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Syed Ghulam Musharraf

Interpretation of Mass Spectrometric Data. 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. Course Outline.

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Syed Ghulam Musharraf

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  1. Interpretation of Mass Spectrometric Data 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

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

  5. E.I. Mass Spectrometric Data • The Mass Spectrum: • Presentation of data • The mass spectrum is presented in terms of ion abundance vs. m/e ratio (mass) • The most abundant ion formed in ionization gives rise to the tallest peak on the mass spectrum – this is the base peak • All other peak intensities are relative to the base peak as a percentage. • 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 Region A Region B Base peak M+.

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

  7. Interpretation of E.I. Mass Spectrometric Data 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: 1-The molecular ion must be the highest mass ion in the spectra, discounting isotope peaks. 2-The compound represented by the molecular ion must be capable of producing the important and logical fragment ions. 3-The ion must be an odd-electron (OE) ion.

  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 For the general formula CxHyNzOn: The total number of rings + double bonds = x - 1/2y + 1/2z + 1 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 For an even electron ion RDB = must end with ½ For an odd electron ion RDB = must end with whole number “This is an important characteristic of even-electron ions-they will never have whole number values for their saturation index” Some Calculations: possible molecular ions? “Words of Caution” CH4 C3H3F C6H6 C7H6O2 C7H5O “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. Interpretation of E.I. Mass Spectrometric Data B- Structural informations extracted from the molecular ion peak (Low resolution analysis) 1-Generate molecular formula tentatively? Example: M = 94, molecular formula = ? Generate base formula by the rule of Thirteen1 When a molecular mass, M+., is known, a base formula can be generated from the following equation: 94/ 13 7 ) 13 94 91 M/13 = n + r/13 M = molecular weight n = number of C and H atoms R = reminder 3 Possible molecular formula = C7H10 Other possible molecular formulas = C6H6O, C5H2O2, C6H8N, C5H2S, CH3Br, CnHn+r 1 = Bright, J. W., and Chen C. M., Journal of Chemical Education, 60 (1983): 557

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

  12. Interpretation of E.I. Mass Spectrometric Data Elements containing only one important isotopic form Element Mass F(A) 19 P(A) 31 I(A) 127 Mass and relative abundance of common organic elements 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 %Abundance Mass %Abundance Mass % Abn. Si(A + 2) 28 100 29 5.1 30 3.4 S(A + 2) 32 100 33 0.80 34 4.4

  13. Interpretation of E.I. Mass Spectrometric Data Different masses used in MS 2- Monoisotopic Mass: “The Exact mass of the most abundant isotope of an element” 1- Nominal Mass: “integer mass of the most abundant naturally occurring stable isotope of an element” SnCl2 (120 + 35 x 2) = 190 u 3- Relative Mass: “Sum of the average weight of the naturally occurring isotopes of an element” Mr =100 x 34.968853 u + 31.96 x 36.965903 u Cl2 = 100 + 31.96 Mr = 35.4528 u

  14. 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: An example: “Number of carbon atoms can be estimated” C = 100 Y/1.1 X C= numbers of carbon X = amplitude of the M ion Y = amplitude of the M+1 ion Peak 0.3% = Absence of S (4.4%), Cl (33%), Br (98%) 2-Information from M +2 Peak: Presence of S or Si Presences of Br and Cl (A characteristics peak intensity pattern observe) For a molecular formula composed of C and H = C4H24 So the probable molecular formula is C4H8O

  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: • insulin (257 carbon atoms) Molecules that are completely 12C are now rare

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

  17. Interpretation of E.I. Mass Spectrometric Data B- Structural in formations extracted from the molecular ion peak (Low resolution analysis) 2-Information from M +2 Peak: • (c)- If a molecule contains a single bromine atom, the molecular ion would appear: • 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+ M+2 The M+2 peak would be about the size of the M+ if one Br is present relative abundance m/e

  18. Interpretation of E.I. Mass Spectrometric Data B- Structural in formations extracted from the molecular ion peak (Low resolution analysis) Presence of multiple Cl or Br atoms? 1-Generation of M+4 and M+6 peaks 2-Change in intensity pattern CH3Cl CH2Cl2 CHCl3

  19. Interpretation of E.I. Mass Spectrometric Data B- Structural in formations extracted from the molecular ion peak (Low resolution analysis) 1-Why M+4 and M+6 peaks are observed? 2-How we can calculate intensity pattern? Example: Br2 By Binomial expression: (a + b)n a and b = abundance of two isotopes of n = number of bromine atom attached For Br2 = total number of combinations = 22 = 4, Br79, Br79; Br79 Br81 + Br81 Br79; Br81 Br81 n=1 (a + b)1 = a+ b n=2 (a + b)2 = a2 + 2ab + b2 n=3 (a + b)3 = a3 + 3a2b + 3ab² + b3 n=4 (a + b)4 = a4 + 4a3b + 6a²b² + 4ab3 + b4 Total number of possible combinations = An A= number of isotopes considered, n = number of atoms of present Pascal intensity Pattern (Only for Br) Calculate number of combinations For CHBr3 http://www.sisweb.com/mstools/isotope.htm

  20. Interpretation of E.I. Mass Spectrometric Data B- Structural informations extracted from the molecular ion peak (Low resolution analysis) One practice Example: S2 32S 32S Total mass: 64, one combination. 32S 33S or 33S 32S Total mass: 65, two combinations. 32S 34S or 34S 32S Total mass: 66, two combinations. 33S 33S Total mass: 66, one combination. 33S 34S or 34S 33S Total mass: 67, two combinations. 34S 34S 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) “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”. Nitrogen is the only common element which has an ODD valency and an EVEN atomic mass Word of Caution: Nitrogen Rule will be “reversed” when you HAVE “protonated molecualr ion peak” like in case of ESI

  22. Interpretation of E.I. Mass Spectrometric Data B- Structural in formations extracted from the molecular ion peak (High resolution analysis) • If sufficient resolution (R > 5000) exists, mass numbers can be recorded to precise values (6 to 8 significant figures) • 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: • C3H6N4 98.0594 • C4H4NO2 98.0242 • C4H6N2O 98.0480 • C4H8N3 98.0719 • C5H6O2 98.0368  gives us the exact formula • C5H8NO 98.0606 • C5H10N2 98.0845 • C7H14 98.1096

  23. Interpretation of E.I. Mass Spectrometric Data B- Structural in formations extracted from the molecular ion peak (High resolution analysis) 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: N2, C2H4, CO How accurate does the mass have to be? xxx.x±0.1? xxx.xx±0.01? xxx.xxx±0.001? 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

  24. Interpretation of E.I. Mass Spectrometric Data A Summary before moving on: • Using the the M+ peak, make any inferences about the approximate formula • Nitrogen Rule • Rule of Thirteen • RDB • 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) • 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)

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