Fragmentation in Mass Spectrometry: - Introduction - Ionization - Separation

Department of Organic Chemistry, IMM, Radboud University Nijmegen
Fragmentation in Mass Spectrometry
• Introduction
• Ionization
• Separation
• Fragmentation
• Isotope Effects
• High Resolution
Instrumental Analysis in Molecular Chemistry

1
Identification of Molecular Ion (1)
As we have seen, it is expected that a chemically pure compound

contains a mixture of isotopes, based on the natural abundance.
Molecular ion assigned on the basis of the most abundant isotope(s).
Conditions for the assignment of M+.:
a) M+. should be the highest m/z in the spectrum, apart from weak
satellite peaks that result from other isotopes.
Problems with unstable M+. or chemical impurities.
b) M+. should be a radical cation in EI as it is ionized by removing 1 e.

In formula CxHyNzOn (where C is any 4-valent element, H any monovalent,
N any 3-valent, and O any divalent)
the number of DBE: x – ½.y + ½.z + 1 should be an integer.
c) Value of M+. is even unless there is an odd number of N.

2
Identification of Molecular Ion (2)
d) If M+. is correctly assigned, the other peaks at high m/z can be

logically explained by loss of neutral parts or molecules
OK: loss of CH3 (M-15), H2O (M-18), CH3O (M-31), CH3C=O (M-43) etc.
Illogical: M-2 till M-14

M-21 till M-25
M-33, M-37, M-38
If M+. cannot be satisfactorily assigned, look for an other assignment,

or try another technique: CI, FAB, MALDI, electrospray …

3
Important Factors for Fragmentation

a) Energy of the molecular ion and the fragments formed from it
b) Stability of the bonds in the ions
c) For rearrangements: steric factors.

It is easier to move an H than a whole group
d) Stability of the formed ions or neutral particles

resonance stabilization such as in an acylium ion
Stevenson’s Rule:
Upon dissociation of AB+. → A+ + B. or A. + B+
A+ will be formed if it has the lower ionization energy
In branched radical cations, the largest group is preferentially lost

CH3 +. CH3 CH3 H CH3
C2H5 C C4H9 C2H5 CH+ > +
HC C4H9 > C2H5 C+ C4H9 > C2H5 C C4H9
H +
4
Stevenson’s Rule, Energy Diagram
A. + B+
A+ + B.
Energy
IB AB+.
IA APB+
APA+
A. + B. IAB
AB DAB
Ionization Energy (I), the energy required to convert radical into cation
Dissociation Energy (D), energy for homolytic dissociation into radicals
Appearance Potential (AP), energy required for cation to appear in MS

5
Ionization Energies for Common Hydrocarbons (1)

Energy of Formation of Aliphatic and Olefinic Radicals
and Carbocations from RH and RCl
250
200
Energy (kcal/Mol)
150
cation
radical
100
50
tBu iPr Et Me Bn All vinyl Ph

R
6
Ionization Energies for Common Hydrocarbons (2)

Formation Energies of Radicals & Carbocations < RH & RCl
350
300
250
Energy (kcal/Mol)
200
cation
150 radical
100
50
0
tBu iPr Bn All Et vinyl Ph Me H
7
Loss of Largest Group from Branched Radical Ion

Stevenson’s Rule:
Upon dissociation of AB+. → A+ + B. or A. + B+
A+ will be formed if it has the lower ionization energy
In branched radical cations, the largest group is preferentially lost
Do not confuse ionization potential and cation/radical stability

CH3 +. CH3
C2H5 C C4H9
H
C2H5 CH+ + .C H 4 9 sec. cation + tert. radical
CH3
+HC C H
4 9
+ .C H 2 5 sec. cation + sec. radical
H
C2H5 C+ C4H9 + .CH 3 sec. cation + prim. radical
CH3
C2H5 C C4H9
+
+ .H .
tert. cation + H
8
Metastable Peaks
Metastable ions: arise because of fragmentation after the ion has
left the ionization chamber
M1+ → M2+ + (M1 - M2)
The kinetic energy of such a M2+ is smaller than when it would have
been formed in the ionization chamber and accelerated there, and it will
be detected at a lower m/z than expected, and moreover broadened.
For fragmentation between electrostatic analyser and magnetic sector:
M* = M22/M1
e.g. C5H9+ (m/z = 69) → C3H5+ (41) + C2H4 (28)
Calculated m* = (41)2/69 = 24.36, observed m* = 24.4
(It is more difficult to solve this problem in the other direction)

9
Types of Fragmentation Reactions (1)
McLafferty & Turecek:
1) Sigma electron ionisation (σ): Alkanes: .

R+ CR3 R.+ +CR
3
Low IE elements .
R+ I R. + +I
2) Radical site initiation (alpha cleavage, α), homolytic dissociation:

