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 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. Lecture 1: Introductory lecture on gas phase ion reactions using Electron Impact (EI) source Mass Spectra EI-MS ESI-MS Which Mass Spectrum You are FAB-MS Going to MALDI-MS Interpretate? CI-MS E.I. Mass Spectrometric Data The Mass Spectrum: A. Presentation of data 1. 2. 3. 4. 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 B Base peak Region A M+. 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: .. : O e .+: O + Molecular ion 2e 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. 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 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? CH4 C3H3F C6H6 C7H6O2 C7H5O “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. Interpretation of E.I. Mass Spectrometric Data B- Structural informations extracted from the molecular ion peak (Low resolution analysis) 1-Generate molecular formula tentatively? 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: M/13 = n + r/13 M = molecular weight n = number of C and H atoms R = reminder CnHn+r Example: M = 94, molecular formula = ? 94/ 13 7 13 )94 91 3 Possible molecular formula = C7H10 Other possible molecular formulas = C6H6O, C5H2O2, C6H8N, C5H2S, CH3Br, 1 = Bright, J. W., and Chen C. M., Journal of Chemical Education, 60 (1983): 557 Lung Cancer: SamplesData Interpretation of E.I.Biological Mass Spectrometric 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: Peak (s) generated due to their naturally occurring heavier isotopes 94 M+. 95 96 M+. + 1 M+. + 2 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: Interpretation of E.I. Mass Spectrometric Data Elements containing only one important isotopic form Element F(A) P(A) I(A) Mass 19 31 127 Mass and relative abundance of common organic elements Elements containing two important isotopic forms Element H(A + 1) C(A + 1) N(A + 1) Cl(A + 2) Br(A + 2) O(A + 2) Mass 1 12 14 35 79 16 % Abundance 100 100 100 100 100 100 Mass 2 13 15 37 81 18 % Abundance 0.01 1.1 0.37 32.5 98.0 0.20a %Abundance 5.1 0.80 Mass 30 34 Elements containing three important isotopic forms Element Si(A + 2) S(A + 2) Mass 28 32 %Abundance 100 100 Mass 29 33 % Abn. 3.4 4.4 Interpretation of E.I. Mass Spectrometric Data Different masses used in MS 1- Nominal Mass: 2- Monoisotopic Mass: “integer mass of the most abundant naturally occurring stable isotope of an element” “The Exact mass of the most abundant 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” Cl2 = Mr =100 x 34.968853 u + 31.96 x 36.965903 u 100 + 31.96 Mr = 35.4528 u 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: “Number of carbon atoms can be estimated” An example: m/z Intensity C = 100 Y/1.1 X 72 M+ 73.0 (X) = 100. 3.3/1.1 . 73 =4 73 M+1 3.3 (Y) 74 M+2 0.2 C = 100 Y/1.1 X C= numbers of carbon X = amplitude of the M ion Y = amplitude of the M+1 ion Peak 2-Information from M +2 Peak: Presence of S or Si Presences of Br and Cl (A characteristics peak intensity pattern observe) 0.3% = Absence of S (4.4%), Cl (33%), Br (98%) For a molecular formula composed of C and H = C4H24 So the probable molecular formula is C4H8O 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 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: relative abundance 1. M+ M+2 m/e The M+2 peak would be 24% the size of the M+ if one Cl is present 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: relative abundance (c)- If a molecule contains a single bromine atom, the molecular ion would appear: a) M+ M+2 The M+2 peak would be about the size of the M+ if one Br is present The effects of multiple Cl and Br atoms m/e is additive. (d)- Sulfur will give a M+2 peak of 4% relative intensity and silicon 3% 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? CH3Cl 1-Generation of M+4 and M+6 peaks 2-Change in intensity pattern CHCl3 CH2Cl2 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? Example: Br2 For Br2 = total number of combinations = 22 = 4, Br79, Br79; Br79 Br81 + Br81 Br79; Br81 Br81 Total number of possible combinations = An A= number of isotopes considered, n = number of atoms of present 2-How we can calculate intensity pattern? By Binomial expression: (a + b)n a and b = abundance of two isotopes of n = number of bromine atom attached n=1 n=2 n=3 n=4 (a + b)1 = a + b (a + b)2 = a2 + 2ab + b2 (a + b)3 = a3 + 3a2b + 3ab² + b3 (a + b)4 = a4 + 4a3b + 6a²b² + 4ab3 + b4 Calculate number of combinations For CHBr3 http://www.sisweb.com/mstools/isotope.htm Pascal intensity Pattern (Only for Br) 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 32S 33S or 33S 32S 32S 34S or 34S 32S 33S 33S 33S 34S or 34S 33S 34S 34S Intensity calculation: Total mass: 64, one combination. Total mass: 65, two combinations. Total mass: 66, two combinations. Total mass: 66, one combination. Total mass: 67, two combinations. Total mass: 68, one combination. Total: nine combinations 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”. Atoms Valency Atomic Weight C H O Br S Cl N 4 1 2 1 2 1 3 12 1 16 79/81 32 35/37 14 Word of Caution: Nitrogen Rule will be “reversed” when you HAVE “protonated molecualr ion peak” like in case of ESI Nitrogen is the only common element which has an ODD valency and an EVEN atomic mass 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: C3 H 6 N 4 C4H4NO2 C4 H 6 N 2 O C4 H 8 N 3 C5H6O2 C5H8NO C5H10N2 C7H14 98.0594 98.0242 98.0480 98.0719 98.0368 gives us the exact formula 98.0606 98.0845 98.1096 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 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) b) c) Nitrogen Rule Rule of Thirteen 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)