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 r2
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¯
.
intensity 5 10 15 20 25 30 35
m/e 8
120
100
80
60
40
20
0
CHO⁺
CH3OH⁺
CH3⁺
CH2OH⁺

9. Fragmentation rules in MS
1. Intensity of MM..++ is LLaarrggeerr ffoorr lliinneeaarr cchhaaiinn than for branched
9
compound
2. Intensity of MM..++ ddeeccrreeaassee with IInnccrreeaassiinngg MM..WW.. (fatty acid is an
exception)
3. Cleavage is ffaavvoorreedd aatt bbrraanncchhiinngg
44.. AArroommaattiicc RRiinnggss,, DDoouubbllee bboonndd,, CCyycclliicc ssttrruuccttuurreess ssttaabbiilliizzee MM..++
55.. DDoouubbllee bboonndd ffaavvoorr AAllllyylliicc CClleeaavvaaggee
6. Saturated Rings lose a Alkyl Chain (case of branching)
7.. AArroommaattiicc CCoommppoouunnddss CClleeaavvee iinn bb
Resonance Stabilized TTrrooppyylliiuumm
8.. CC--CC NNeexxtt ttoo HHeetteerrooaattoomm cleave leaving the cchhaarrggee oonn tthhee
HHeetteerrooaattoomm
99.. CClleeaavvaaggee ooff ssmmaallll nneeuuttrraall mmoolleeccuulleess ((CCOO22,, CCOO,, oolleeffiinnss,, HH22OO ……..))..
RReessuulltt oofftteenn ffrroomm rreeaarrrraannggeemmeenntt -- MMccLLaaffffeerrttyy rreeaarrrraannggeemmeenntt 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 (CnH2n+1) peaks decreases with increasing mass.
10

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

12. CH3
1 2 3 4 5 6 7 8
+
cleavage at 6-1
cleavage at 6-2
cleavage at 6-3
C4H9
C H
C3H7
+
CH3
C H
C4H9
+
CH3
C H
C3H7
+
(F1)
(F2)
(F3)
H3C CH2 CH2 C
H
CH2 CH2 CH2 CH3
Eg.
Produces thre secondary cations, the most favored fragments
at C-4 of
4- methyl octane.
Note that C4 is common for fragments (F1)(F2) And (F3). 12

13. General rules of Fragmentation
3.Rule of b cleavage
Most important rule covers 70% of mass fragmentation.
X C1 C2 R X CH
a b
Cleavage favored at b bond leaving positive charge on C1.
13

14. H3C CH2 O CH2 CH3
H3C CH2 O CH2 CH3
CH2 O CH2
m/e = M-15
1.
H3C
2.
CH2 CH2 CH3
H3C CH2 N CH2
N
C2H5
C3H7
H2C
N
C2H5
H2C
H2C
m-57 m-29
N
CH2
C3H7
m-15
CH2
tert.amine
B1
B3 B2
e.g.: A) (x) = O, N, S.
14

15. 3.
CH2 S CH2 CH2 CH3
H2C S
CH2
H2C S
M-71 C3H7
M-29
B2 B1
B1
B2

16. R
CH2
CH2
+
+
m/e = ( M-R ) Stable
benzylic cation
+
m/e = 91
Tropylium cation
b) Benzylic clevage
+
b)
m/e = 65
cyclopentadienyl
cation
-(x)- =
16

17. O
+
O C CH + 3
m/e = M-R m/e = M-15
Simarly for x= N & S
+
Very common fragment for ester
C. Allylic Cleavage
H2C
M-31 = methyl ester
M-45 = ethyl ester
R
m/e = M-R stable allyliz cation
O
H3C CH3
R CH3
R C O
i)
ii)
O
R C OCH3
R C O
m/e = M-31
+
17

18. General rules of Fragmentation
4 Rule of elimination of small neutral molecule
A) b - Elimination
The high temperature and high vacuum are quite favourable for elimination reaction
H
C
C
OH
C C
+
+ H2O
m/e M - 18
and hence
i)Loss of water (H2O) for alcohols (M-18) is a prominent fragment.
Tertiary alcohols lose the water so fast that in many cases M.I. Peak is absent.
18

