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BW Mass Spectrometry Zeeshan
BW Mass Spectrometry Zeeshan
distribution
Protein sequence (MS-MS)
Rule of Thirteen
Calculating Mass
The Rule of Thirteen can be used to identify
possible molecular formulas for an unknown
hydrocarbon, CnHm.
Step 1: n = M+/13 (integer only, use remainder in
step 2)
Step 2: m = n + remainder from step 1
Rule of Thirteen
Example: The formula for a hydrocarbon with
M+ =106 can be found:
Step 1: n = 106/13 = 8 (R = 2)
Step 2: m = 8 + 2 = 10
Formula: C8H10
Nitrogen Rule
This rule states that if a compound has an even no.of N
atoms (or no N atom) , its molecular ion will appear at
an even mass value.
On the other hand , a molecule with an odd no.of N
atom will form a molecular ion with an odd mass.
The N rule stems from the fact that N , although it has
an even mass , has an odd numbered valence.
Consequently , an extra hydrogen atom is included as a
part of a molecule , giving it an odd mass.
Nitrogen Rule
To picture this effect, consider Ehtylamine,
C2H5NH2.
This substance has one N atom, and its mass is
an odd number (45), whereas
EhtyleneDiamine H2N-CH2-CH2-NH2, has 2 N
atoms, and its mass is an even number (60).
Mass Spectrometry
An outline of what happens in a mass
spectrometer
Atoms can be deflected by magnetic fields provided the atom is first turned into an ion.
Electrically charged particles are affected by a
magnetic field although electrically neutral
ones aren't.
The sequence is :
Ionization
Mass Spectrometry
Slightly +ve
To repel +ve ions
Mass Spectrometry
Mass Spectrometry
Assume all streams having +1 charge
Mass Spectrometry
Detection
Separate Based on
Mass/Charge
Sample
Detector
Mass Spectrometry
The Mass Spectrometer
Fundamental operating principle
Determine mass by manipulating flight path of an ion in a magnetic field
Electron gun
Ionization
m/z
m/ztoo
toosmall
large
sample
introduction
+ Accelerator
plates
Detector quiet
Detector fires
Detector quiet
Detector
14
Isotopes
Isotopes: atoms with same number of protons and same number of electrons
but different numbers of neutrons
Aston mass spectrum of neon (1919)
Ne empirical atomic weight = 20.2 amu
Ne mass spectrum: predict single peak at m/z = 20.2
Results
m/z
20.2
20.0
22.0
relative intensity
no peak
90%
10%
Conclusions
Neon is a mixture of isotopes
Weighted average: (90% x 20.0 amu) + (10.0% x 22.0 amu) = 20.2 amu
Nobel Prize in Chemistry 1922 to Aston for discovery of stable element isotopes
15
M contributors
M+1 contributors
M+2 contributors
Isotope
Natural
Abundance
Isotope
Natural
Abundance
Isotope
Natural
Abundance
1H
99.9855%
2H
0.015%
3H
ppm
12C
98.893
13C
1.107
14C
ppm
14N
99.634
15N
0.366
16O
99.759
17O
0.037
18O
0.204
19F
100.0
32S
95.0
33S
0.76
34S
4.22
35Cl
75.77
37Cl
24.23
79Br
50.69
81Br
49.31
127I
100.0
16
Ionization Methods
Electron bomb Ionization (EI)
Liquid Sample
Gas Sample
Chemical Properties
of analyte in gas
phase ?
Chemical Properties
of analyte in
solution phase ?
MALDI
APCI
APPI
ESI
CI
EI
Electron Impact
e-
e-
e-
M(g) + e- M+(g) + 2eThis reaction creates the molecular ion so is very useful.
However, the excess energy from the electron can cause
the molecular ion to fall apart:
Neutral
Molecule
IP2
Excess Energy get
redistributed
throughout ion
to cause
fragmentation.
s1
IP
s0
s1
s0
Ionized
Molecule
Electron Impact
e-
e-
e-
A+
M(g) + (g) +
M+(g) A+Fragment 1 (g) + BFragment 2 (g)
e-
M+
2e-
Properties of EI
Hard ionization
Chemical Ionization
EI is not appropriate for some molecules (it
causes too much fragmentation)
Instead, ionize a reagent gas (by EI) then react
it with a analyte molecules
Typically use methane or ammonia for reagent
gas
Properties of CI
Advantages
Disadvantages
Parent Ion
Interface to GC
Insoluble Samples
CI is lower energy
process than EI
No Fragment Library
Need Volatile Sample
Need Thermal Stability
Low Mass Compounds
(<1000 amu)
Probe
+
+
Probe
+
+ +
d<1mm
+
+
+
+ + +
+
Ionization
Difference
There are three practical differences between CI and FI: there is less
fragmentation in FI
There is no high-resolution FI, and FI is less sensitive.
