CHMBD 449 Mass Spectrometry

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_at_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.

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Lecture 1 Introductory lecture on gas phase
ion reactions using Electron Impact (EI) source
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Mass Spectra
Which Mass Spectrum You are Going
to Interpretate?
EI-MS
ESI-MS
FAB-MS
MALDI-MS
CI-MS
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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.
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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
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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.
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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
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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.
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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
)
94
13
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,
CnHnr
1 Bright, J. W., and Chen C. M., Journal of
Chemical Education, 60 (1983) 557
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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-X1 Element
• 12C, 13C 14N, 15N 1H, 2H
• b-X2 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
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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
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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
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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
m/z Intensity C 100 Y/1.1 X
72 M 73.0 (X) 100. 3.3/1.1 . 73 4
73 M1 3.3 (Y)
74 M2 0.2
C 100 Y/1.1 X C numbers of carbon X
amplitude of the M ion Y amplitude of the M1
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
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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
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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 M2 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 M2 peak would be 24 the size of the M if
one Cl is present
relative abundance
M2
m/e
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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 M2 peak of 4
  relative intensity and silicon 3

M
M2
The M2 peak would be about the size of the M if
one Br is present
relative abundance
m/e
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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 M4 and M6 peaks 2-Change in
intensity pattern
CH3Cl
CH2Cl2
CHCl3
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Interpretation of E.I. Mass Spectrometric Data
B- Structural in formations extracted from the
molecular ion peak (Low resolution analysis)
1-Why M4 and M6 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
n1 (a b)1 a b n2 (a b)2 a2 2ab
b2 n3 (a b)3 a3 3a2b 3ab² b3 n4 (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
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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
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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
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
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Interpretation of E.I. Mass Spectrometric Data
B- Structural in formations extracted from the
molecular ion peak (High resolution analysis)

• If sufficient resolution (R gt 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

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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 Element
al 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.x0.1?
xxx.xx0.01? xxx.xxx0.001?
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
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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 M1 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 M1)
• If M2 becomes apparent, analyze for the presence
  of one or more Cl or Br atoms (sulfur and
  silicon can also give prominent M2)