19. ii)Loss of Ammonia (NH3)(M-17) for primary amines and primary
and secondary alkyl ammonia derivatives
For
C C
NH
C C + NH2
M - 46
C2H5
C2H5
H
C
C
NH2
C C +
M - 17
NH3
19

20. iii)Elimination at Hydrogen sulphide (H2S)[M-34] confirms thiols
(mercaptons)
H
C
C
SH
C C + H2S
M - 34
iv)Elimination of Hydrogen cyanide (HCN)[M-27] confirms nitriles.
H
C
C
CN
C C + HCN
M - 27
20

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

22. General rules of Fragmentation
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
22

23. MCLAFFERTY REARRANGEMENT:-
Rearrangement ions are fragments, they are formed
due to the result of intermolecular atomic
MMccLLaaffffeerrttyy
rearrangement H
during fragmentation
x
To undergo this CH2
rearrangement the molecule must
posses heteroatom, CH2
one double bond and hydrogen atom
CH2
O
C
Y
+
YY  HH,, RR,, OOHH,, NNRR22
IIoonn SSttaabbiilliizzeedd
bbyy rreessoonnaannccee
x
CH2
CH2
H
CH2
O
C
Y
-- CCHH22==CCHH22
x
CH2
O
C
Y
H
x
+
CH2
O+
C
Y
H
x
CH2
O
C+
Y
H
23

24. NITROGEN RULE:-
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.
H3C
Molecules having even mass number contain even no
H3C of nitrogen CH3
atoms.
H
MMWW == 5599
((oodddd))
MMWW == 5588
((eevveenn))
IIoonniissaattiioonn
[[MM++HH]]
[[MM++HH]]
MMWW == 6600
MMWW == 5599
CH3
N
H3C CH3
24

25. Nitrogen:
Odd number of N = odd MW
CH3CN
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. Fragmentation Patterns
H3C CH3
H3C CH3
H3C CH3
CH2
H3C CH3
H3C CH3
+
H2C
CH2
H2C
H3C CH3
H3C CH3
+
H2C
+
CH2
H2C
CH2
CH
CH2
CH2
Aklenes (olefins)
CH2
CH2
m/z 69 mm//zz 6 973

30. Fragmentation Patterns

31. Fragmentation Patterns
HHyyddrrooxxyy ccoommppoouunnddss::
R1
R2 C
R3
x
O H
LLoossss ooff llaarrggeesstt ggrroouupp
- R3

R2
–– ((HH22OO))
C
R1
CHR
CHR CHR+
O+ H
CR1 +
R2
O H
MM –– ((HH22OO))
IIff RR11==HH mm//ee 4455,, 5599,, 7733 ……
IIff RR11==aallkkyyll mm//ee 5599,, 7733,, 8877 ……
xOH
CHR
H
CHR
CHR
CHR
OH+
CHR
CHR
CHR
CHR
H
CHR+
CHR
CHR
CHR
CHR
xOH
CHR
H
CHR
CHR
CHR
CHR
CHR
- H2O
- CHR=CHR
MM –– ((HH22OO)) –– ((CC11==CC22)) AAllkkeennee

32. Fragmentation Patterns

33. Fragmentation Patterns
Aromatics may also have a peak at m/z = 77 for the
benzene ring.
NO2
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.
1o alcohol usually has prominent peak at m/z =
31 corresponding to H2C=OH+

35. Fragmentation Patterns
 MS for 1-propanol
M+-18 M+
CH3CH2CH2OH
H2C OH
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
RC O
 Common fragments:
M+ - 1 for
M+ - 29 for
RC O
R (i.e. RCHO - CHO)

39. Fragmentation Patterns
 Ketones
O
 Fragmentation leads to formation of
acylium ion:
Loss of R forming
Loss of R’ forming
R'C O
RC O
RCR'

40. Fragmentation Patterns
 MS for 2-pentanone
O
CH3CCH2CH2CH3
CH3CH2CH2C O
M+
CH3C O
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/28/09)

41. Fragmentation Patterns
 Esters (RCO2R’)
 Common fragmentation patterns
include:
Loss of OR’
 peak at M+ - OR’
Loss of R’
 peak at M+ - R’

42. Fragmentation Patterns
M+ = 136
77 105
O
C
O CH3
105
77
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/28/09)

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Any Q. Thank You