N > S, R, O, π > Cl, Br > H
Saturated R CR2
+
YR
. R . + R2C Y+R
Donates an electron,
Y+R
.CH +.
+
forms new bond to an CH2 2 YR CH2 CH2
+.
adjacent atom
Unsaturated
heteroatom R CR Y R. + RC Y+
concomitant with . R. + H C
Allylic + +CH
cleavage of other R CH2 CH2 CH2 2 CH 2
bond to that atom,

moves . site,
Retro-
Diels-Alder R R . α2 RHC
+
loss of largest
(double α-
-e
+
H2C
. +
alkyl favoured
cleavage)
α,i charge RC2H3
+ . + C4H6
migration
10
Types of Fragmentation Reactions (2)

McLafferty & Turecek:
3) Charge site initiation (inductive effect, i), heterolytic dissociation:

Cl, Br, NO2 > O, S >> N, C
Attracts e pair, +
OE : R
+
Y R. .
R+ + YR .
cleaves bond,
moves + site, +
EE : R
+
YH2 R+ + YH2 .
most stable + formed
4) Rearrangements (r):
Example with H
H+
Y
. rH .HY + α
+
HY
+
and unsaturated
receptor site in ring
.
Charge retention, ion gains H
or
Also with saturated .HY +

. HY
i
receptor site, +
+
2H, displacement (rd)
Charge migration, ion loses H
and elimination (re)
11
Fragmentation Reactions
(Hesse, Meier, & Zeeh)
Alpha splitting
Benzyl- and Allyl splitting
Splitting of non-activated bonds
Retro-Diels-Alder reaction
McLafferty rearrangement
Onium reaction
Loss of CO

12
α-Splitting (Homolytic Dissociation) (1)
2-butanone
The acylium ion formed by loss

of the largest alkyl radical is
preferred
O .O +
.
O+
or
O+ O+
CH3 .+ CH3
H3C
+ CH3CH2 .
(15) (29)
m/z 57 m/z 43

13

100
O H 45
2-butanol CH
75 H3C
splitting 50 OH OH
of Et. CH 2
H3C
CH
CH 2 OH
•
preferred 25 27
29
31
43
H3C
CH C H 3 59
CH 2 CH CH 3
41 H3C CH 2
19 28 57
0
20 25 30 35 40 45 50 55 60 65 70
100 59 H O
CH 3
C
t-butanol 75 CH 3
only Me . 50
CH 3 •
splitting 31 C HO
25 CH 3
41 H3C C
CH 3
possible 15
27 29 39
43
57
60
H3C
CH 3
0
15 20 25 30 35 40 45 50 55 60 65 70

14
2-Aminoethanol
Ionization by loss of n-electron
from N
(less electronegative than O)
which is also a likely starting
point for radical-site initiated
fragmentation
-e
H2N OH H2N
+ . OH
m/z 61
.CH OH 2
H2C N+H2 (31)
m/z = 30

15
α-splitting, mainly of Me, but also of H,

is the main mechanism of fragmentation
in amines because of the electron-donating
ability of N

16
α-Splitting in MS of Protected Steroid
Me
Me Me
.
+
O O+ O+
.
Me
O+
O+
O O
.H
O
Me
O
m/z 99 O
m/z 125
or
Me
Me Me Me
.
+
O+
.H Me
Me
Me .H Me
Me
O O+
. O+ O+
.
O O O O O

17
Formation of Stable Allylic Carbocation

EI of 1-heptene
+
m/z 41
R
+
CH2 CH2 CH2
. . + HC
R 2 CH +CH
2
EI of 4-methyl-1-hexene

18
Formation of Stable Benzylic Carbocation (1)
EI of butylbenzene
.+
+
.C H 3 7 C2H2
(43) (26)
+ + +
m/z = 91 m/z = 65
α + +
or .+ .C(57)H m/z = 51
4 9 m/z = 77 C2H2
(26)

19
Formation of Stable Benzylic Carbocation (2)

The EI-MS of o-Cl-toluene and
benzyl chloride are similar, both
are dominated by the tropylium
ion

20
σ-Electron Ionization, Non-activated Bonds

EI-MS of an unbranched
alkane shows an almost
random fragmentation
pattern (homologous
series, differing in the
no. of CH2 groups), every
σ-bond is as likely to break
as the other one.
In the branched alkane,

the possibility to have
relatively stable secondary
carbocations as opposed
to primary ones influences
the fragmentation pattern.
Stevenson’s rule,
loss of largest alkyl
21
Alkyl halide spectra (1)

n-heptyl F: strong C-F bonds,
electronegativity of F
makes + on F unattractive,
HF formation attractive
No M+. observed
C7H14 F+ . C7H14 F+ C7H14+.

