Information from M +1 Peak: Molecules that are completely 12 C are now rare insulin (257 carbon atoms)

16. Interpretation of E.I. Mass Spectrometric Data 2-Information from M +2 Peak: B- Structural in formations extracted from the molecular ion peak (Low resolution analysis) 1.For molecules that contain Cl or Br, the isotopic peaks are diagnostic (a)- In both cases the M+2 isotope is prevalent:  35 Cl is 75.77% and 37 Cl is 24.23% of naturally occurring chlorine atoms  79 Br is 50.52% and 81 Br is 49.48% of naturally occurring bromine atoms (b)- If a molecule contains a single chlorine atom, the molecular ion would appear: The M+2 peak would be 24% the size of the M + if one Cl is present m/e relative abundance M+M+ M+2

17. Interpretation of E.I. Mass Spectrometric Data 2-Information from M +2 Peak: B- Structural in formations extracted from the molecular ion peak (Low resolution analysis) (c)- If a molecule contains a single bromine atom, the molecular ion would appear: a)The effects of multiple Cl and Br atoms is additive. (d)- Sulfur will give a M+2 peak of 4% relative intensity and silicon 3% m/e relative abundance M+M+ M+2 The M+2 peak would be about the size of the M + if one Br is present

18. Interpretation of E.I. Mass Spectrometric Data Presence of multiple Cl or Br atoms? 1-Generation of M+4 and M+6 peaks 2-Change in intensity pattern B- Structural in formations extracted from the molecular ion peak (Low resolution analysis) CH 3 Cl CH 2 Cl 2 CHCl 3

19. Interpretation of E.I. Mass Spectrometric Data Total number of possible combinations = A n A= number of isotopes considered, n = number of atoms of present For Br 2 = total number of combinations = 2 2 = 4, Br79, Br79; Br79 Br81 + Br81 Br79; Br81 Br81 http://www.sisweb.com/mstools/isotope.htm 1-Why M+4 and M+6 peaks are observed? B- Structural in formations extracted from the molecular ion peak (Low resolution analysis) 2-How we can calculate intensity pattern? Example: Br 2 Calculate number of combinations For CHBr 3 By Binomial expression: (a + b) n a and b = abundance of two isotopes of n = number of bromine atom attached n=1 (a + b) 1 = a + b n=2 (a + b) 2 = a 2 + 2ab + b 2 n=3 (a + b) 3 = a 3 + 3a 2 b + 3ab² + b 3 n=4 (a + b) 4 = a 4 + 4a 3 b + 6a²b² + 4ab 3 + b 4 Pascal intensity Pattern (Only for Br)

20. Interpretation of E.I. Mass Spectrometric Data B- Structural informations extracted from the molecular ion peak (Low resolution analysis) One practice Example: S 2 32 S 32 S Total mass: 64, one combination. 32 S 33 S or 33 S 32 S Total mass: 65, two combinations. 32 S 34 S or 34 S 32 S Total mass: 66, two combinations. 33 S 33 S Total mass: 66, one combination. 33 S 34 S or 34 S 33 S Total mass: 67, two combinations. 34 S 34 S Total mass: 68, one combination. Total: nine combinations Intensity calculation:

21. Interpretation of E.I. Mass Spectrometric Data B- Structural informations extracted from the molecular ion peak (Low resolution analysis) Presences of nitrogen or not: (Nitrogen rule) Word of Caution: Nitrogen Rule will be “reversed” when you HAVE “protonated molecualr ion peak” like in case of ESI “A molecule containing an odd number of nitrogens will have an odd molecular weight, while a compound containing no nitrogens or an even number of nitrogens will have an even molecular weight”. AtomsValencyAtomic Weight C H O Br S Cl N 41212134121213 12 1 16 79/81 32 35/37 14 Nitrogen is the only common element which has an ODD valency and an EVEN atomic mass

22. Interpretation of E.I. Mass Spectrometric Data B- Structural in formations extracted from the molecular ion peak (High resolution analysis) 1.If sufficient resolution (R > 5000) exists, mass numbers can be recorded to precise values (6 to 8 significant figures) 2.From tables of combinations of formula masses with the natural isotopic weights of each element, it is often possible to find an exact molecular formula from HRMS Example: HRMS gives you a molecular ion of 98.0372; from mass 98 data: C 3 H 6 N 4 98.0594 C 4 H 4 NO 2 98.0242 C 4 H 6 N 2 O98.0480 C 4 H 8 N 3 98.0719 C 5 H 6 O 2 98.0368  gives us the exact formula C 5 H 8 NO98.0606 C 5 H 10 N 2 98.0845 C 7 H 14 98.1096

23. Problems overcome by HR analysis Number of carbon atom---------------Solved Elemental composition-----------------Solved Presence of N, Halogen----------------Solved But you need to calculate OE ions for molecular ion peaks Compounds with molecular wt 28: N 2, C 2 H 4, CO Interpretation of E.I. Mass Spectrometric Data Goal is to measure ion mass with an accuracy of ± 1-10 ppm m/z 100 mu m/z 500 mu m/z 1000 mu ±1 ppm ±0.0001 ±0.0005 ±0.001 ±10 ppm ±0.001 ±0.005 ±0.0 ±1 How accurate does the mass have to be? xxx.x±0.1? xxx.xx±0.01? xxx.xxx±0.001? B- Structural in formations extracted from the molecular ion peak (High resolution analysis)

24. Interpretation of E.I. Mass Spectrometric Data A Summary before moving on: 1.Using the the M + peak, make any inferences about the approximate formula a)Nitrogen Rule b)Rule of Thirteen c)RDB 2.Using the M+1 peak (if visible) make some inference as to the number of carbon atoms (for small molecules this works as H, N and O give very low contributions to M+1) 3.If M+2 becomes apparent, analyze for the presence of one or more Cl or Br atoms (sulfur and silicon can also give prominent M+2)