Syed Ghulam Musharraf

Syed Ghulam Musharraf

Assistant ProfessorH.E.J. Research Institute of Chemistry

International Centre for Chemical and Biological Sciences (ICCBS)University of Karachi, Karachi-75270E mail: [email protected]

Interpretation of Mass Spectrometric Data

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

CI-MS

FAB-MS

ESI-MS

MALDI-MS

Which

Mass

Spectrum

You are

Going to

Interpretate?

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 peak3. 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 peakRegion ARegion B

Interpretation of E.I. Mass Spectrometric Data

A- Find out the molecular ion peak:

B- Structural information extracted from the molecular ion peak:

O..

: e O.+

:+ 2 e

Molecular ion

1st Step for Mass Spectral Interpretation


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:


How we can know that ion must be odd-electron (OE)?

By the calculation of saturation index :

saturation index: (R + DB) R = number of ringsDB = number of double bonds

The total number of rings + double bonds = x - 1/2y + 1/2z + 1

For the general formula CxHyNzOn:

Si is treated as CP is treated as NS is treated as OF, Cl, Br and I are treated as H


Some Calculations: possible molecular ions?

CH4

C3H3FC6H6

C7H6O2

C7H5O

“This is an important characteristic of even-electron ions-they will never havewhole 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 beodd-electron ions, not all odd-electron ions are molecular ions”.

Many compounds can form odd-electronions by breaking two chemical bonds, like in

McLafferty rearrangement.

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

M/13 = n + r/13

M = molecular weight n = number of C and H atoms

R = reminder

CnHn+r

Example:

1 = Bright, J. W., and Chen C. M., Journal of Chemical Education, 60 (1983): 557

M = 94, molecular formula = ?

94/ 13

)941391

7

3

Possible molecular formula = C7H10

Other possible molecular formulas =

C6H6O, C5H2O2, C6H8N, C5H2S,

CH3Br,


When a molecular mass, M+., is known, a base formula can be generated

from the following equation:

Lung Cancer: Biological Samples

94

9596

M+. + 1M+. + 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)


1-Monoisotopic: A or X elements 19F, 23Na, 31P, 127IOthers are 27Al, 45Sc, 55Mg, 59Co, 103Rh, 133Cs

2-Di-isotopic element:a-X+1 Element

12C, 13C; 14N, 15N; 1H, 2Hb-X+2 Element

35Cl, 37Cl; 79Br, 81Br; 63Cu, 65Cu; 69Ga, 71Ga; 107Ag, 109Ag; 113In, 115In; 121Sb, 123Sb.

c-X-1 Elements6Li, 7Li; 10B, 11B; 50V, 51V

3-Polyisotopic element:

Isotopic Classification of the Element:

Elements containing only one important isotopic form

Element MassF(A) 19P(A) 31

I(A) 127

Elements containing two important isotopic forms

Element Mass % Abundance Mass % AbundanceH(A + 1) 1 100 2 0.01C(A + 1) 12 100 13 1.1N(A + 1) 14 100 15 0.37Cl(A + 2) 35 100 37 32.5Br(A + 2) 79 100 81 98.0O(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 3.4S(A + 2) 32 100 33 0.80 34 4.4

Mass and relative abundance of common

organic elements



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 isotopesof 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 uCl2 =



“Number of carbon atoms can be estimated”

1-Information from M +1 Peak:

C = 100 Y/1.1 X

C= numbers of carbonX = amplitude of the M ionY = amplitude of the M+1 ion Peak

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

0.3% = Absence of S (4.4%), Cl (33%), Br (98%)

So the probable molecular formula is C4H8O

For a molecular formula composed of C and H = C4H24

An example:


Presence of S or Si Presences of Br and Cl (A characteristics peak intensity pattern observe)




Molecules that are completely 12C are now rare

insulin (257 carbon atoms)




1. 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:

The M+2 peak would be 24% the size of the M+

if one Cl is present

m/e

rela

tive a

bu

nd

an

ce M+

M+2




(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

rela

tive a

bu

nd

an

ce M+

M+2

The M+2 peak would be about the size of the M+

if one Br is present


Presence of multiple Cl or Br atoms?

1-Generation of M+4 and M+6 peaks2-Change in intensity pattern


CH3Cl

CH2Cl2CHCl3


Total number of possible combinations = An

A= number of isotopes considered, n = number of atoms of present

For Br2 = total number of combinations = 22 = 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?


2-How we can calculate intensity pattern?

Example: Br2

Calculate number of combinations For CHBr3

By Binomial expression:(a + b)n a and b = abundance of two isotopes ofn = number of bromine atom attached

n=1 (a + b)1 = a + bn=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

Pascal intensity Pattern

(Only for Br)



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:



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 compoundcontaining no nitrogens or an even number of nitrogens will have an even molecular weight”.

Atoms Valency Atomic Weight

CHOBrSClN

4121213

1211679/813235/3714

Nitrogen is the only common element which has an ODD valency and an EVEN atomic mass


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:

C3H6N4 98.0594

C4H4NO2 98.0242

C4H6N2O 98.0480

C4H8N3 98.0719

C5H6O2 98.0368 gives us the exact formulaC5H8NO 98.0606

C5H10N2 98.0845

C7H14 98.1096

Problems overcome by HR analysis

Number of carbon atom---------------SolvedElemental composition-----------------SolvedPresence of N, Halogen----------------Solved

But you need to calculate OE ions for molecular ion peaks

Compounds with molecular wt 28: N2, C2H4, CO


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)


A Summary before moving on:

1. Using the the M+ peak, make any inferences about the approximate formula

a) Nitrogen Rule b) Rule of Thirteenc) 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)

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Syed Ghulam Musharraf