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PRINCIPLES
P
NCIPLES OF
MASS
S SPECTROMETRY
BACKGROUND
BACKGROUND
H
H C H
H
H C H
+ 2e
H
Molecular ion (M+)
m/z = 16
(C) HKD 2013
BACKGROUND
H H
+ e
H C C
H H
+ H
H H
m/z = 29
H
H C
H
H
C 2013
H
+(C) HKD
H
BACKGROUND
H
H C H
H C C H
H H
BACKGROUND
M+
BACKGROUND
MASS RESOLUTION
C3H6O and C3H8O have nominal masses of 58 and 60, and
can be distinguished by low-resolution MS.
C3H8O and C2H4O2 both have nominal masses of 60.
Distinguish between them by high-resolution MS.
Molecular Nominal Precise
Formula
Mass
Mass
C3 H8 O
60
60.05754
C2 H4 O2
60.02112
60
BACKGROUND
Ionization
Source
Inlet
Mass
Analzyer
Detector
selected
ions
all ions
Data
System
BACKGROUND
BACKGROUND
ESI
EI
(C) HKD 2013
BACKGROUND
FAB
MALDI
(C) HKD 2013
BACKGROUND
ICP
(C) HKD 2013
BACKGROUND
IONIZATION METHODS
1. Electron Ionization (EI): most common
ionization technique, limited to relatively low
MW compounds (<600 amu).
2. Chemical Ionization (CI): ionization with very
little fragmentation, still for low MW
compounds (<800 amu).
3. Desorption Ionization (DI): for higher MW or
very labile compounds.
4. Spray ionization (SI)
for LC-MS, biomolecules, etc.
(C) HKD 2013
BACKGROUND
Electron ionization
vaporized sample is bombarded with high
energy electrons (typically 70 eV).
hard ionization method leads to significant
fragmentation.
ionization is efficient but non-selective.
eM + e-
M+
ABCD+
ABC+
E << 70 eV
E = 70 eV
fragmentation
. + 2e-
M+
ABCD+
+N
ABC+ + C
BC+ + A
M+
e-
Neutral
molecule (C) HKD 2013
Molecular
ion
+ + +
+ +
Fragment
ions
BACKGROUND
Electron ionization
Advantages
inexpensive, versatile and reproducible.
fragmentation gives structural information.
large databases if EI spectra exist and are
searchable.
Disadvantages
fragmentation at expense of molecular ion.
sample must be relatively volatile.
(C) HKD 2013
BACKGROUND
BACKGROUND
eHigh
energy
electrons
CH4
CH4+
CH4
CH4
CH3+
CH2
CH4
CH5
MH+
Protonation
Reaction
C2H3+
C2H5+ C2H5
Primary
reagent ions
Reagent gas
Secondary
reagent ions
Analytes
C 2H 4
BACKGROUND
eHigh energy
electrons
(from the
filament)
CH4
e-
MX
MX
CH4
MX
MX
CH4
H
CH4+
Analytes
Low energy
electrons
CH4
Associative resonance
capture
CH3+
Dissociative
resonance capture
BACKGROUND
m/z = B2r2/2V
(C) HKD 2013
BACKGROUND
9 High resolution.
9 High sensitivity.
9 10,000 Mass Range.
9 Requires Skilled Operator
9 Usually larger and higher cost than other mass analyzers.
9 Difficult to interface to ESI.
HKD 2013 analyzers.
9 Low resolution MS/MS without(C)multiple
BACKGROUND
BACKGROUND
m2+
m1+
m2
+
m3
+
+
mmass number
z charge
m3
m/z = k.V
1
m1/z=kV1
m2/z=kV2
m3/z=KV3
k constant
V :voltage applied to rods
We can select mass
BACKGROUND
m/z = K.V/(r22)
(C) HKD 2013
BACKGROUND
BACKGROUND
BACKGROUND
BACKGROUND
BACKGROUND
BACKGROUND
BACKGROUND
9 Fastest scanning.
9 Requires pulsed ionization method or ion beam switching (duty cycle is
a factor).
