INTRODUCTION of Mass Spectrometry, Applications of Mass Spectrometry,Principle of Mass Spectrometry, Mass Spectrum,MOLECULAR ION PEAK, MOLECULAR ION / PARENT PEAK, BASE PEAK, Metastable ion ,Instrumentation of Mass Spectrometry, Electron impact spectra, CHEMICAL IONIZATION, ELECTROSPRAY IONISATION, MATRIX ASSISTED DESORPTION / IONISATION(MALDI), FAST ATOM BOMBARDMENT SOURCE,Ion separator (analyzer), Types of mass spectrometers, Single focussing spectrometers,Time of flight systems,FRAGMENTATION of Mass Spectrometry.

1. MASS SPECTROSCOPY
Presented by
Prof. Mrs. Hemlata Manohar Nimje

2. - +--
INTRODUCTION
Micro Analytical technique -structure and
molecular weight
Only pico molar concentrations required

3. MS measures MASS OF INDIVIDUAL IONS
produced by fragmentation of the analyte
molecule.
Does not involve absorption or emission of
electromagnetic radiation
Destructive technique
Contnd…

4. Applications
Determination of molecular weight &
molecular formula
Fragmentation-Functional groups
Isotopic composition of elements in a
compound
Kinetics and mechanisms of uni-
molecular decomposition reactions.

5. Principle
M + e M+ + 2e
Electric field, V
½ mv2 = eV
v = √2eV/m
Magnetic field, H
mv2/r = Hev
r = mv/He
m = H2r2
e 2V

6. A graph of m/z Vs. Relative abundance.
Where,
→ M is the mass of the ion and z is the
charge of the ion.
→ Relative abundance is the number of
times an ion of that m/z ratio strikes the
detector.
Mass Spectrum : Presentation

7. MASS SPECTRUM
PRESENTATION
REPRESENTED BY BAR GRAPH
PRODUCES A SPECTRUM OF MASSES
BASED ON STRUCTURE OF MOLECULE

8. m/z
M•+ (EI)
“molecular ion”
Fragment Ions
isotope peak
base
peak

9. X-AXIS
REPRESENTS m/z VALUES
m –unified atomic mass
z- charge on the ion (positive value)
m/z values give mol.wt of ion

10. Y-AXIS
REPRESENTS SIGNAL INTENSITY
ie, relative abundance of each ion
produced
most intense peak is base peak(100%)

11. CHARACTERISTICS
MOLECULAR ION PEAK
 REPRESENTED BY [M]+.
MOLECULE BOMBARDED WITH
ELECTRON BEAM PRODUCES [M]+.
GIVES MOL.WT OF COMPOUND

12. m/e
Relativeabundance
20 30 40 50 60 70 80 90 100
20406080100
Base peak
CH2Cl+
49
84
M+
CH2Cl2

13. MW = 86
CH3
CH3-CH-CH2-CH2-CH3
CH3
86(M+)
71 (C5H11
+)
57(C4H9
+)
43(C3H7
+)
29(C2H5+)

14. MOLECULAR ION / PARENT PEAK
When a sample substance is bombarded
with electrons of energies of 9-15 eV, the
molecular ion is produced by loss of a
single electron.
This will give rise to a very simple mass
spectrum with essentially all of the ions
appearing in one peak…
+ e
-
+
9-15ev

15. π electron-stabilises more easily
σ-stabilises slowly
Aromatics>conjugated
olefines>alicyclics>sulphides>unbranched
hydrocarbons>ketones>amines>esters>
ethers>carboxylic acids>branched
hydrocarbons and alcohols.
Parent peak-molecular weight
Contnd…

16. BASE PEAK
•If an electron beam of energy of 70 eV is
used in a mass spectrometer, the
molecular ion is produced by the loss of a
single electron which undergoes splitting to
form many fragments.
•The height of all other peaks are
measured with respect to base peak(100%
abundance).
+ e-
70ev +

17. Metastable ion
Short life time
Ion source
Broad peaks at non integral mass numbers
For fragmentation studies.
m1 m2+m0
M* =m2
2/ m1
m1=parent ion,m2=daughter ion, m*=apparent
mass of metastable ion ,m0=mass of neutral
fragment
Decomposition
Magnetic analyser

19. Instrumentation
Mass spectrometer is an instrument which
provides charged ions consisting of the parent
ion and ionic fragments of the original
molecules and sorts these according to their
m/e ratio.
1912:first mass spectrometer(JJ Thomson)
1919:modified form(Aston).

20. Inlet
system
Ion
source
Mass
analyzer
Detector
Signal
processor
Read out
Vacuum
system
Sample
Electric field
Magnetic field

22. Components
The inlet system (sample handling
system)
The ion source
The electrostatic accelerating system
The magnetic field
The ion separator
The ion collector (detector & readout
system)
The vacuum system

23. Different according to physical
properties
Batch inlet system
Direct probe inlet
Chromatographic & capillary
electrophoretic inlet system.
Sample inlet system

24. Batch inlet system These systems are the simplest and
simply involve the volatilization of the
sample externally and then the
gradual leakage of the volatilized
sample into the evacuated ionization
chamber. For gases, the sample is
introduced into the metering volume
container and then expanded into the
reservoir flask where it is then leaked
into the ionization chamber. For
liquids, a small quantity of sample is
introduced into the reservoir and the
pressure of the system is reduced to
about 10-5 torr. The inlet system is
lined with glass to avoid losses of
polar analytes by adsorption.

