Mass spectroscopy

1. Mass
Spectrometry
1

2. ?
Why it is use Mass Spectroscopy ?
2

3. Introduction to Mass Spectrometry
Sample
introduction
Ionization
Minimize
collisions,
interferences
Separate
masses
Count ions
Collect results
3

4. Principle
It is also called as positive ion spectra or line spectra Sample is
bombarded with the high electron beam produce the positive
ions.
They travel in straight path
When a magnetic field or electric field is applied then travels
in curved path
The fragments of different masses are separated based on the
radius of curvature.
m/e α r
2

4

5. How does a mass spectrometer work?
Sample Plate
Target
HPLC
GC
Solids probe
MALDI
ESI
IonSpray
FAB
EI/CI
SFA
DFA
Quadrupole
FTMS
Faraday cup .
Electron Mult.
Photomultiplier
5

6. V i d e o
6

7. Mass Spectrometry Needs
Ionization-How the protein is injected in to the MS
machine
Separation-Mass and Charge is determined
Activation-Protein are broken into smaller fragments
(peptides/AAs)
Mass Determination- m/z ratios are determined for the
ionized protein fragments/peptides
?
7

8. FRAGMENTATION
The process of Breaking Molecules /ions into
fragments is known as fragmentation.
This can be seen in the form of peaks in mass spectra
Methanol can be divided in to 4fragments
e.g.
CH3OH CH3OH +e¯
⁺
CH3OH CH3 + OH¯
⁺
CH3OH CH2OH + H¯
⁺
CH3OH CHO + H2¯
⁺

.
5 10 15 20 25 30 35
120
100
80
60
40
20
0
CHO⁺
CH3OH⁺
CH3⁺
CH2OH⁺
m/e
i
n
t
e
n
s
i
t
y
8

9. Fragmentation rules in MS
9
1.Intensity of MM
.+.+
is Larger for linear chainLarger for linear chain than for branched
compound
2.Intensity of MM
.+.+
decreasedecrease with IncreasingIncreasing M.W.M.W. (fatty acid is an
exception)
3.Cleavage is favored at branchingfavored at branching
4.4.Aromatic Rings, Double bond, Cyclic structures stabilizeAromatic Rings, Double bond, Cyclic structures stabilize MM
.+.+
5. 5. Double bond favor Allylic CleavageDouble bond favor Allylic Cleavage
6. Saturated Rings lose a Alkyl Chain (case of branching)
7. 7. Aromatic Compounds Cleave in b Aromatic Compounds Cleave in b
Resonance Stabilized TropyliumTropylium
8. 8. C-C C-C Next to HeteroatomNext to Heteroatom cleave leaving the charge on the charge on the
HeteroatomHeteroatom
9. 9. Cleavage of small neutral molecules (COCleavage of small neutral molecules (CO
22, CO, olefins, H, CO, olefins, H
22O ….).O ….).
Result often from rearrangement - McLafferty rearrangement Result often from rearrangement - McLafferty rearrangement
9

10. General rules of Fragmentation
1.Hydrocarbons
•Hydrocarbons give clusters of peaks.
•Molecular ion peaks of very low abundance are observed for linear hydrocarbons.
•For branched hydrocarbons give a low intensity at M
+
.
•Intensity of (C
n
H
2n+1
) peaks decreases with increasing mass.
10

11. 2.Cleavage at Branched carbon
C>
C
H
>
C
H
H
>
H
C
H
H
tert.
sec.
primary
methyl
Cleavage at branched carbon is favored due to higher stability
at tertiary carbocation.
General rules of Fragmentation
11

12. +
cleavage at 6-1
cleavage at 6-3
cleavage at 6-2
C H
C
4H
9
C
3H
7
C H
CH
3
C
4H
9
+
+
C H
CH
3
C
3H
7
+
(F1)
(F2)
(F3)
H
3C CH
2 CH
2 C
CH
3
H
CH
2 CH
2 CH
2 CH
3
1 2 3 4 5 6 7 8
Eg.
Produces thre secondary cations, the most favored fragments
at C-4 of
4- methyl octane.
Note that C
4
is common for fragments (F1)(F2) And (F3).
12