Sensitivity is not an issue unless there is an extremely small amount of
sample. FI can be performed by direct probe and GC/MS.
Properties of MALDI
Good solubility
Principle of MALDI
2. Principle
Properties of ESI
Advantages
Electrospray Ionization can be
easily interfaced to LC.
Absolute signals from
Electrospray are more easily
reproduced, therefore, better
quantitation.
Mass Accuracy is considered
better.
Multiple charging is more
common then MALDI.
Disadvantages
No Fragmentation
Need Polar Sample
Need Solubility in Polar
Solvent (MeOH, ACN, H2O,
Acetone are best)
Sensitive to Salts
Suppression
m/z = (1 x 12) + (4 x 1) = 16
C
H
Fragmentation Patterns
The impact of the stream of high energy
electrons often breaks the molecule into
fragments, commonly a cation and a radical.
Bonds break to give the most stable cation.
Stability of the radical is less important.
Fragmentation Patterns
Alkanes
Fragmentation often splits off simple alkyl groups:
Loss of methyl
Loss of ethyl
Loss of propyl
Loss of butyl
M+ - 15
M+ - 29
M+ - 43
M+ - 57
Fragmentation Patterns
Mass spectrum of 2-methylpentane
Fragmentation Patterns
Alkenes:
Fragmentation typically forms resonance
stabilized allylic carbocations
Fragmentation Patterns
Aromatics:
Fragment at the benzylic carbon, forming a resonance
stabilized benzylic carbocation (which rearranges to the
tropylium ion)
H
H C Br
H
H C
H
H C
or
M+
Fragmentation Patterns
Alcohols
Fragment easily resulting in very small or missing
parent ion peak
Commonly losses H2O or OH
M+ - 17 or M+ - 18
Commonly lose an alkyl group attached to the
carbinol carbon forming an oxonium ion.
1o alcohol usually has prominent peak at m/z = 31
corresponding to H2C=OH+
Fragmentation Patterns
MS for 1-propanol
CH3CH2CH2OH
H2C OH
M+-18
M+
Fragmentation Patterns
Amines
Odd M+ (assuming an odd number of nitrogens
are present)
a-cleavage dominates forming an iminium ion
CH3CH2
CH2
N CH2
H
CH2CH2CH3
CH3CH2CH2N CH2
H
m/z =72
iminium ion
Fragmentation Patterns
86
CH3CH2
CH2
N CH2
H
72
CH2CH2CH3
Fragmentation Patterns
Aromatics may also have a peak at m/z = 77 for the benzene
ring.
NO2
77
77
M+ = 123
Fragmentation Patterns
Aldehydes (RCHO)
Fragmentation may form acylium ion
RC O
Common fragments:
RC O
M+ - 1 for
M+ - 29 for
Fragmentation Patterns
MS for hydrocinnamaldehyde
105
91
H H O
C C C H
H H
133
91
M+ = 134
105
Fragmentation Patterns
O
Ketones
RCR'
R'C O
Loss of R forming
RC O
Fragmentation Patterns
O
MS for 2-pentanone
CH3CCH2CH2CH3
CH3C O
CH3CH2CH2C O
M+
Fragmentation Patterns
Esters (RCO2R)
Common fragmentation patterns include:
Loss of OR
peak at M+ - OR
Loss of R
peak at M+ - R
Frgamentation Patterns
105
77
O
C O CH3
77
105
M+ = 136
2. Rearrangement
McLafferty rearrangement
Pattern I
H
+
C
D
H2C
D
C
54
55
-e
R
+
R
+
Examples:
CH3
CH3
+
56
H2O + C6H13
H HO
OH
H
O
- CO
- CO
H
O
O
+
H2O
57
CH2 CH3
- CH3
CH3 CH2 O
CH2 =
H2C
H2C
O CH2
HO CH2
Other rearrangement
X
R +
58
oConcluding remarks
oSoft techniques produces Molecular ion peaks
whereas hard techniques produces Fregmentation.
oBy Soft techniques exact molecular ion peak can be
obtained for large and small molecules.
oBy Fregmentation connection pattern of a molecule
can be concluded, ehich helps in structure
elucidation.