9 Low resolution (4000).
(C) HKD
9 Limited precursor-ion selectivity
for 2013
most MS/MS experiments
BACKGROUND
BACKGROUND
BACKGROUND
= qB/m = v/r
The decay over time of the image current resulting after applying a
short radio-frequency sweep is transformed from the time domain into a
(C) HKD 2013
frequency domain signal by a Fourier
transform
BACKGROUND
BACKGROUND
9 Expensive.
BACKGROUND
BACKGROUND
BACKGROUND
Fragment Ions
Derived from
molecular ion
or higher
weight
fragments
[M+H]+(CI)
Or M+ (EI)
molecular ion
Unit mass
spacing
High
mass
(C)and
HKD 2013
Mass, as m/z. Z is the charge,
for doubly charged ions (often seen in
macromolecules), masses show up at half their proper value
BACKGROUND
M
Intensity (% of Base Peak)
Molecule
e-
High Energy
Electron
M+
2e-
Molecular
Ion
(Radical Cation)
Base Peak
100
90
80
70
60
50
40
30
20
10
0
CH2OH+
1-Pentanol - MW 88
CH3(CH2)3 CH2OH
M - H2O
20
30
40
50
60
70
80
90
m/z
BACKGROUND
BACKGROUND
BACKGROUND
Bromine:
M+ ~ M+2 (50.5%
79Br/49.5% 81Br)
2-bromopropane
M+ ~ M+2
BACKGROUND
Chlorine:
M+2 is ~ 1/3 as large as M+
M+
Cl
M+2
BACKGROUND
Sulfur:
M+2 larger than usual (4% of M+)
M+
S
Unusually
large M+2
BACKGROUND
Iodine
I+ at 127
Large gap
Large gap
ICH2CN
(C) HKD 2013
FRAGMENTATION PATTERNS
There are 3 type of fragmentations
1) Cleavage off s bond
+.
+.
---- C +
. C ----
---- C +
. Z ----
C=Z +
---- C .
Z+.
---- C = C
D to heteroatom
+.
---- C - C Z ----
+.
---- C - C Z ----
M+
FRAGMENTATION PATTERNS
There are 3 type of fragmentations
2) Cleavage of 2 s bond (rearrangements)
---- HC C Z ----
+.
---- C=C + +
Retro Diels-alder
CH2
+.
HZ
CH2
CH2
CH2
McLafferty
H
Z
+.
+.
R
CH2
CH2
FRAGMENTATION PATTERNS
RULES
1.
2.
3.
Branched alkanes
H3C
H3C
H3C
H3C
CH3
CH3
H3C
MW=170
ALKANES
Rule 3
FRAGMENTATION PATTERNS
RULES
4.
5.
H2C+
CH
CH2
-R
H2C
H2C
CH
CH
CH2
CH+2
FRAGMENTATION PATTERNS
RULES
6. a) Saturated Rings lose a Alkyl Chain (case of branching)
R
+.
+
-R.
CH2
+.
CH2
+
CH2
(C) HKD 2013
CH2
Retro Diels-alder
+.
+.
FRAGMENTATION PATTERNS
RULES
7.
CH2
CH2
-R.
CH
CH
Tropylium ion
+
m/z 91
FRAGMENTATION PATTERNS
RULES
8. C-C next to Heteroatom cleave leaving the charge on the
Heteroatom
- [RCH2]
CH2
CH2
H2C
H2C
R2
C
R1
- [R2]
R1
R1
+
larger
FRAGMENTATION PATTERNS
RULES
9. Cleavage of small neutral molecules (CO2, CO, olefins, H2O .)
results often from rearrangement.