13. 3.Rule of b cleavage
X
C
1
C2
R X
CH
a b
Most important rule covers 70% of mass fragmentation.
Cleavage favored at b bond leaving positive charge on C
1.
General rules of Fragmentation
13

14. H
3C CH
2 O CH
2 CH
3
H
3C CH
2 O CH
2 CH
3
CH
2O
CH
2
m/e = M-15
1.
H
3C
2.
H
3C CH
2 N CH
2
CH2 CH
2 CH
3
N
C
2H
5
C
3H
7
H
2C
m-57
m-29
N
C
2H
5
H
2C
H
2C
N
CH
2
C
3H
7
m-15
CH
2
tert.amine
B1
B2B3
e.g.: A) (x) = O, N, S.
14

15. 3.
CH
2 S CH
2 CH
2 CH
3
SH
2C
CH
2
SH
2C
C
3H
7
M-71
M-29
B2 B1
B1
B2

16. R
CH
2
CH
2
+
+
m/e = ( M-R )
Stable
benzylic cation
+
m/e = 91
Tropylium cation
m/e = 65
cyclopentadienyl
cation
+
b)
b) Benzylic clevage
-(x)- =
16

17. Very common fragment for ester
M-31 = methyl ester
M-45 = ethyl ester
C. Allylic Cleavage
H
2C
R
m/e = M-R stable allyliz cation
CH
3H
3C
O
R CH
3
O
+
R C O
+
CO CH
3
m/e = M-R m/e = M-15
Simarly for x= N & S
i)
ii)
CR OCH
3
O
CR O
+
m/e = M-31
+
17

18. 4 Rule of elimination of small neutral molecule
C
H
C
OH
C C
+
+H
2O
m/e M - 18
A)b - Elimination
The high temperature and high vacuum are quite favourable for elimination reaction
and hence
i)Loss of water (H
2
O) for alcohols (M-18) is a prominent fragment.
Tertiary alcohols lose the water so fast that in many cases M.I. Peak is absent.
General rules of Fragmentation
18

19. C C
NH
C C +NH
2
M - 46
C
2H
5
C
2H
5
ii)Loss of Ammonia (NH
3
)(M-17) for primary amines and primary
and secondary alkyl ammonia derivatives
For
C
H
C
NH
2
C C +
M - 17
NH
3
19

20. iii)Elimination at Hydrogen sulphide (H
2
S)[M-34] confirms thiols
(mercaptons)
C
H
C
SH
C C +H
2S
M - 34
iv)Elimination of Hydrogen cyanide (HCN)[M-27] confirms nitriles.
C
H
C
CN
C C +HCN
M - 27
20

21. v)Elimination of Hydrogen halide(HX),
Common for tertiary halides.
C
H
C
X
C C
m/e = M - HX
X = F, Cl, Br, I
21

22. 5.Rule – retro Diel’s Alder reaction
High temperature high vacuum highly favorable for(DA) common for all
these six membered cyclic mono olefins.

+
O
O
O
+
O
O
O
diene dienophile
General rules of Fragmentation
22

23. MCLAFFERTY REARRANGEMENT:-
Rearrangement ions are fragments, they are formed
due to the result of intermolecular atomic
rearrangement during fragmentation
To undergo this rearrangement the molecule must
posses heteroatom, one double bond and hydrogen atom
McLaffertyMcLafferty
x
CH
2
CH
2
H
CH
2
O
C
Y
Y Y  H, R, OH, NR2 H, R, OH, NR2
Ion Stabilized Ion Stabilized
by resonanceby resonance
x
CH
2
CH
2
H
CH
2
O
C
Y
- CH- CH
22=CH=CH
22
x
CH
2
O
C
Y
H
x
CH
2
+
O
+
C
Y
H
x
CH
2
+
O
C
+
Y
H
23

24. It is used for determination of molecular mass of
compounds and its elemental composition
Molecules having odd mass number contain odd
number of nitrogen atoms.
Molecules having even mass number contain even no
of nitrogen atoms.
NITROGEN RULE:-
CH
3
CH
3
CH
3
H
MW = 59 MW = 59
(odd)(odd)
MW = 58 MW = 58
(even)(even)
Ionisation Ionisation