H . H HF O.E. ion
(118) (20)
various
fragments
n-heptyl I: less electronegative, less basic,
.
I+ (m/z 127) or I (with R+, m/z 99) feasible,
but base peak at m/z 57 (C4H9+) ?!
σ
.
R+ I .
R + +I (m/z127)
m/z 226
or R+ (m/z 99) + I .
22
Alkyl halide spectra (2)

Fragments containing Br, Cl (X = F, 53)
recognized by satellite peaks .X +
i + + X . (X = Cl, 77/79)
(X = Br, 121/123)
An α splitting with cyclization is m/z = 57 (X = I, 169)
important (base peak for Cl) .

X+ α . X+ (X = F, 67)
+ X = Cl, 91/93
An i splitting with cyclization is 43 X = Br, 135/137
m/z = M - 43 (X = I, 183)
also important (base peak for
I, Br)

23
Retro-Diels-Alder (1)
Retro-
R
-e
R . α
R . or
R .
Diels-Alder + + +
α i
Retro-Diels-Alder, not pericyclic
R
HC
R
HC .+
+
Charge retention or migration, H2C+
depending on R
H2C
. +
charge charge
retention migration
R = H, 80 % R = Ph, 100 %
NB: α followed by α or i gives another OE ion
See the example of

4-phenyl-
cyclohexene

24
Retro-Diels-Alder (2)
See the example of the ionones

α-ionone gives the expected Retro-DA
β-ionone has a competing loss of methyl group
O O
.
+ CH3
α-ionone β-ionone
.O+ O
+

25
1,2,3,4-Tetrahydrocarbazole
H
N
N
.
H+ H
N +
.
m/z 171
C2H4 C2H4
H+
N . H
N + .
m/z 143

26
5,7-dihydroxy-4’-methoxyisoflavonone

27
McLafferty Rearrangement
In McLafferty rearrangement H+ . .HY + α
+
HY
(H rearrangement with unsaturated Y rH
+
receptor site) a H from the carbon in .
Charge retention, ion gains H
γ position relative to a C=Y double bond is or
transferred to the Y atom, accompanied by .HY +
. HY
i
a β-splitting. This can be with charge +
+
retention (α) or charge migration (i). Charge migration, ion loses H
Note that McLafferty rearrangements are relatively exceptional

(cf. retro Diels-Alder, CO loss) in producing another OE ion.
Example: a relatively long ketone
CH3 H+
O
. rH
H3C . HO + α
H3C
+
HO
m/z 62
20 %
+
CH2
CH2 C2H5 C2H5 (42)
. C2H5
m/z 104 Charge retention, ion gains H

28
EI-MS of methyl butanoate
The alkyl chain of the acid part H+

CH2 O rH
. . HO + α
+
HO
of the ester is long enough to +
give a McLafferty rearrangement,
CH2
CH2 OCH3 OCH3 (28)
. OCH3
m/z 74
in addition to α-fragmentation or
and subsequent loss of CO. CH3 O
+
α
. O+
CH2 CH3CH2CH2+
m/z 102
CH2 OCH3
CH3O . m/z 71 CO
(28)
m/z 43
(31)
or
+
CH3 O
.
α O+
CH3O+
CH2
CH2 OCH3 OCH3 CO m/z 31
CH3CH2CH2 . m/z 59 (28)
(43)

29
Loss of CO H
O
.
+ .
+
O H H
H
H +
CO .
H m/z 65
m/z 94 m/z 66

30
Some Loss of CO
O
.
+
m/z 192 O
O
+. +. .
+ + .
C O +
m/z 96 m/z 68
m/z 164 m/z 136 m/z 65

31
No Loss of CO H
O
+. O +
O+ O+
+O
. . Me
H CH2
CH2
m/z 98 .C H3 7 m/z 55
m/z 57

32
Cyclohexylamine
Et
+N
CH2
m/z 84

33
Onium Reaction
In the Onium reaction

(H rearrangement with
+ . +
.
saturated receptor site)
H YR
. HYR +
HYR
a H is transferred from
somewhere in an alkyl
chain to the heteroatom
of initial ionization. +
H YR HYR +
+ HYR
Y can be O (oxonium),
N (nitronium), etc.
In OE ions, H is transferred
with one e; in EE ions it is a
hydride shift, H as H- with 2 e.