McLafferty
CH2
CH2
CH2
CH2
CH2
- CH2=CH2
C
Y
CH2
CH2
Ion Stabilized
by resonance
Y as H, R, OH, NR2
(C) HKD 2013
FRAGMENTATION PATTERNS
SUMMARY
Alkanes
Fragmentation often
alkyl groups:
Loss of methyl
Loss of ethyl
Loss of propyl
Loss of butyl
15
29
43
57
FRAGMENTATION PATTERNS
SUMMARY
FRAGMENTATION PATTERNS
SUMMARY
Alkenes:
Fragmentation typically forms
resonance stabilized allylic carbocations
Alkenes
Most intense peaks are often:
CH2 + CH
Me
Et
CH
Et
-Et
-29
+CH
CH
CH2
CH
Me
M+ = 112
CH
Et
Me
m/z = 83
FRAGMENTATION PATTERNS
SUMMARY
Aromatics:
Fragment at the benzylic carbon, forming a
resonance stabilized benzylic carbocation
(which rearranges to the tropylium ion)
H
H C Br
H C
H C
or
M+
(C) HKD 2013
CH +
Et
FRAGMENTATION PATTERNS
SUMMARY
Aromatics may also have a peak at m/z = 77 for
the benzene ring.
77
NO2
M+ = 123
77
FRAGMENTATION PATTERNS
SUMMARY
Alcohols
Fragment easily resulting in very small or
missing parent ion peak
May lose hydroxyl radical or water
M+ - 17 or M+ - 18
Commonly lose an alkyl group attached to
the carbinol carbon forming an oxonium
ion.
9 1o alcohol usually has prominent peak at
m/z = 31 corresponding to H2C=OH+
(C) HKD 2013
FRAGMENTATION PATTERNS
SUMMARY
CH3CH2CH2OH
H2C OH
M+-18
M+
FRAGMENTATION PATTERNS
SUMMARY
Amines
Odd M+ (assuming an odd number of
nitrogens are present).
D-cleavage
dominates
forming
an
iminium ion.
CH3CH2
CH2
N CH2 CH2CH2CH3
H
CH3CH2CH2N CH2
H
m/z =72
iminium ion
(C) HKD 2013
FRAGMENTATION PATTERNS
SUMMARY
CH3CH2
86
CH2
N CH2 CH2CH2CH3
H
72
FRAGMENTATION PATTERNS
SUMMARY
Ethers
D-cleavage forming oxonium ion
Loss of alkyl
carbocation
group
forming
FRAGMENTATION PATTERNS
SUMMARY
MS of diethylether (CH3CH2OCH2CH3)
H O CH2
CH3CH2O CH2
H O CHCH3
FRAGMENTATION PATTERNS
SUMMARY
Aldehydes (RCHO)
Fragmentation may form acylium ion
RC O
Common fragments:
9 M+ - 1 for RC O
9 M+ - 29 for R
FRAGMENTATION PATTERNS
SUMMARY
105
H H O
C C C H
M+ = 134
H H
133
91
FRAGMENTATION PATTERNS
SUMMARY
Ketones
Fragmentation leads to formation of
acylium ion:
FRAGMENTATION PATTERNS
SUMMARY
CH3C O
O
CH3CCH2CH2CH3
CH3CH2CH2C O
M+
FRAGMENTATION PATTERNS
SUMMARY
Esters (RCO2R)
Common fragmentation patterns include:
9 Loss of OR: peak at M+ - OR
9 Loss of R: peak at M+ - R
FRAGMENTATION PATTERNS
SUMMARY
77
105
O
C O CH3
77
M+ = 136
105
RULES OF THIRTEEN
RULES OF THIRTEEN
RULES OF THIRTEEN
If a heteroatom is present,
Subtract the mass of each heteroatom
from the MW.
Calculate
the
formula
for
the
corresponding hydrocarbon.
Add the heteroatoms to the formula.
Example: A compound with a molecular ion
peak at m/z = 102 has a strong peak at
1739 cm-1 in its IR spectrum. Determine
its molecular formula.
(C) HKD 2013