[M+H][M+H]
[M+H][M+H]
MW = 60MW = 60
MW = 59MW = 59
CH
3
N
CH
3
CH
3
24

25. Nitrogen:
Odd number of N = odd MW
CH
3CN
M
+
= 41
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/2/09)
25

26. Problems and General pattern for
individual Families
26

27. Fragmentation Patterns
Alkanes:
Fragmentation often splits off simple alkyl groups:
Loss of methyl M
+
- 15
Loss of ethyl M
+
- 29
Loss of propyl M
+
- 43
Loss of butyl M
+
- 57
Branched alkanes tend to fragment forming the
most stable carbocations.
27

28. Fragmentation Patterns
Mass spectrum of 2-methylpentane

29. CH
2
CH
3
CH
3
CH
2
CH
2
CH
2
+
CH
3
CH
3
CH
2
+
CH
2
CH
2
CH
3
CH
3
CH
2
CH
3
CH
3
CH
2
+
CH
2
CH
CH
3
CH
3
Aklenes (olefins)
CH
2
CH
3
CH
3
CH
2
CH
3
CH
3
m/z 69
m/z 67
m/z 93
Fragmentation Patterns

30. Fragmentation Patterns

31. Hydroxy compounds:Hydroxy compounds:
R
2
C
R
3
R
1
O H
x
Loss of largest groupLoss of largest group
- R
3

R
2
C
R
1
O
+
H
R
2
C
+
R
1
O H
If RIf R
11=H m/e 45, 59, 73 …=H m/e 45, 59, 73 …
If RIf R
11=alkyl m/e 59, 73, 87 …=alkyl m/e 59, 73, 87 …
x
OH
CHR
H
CHR
CHR
CHR
OH
+
CHR
CHR
CHR
CHR
H
CHR
+
CHR
CHR
CHR
CHR
+
CHR
CHR
CHR
x
OH
CHR
H
CHR
CHR
CHR
CHR
CHR
M – (HM – (H
22O) – (C1=C2) AlkeneO) – (C1=C2) Alkene
- H
2
O
- CHR=CHR
M – (HM – (H
22O)O)
– – (H(H
22O)O)
Fragmentation Patterns

32. Fragmentation Patterns

33. Fragmentation Patterns
Aromatics may also have a peak at m/z = 77 for the
benzene ring.
NO
2
77
M
+
= 123
77

34. Fragmentation Patterns
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.
1
o
alcohol usually has prominent peak at m/z =
31 corresponding to H
2
C=OH
+

35. Fragmentation Patterns
MS for 1-propanol
M
+
M
+
-18
CH
3CH
2CH
2OH
H
2COH
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/28/09)

36. Fragmentation Patterns
Ethers
a-cleavage forming oxonium ion
Loss of alkyl group forming oxonium ion
Loss of alkyl group forming a
carbocation

38. Fragmentation Patterns
Aldehydes (RCHO)
Fragmentation may form acylium ion
Common fragments:
M
+
- 1 for

M
+
- 29 for
RCO
R (i.e. RCHO - CHO)
RCO

39. Fragmentation Patterns
Ketones
Fragmentation leads to formation of
acylium ion:
Loss of R forming
Loss of R’ forming

RCO
R'CO
RCR'
O

40. Fragmentation Patterns
MS for 2-pentanone
CH
3CCH
2CH
2CH
3
O
M
+
CH
3CH
2CH
2CO
CH
3CO
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/28/09)

41. Fragmentation Patterns
Esters (RCO
2
R’)
Common fragmentation patterns
include:
Loss of OR’
peak at M
+
- OR’
Loss of R’
peak at M
+
- R
’

42. Fragmentation Patterns
M
+
= 136
C
O
OCH
3
105
77 105
77
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/28/09)

43. ?
?
??
?
?
?
? ?
?
??
??
?
?
? ?
?
?
?
?
?
?
?
?
?
?
?
?
?
? ??
?
?
?
??
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
Any Q.
Thank You