Transcript CHMBD 449 Mass Spectrometry

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: [email protected]
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)