34
Onium reaction, Ether
m/z 102
+
O
.
.CH 3 .C H
3 7
α α
m/z 87
H Me
O+ or m/z 59
+O O+
CH2
H
Mc Onium
Onium C4H8 (56) C3H6 (42)
Lafferty C2H4 (28)
m/z 31 m/z 45 m/z 31

HO+ O+ HO+
CH2 Me CH2 CH2
35
Onium reaction, Amine
Me Me
N
+. m/z 129
.CH 3
Me
.C H
3 7
α α
Me m/z 114
Me H Me Me Me
N+ or m/z 86
Me
N+ N+
Me Me CH2
H
Onium Mc Onium
C4H8 (56) Lafferty C3H6 (42) C3H6 (42)
Me m/z 58 Me m/z 44
m/z 72 H+
N+ N+ N
Me H Me CH2 Me CH2

36
Standard Interpretation Procedure

(McLafferty & Turecek) (1)
1) Study all available information (spectroscopic, chemical, sample

history). Give explicit directions for obtaining spectrum.
Verify m/z assignments.
2) Using isotopic abundances, where possible deduce the elemental

composition of each peak in the spectrum; calculate rings plus
double bonds.
3) Test molecular ion identity; must be highest peak in mass spectrum,

odd-electron ion, and give logical neutral losses. Check with CI or other
soft ionization.
4) Mark ‘important’ ions: odd electron and those of high abundance,

highest mass, and/or highest mass in a group of peaks.

37
Standard Interpretation Procedure

(McLafferty & Turecek) (2)
5) Study general appearance of the spectrum; molecular stability,

labile bonds.
6) Postulate and rank possible structural assignments for:

- important low-mass ion series;
- important primary neutral fragments from M+. indicated by
high-mass ions (loss of largest alkyl favoured) plus those from
collision activation;
- important characteristic ions.
7) Postulate molecular structures; test again reference spectrum,

against spectra of similar compounds, or against spectra predicted
from mechanisms of ion decompositions.

38

Fragmentation in Mass Spectrometry: - Introduction - Ionization - Separation

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Fragmentation in Mass Spectrometry: - Introduction - Ionization - Separation

Uploaded by

Copyright:

Available Formats

Department of Organic Chemistry, IMM, Radboud University Nijmegen

Fragmentation in Mass Spectrometry

Instrumental Analysis in Molecular Chemistry

Identification of Molecular Ion (1)

As we have seen, it is expected that a chemically pure compound

Conditions for the assignment of M+.:

b) M+. should be a radical cation in EI as it is ionized by removing 1 e.

c) Value of M+. is even unless there is an odd number of N.

Instrumental Analysis in Molecular Chemistry

Identification of Molecular Ion (2)

d) If M+. is correctly assigned, the other peaks at high m/z can be

Illogical: M-2 till M-14

If M+. cannot be satisfactorily assigned, look for an other assignment,

Instrumental Analysis in Molecular Chemistry

Important Factors for Fragmentation

b) Stability of the bonds in the ions

c) For rearrangements: steric factors.

d) Stability of the formed ions or neutral particles

In branched radical cations, the largest group is preferentially lost

Stevenson’s Rule, Energy Diagram

Dissociation Energy (D), energy for homolytic dissociation into radicals

Appearance Potential (AP), energy required for cation to appear in MS

Instrumental Analysis in Molecular Chemistry

Ionization Energies for Common Hydrocarbons (1)

tBu iPr Et Me Bn All vinyl Ph

Ionization Energies for Common Hydrocarbons (2)

Loss of Largest Group from Branched Radical Ion

Do not confuse ionization potential and cation/radical stability

M1+ → M2+ + (M1 - M2)

For fragmentation between electrostatic analyser and magnetic sector:

e.g. C5H9+ (m/z = 69) → C3H5+ (41) + C2H4 (28)

Calculated m* = (41)2/69 = 24.36, observed m* = 24.4

(It is more difficult to solve this problem in the other direction)

Types of Fragmentation Reactions (1)

McLafferty & Turecek:

1) Sigma electron ionisation (σ): Alkanes: .

2) Radical site initiation (alpha cleavage, α), homolytic dissociation:

bond to that atom,

Types of Fragmentation Reactions (2)

3) Charge site initiation (inductive effect, i), heterolytic dissociation:

Also with saturated .HY +

Benzyl- and Allyl splitting

Splitting of non-activated bonds

Instrumental Analysis in Molecular Chemistry

α-Splitting (Homolytic Dissociation) (1)

The acylium ion formed by loss

Instrumental Analysis in Molecular Chemistry

α-Splitting (Homolytic Dissociation) (2)

Instrumental Analysis in Molecular Chemistry

α-Splitting (Homolytic Dissociation) (3)

H2C N+H2 (31)

Instrumental Analysis in Molecular Chemistry

α-Splitting (Homolytic Dissociation) (4)

α-splitting, mainly of Me, but also of H,

Instrumental Analysis in Molecular Chemistry

Instrumental Analysis in Molecular Chemistry

Formation of Stable Allylic Carbocation

Instrumental Analysis in Molecular Chemistry

Formation of Stable Benzylic Carbocation (1)

Instrumental Analysis in Molecular Chemistry

Formation of Stable Benzylic Carbocation (2)

Instrumental Analysis in Molecular Chemistry

σ-Electron Ionization, Non-activated Bonds