25. Direct inlet probe
These systems are used for
solids and non-volatile liquids.
In these systems the sample is
introduced into the ionization
region by means of a sample
holder, or probe, which is
inserted through a vacuum
lock. Probes are also used
when the amount of the
sample to be analyzed is
small.
With a probe, the sample is
generally held on the surface
of a glass or aluminum
capillary tube and positioned
within a few meters of the
ionization source.

26. Ion source
Hard Soft
Excitation
Relaxation
Fragmentation
No. of peaks high
-functional groups
Little fragmentation
Molecular ion peak
-molecular weight.

27. GAS PHASE
Vaporised
Ionised
<10 3D
DESORPTION
(Solid/liquid)
Ionised
≥10 5D

28. Ion source
Gas phase
Electron impact
Chemical ionization
Field ionization
Desorption
Field desorption
Electrospray ionization
MALDI
Plasma desorption
Fast atom bombardment
Secondary ion mass spectrometry
Thermospray ionization

29. Electron impact
At low energy,
m +e m +2e
At increased energy,
m +e m2+ +2e-
m2+ +e- m3+ +2e-

30. Electron impact spectra
m/e
Relativeabundance
20 30 40 50 60 70 80 90 100
20406080100
Base peak
CH2Cl+
49
84
M+
CH2Cl2

31. CHEMICAL IONIZATION
Carrier gas is ionized by electron impact to
primary ions.
These react with the excess rgt to
secondary ions.
Common reagent gases
–methane, isobutane, ammonia
P= 1 torr
Mass spectrometer
Vaporised sample + reagent gas

32. CH4+e CH4+, CH3+ ,CH2
+
CH4+ + CH4 CH5+ + CH3
CH3+ + CH4 C2H5+ + H2
CH5+ + MH [MH]2
+ + CH4
C2H5+ + MH [MH]2
+ + C2H4
M++C2H6C2H5
+ + MH
Methane ionized by EI

33. ELECTROSPRAY IONISATION
For biomolecules –
polypeptides,proteins,oligonucleotide-having
mol.wt.100000 Da.
Used in atmospheric pressure and temperature.
Little fragmentation of thermally fragile
molecules.
Multiple charged ions- m/e value small- easy
detection.

35. MATRIX ASSISTED DESORPTION /
IONISATION(MALDI)
New ionisation method
Polar bio polymers- Few thousands to several
hundred thousands
Aqueous/alcoholic soln of sample +radiation
absorbing matrix (nicotinic acid)
evaporated on the tip of a probe + laser
beam pulsed ions time of flight
mass analyser.

36. FAST ATOM BOMBARDMENT
SOURCE
Energetic Argon/xenon + chamber with
argon argon+ electrons
removed+ samples in condensed
state in glycerol matrix.
Organic/biochemical compounds-
mol.wt.10000.
Detailed structural information upto
3000Dalton.

37. Fast atom bombardment
source

38. The Electrostatic
Accelerating System
Positive ions from ionisation source-withdrawn
by electric field between first Accelerator plate
and repeller plate.
m1,m2,m3
m1v1
2=m2v2
2=m3v3
2……..
Second accelerator is charged to a potential
of about 4000.
charge allowed to leak out to ground.

39. Magnetic field
Ionised molecule enter into
magnetic field
r = mV/eH
r,m/e two properties on which
mass spectrometry is based.

40. Ion separator
(analyzer)
Separates ions according to
their masses.
Characteristics
High resolution
High rate of transmission of
ions

41. Resolution of mass
spectrometers
Ability to distinguish between ions of nearly
equal masses.
R=m/Δm
Resolution depends on its application.
With same nominal mass-high resolution
C2H4
+,CH2N+ (28.0313,28.0187)
Different nominal mass-low resolution
NH3
+&CH4
+ (17,16)
-500-500000-

42. Types of mass
spectrometers
Single focussing
Double focussing
Cycloidal focussing
Quadrupole
Time of flight systems
radiofrequency analyzer
Others
omegatron
iontrap analyzer

43. Single focussing spectrometers

44. Double focussing type
m/e = H2r2/2V
Electrostatic field +magnetic field
Select with certain V&K.E
Precise molecular weight.

46. Quadrupole mass
spectrometer

47. Time of flight systems
Non-magnetic separator.
Ion beam produced as pulses by
placing control grid in electron beam
path.
Pulses last for about 0.25msec.
Frequency-10,000times/sec.

48. Ions travel through a magnetic free straight
tube (drift tube) take different times to travel
given distance-
t =k √m/e
K=lengh of flight path(proportionality
constant).
Oscilloscope record the current.

50. Electron multiplier
Discrete dynode electron multiplier

51. Vacuum system
Inlet system-0.015 torr.
Ion source-10-5 torr.
Analyser-10-7
Oil diffusion and mercury diffusion
pumps.

52. FRAGMENTATION
CHEMICAL PROCESS RESULTING IN BOND
BREAKING
INTIATED BY ELECTRON IMPACT
MOLECULAR ION UNDERGO FRAGMENTATION
CATION –RADICAL CHARACTER IS THE MAJOR
DRIVING FORCE
[M·]+ A+ + B· (neutral)
or
B+ + A·
EI

53. Example: The spectrum of hexane
13

54. TYPES OF FRAGMENTATION
 SIMPLE CLEAVAGE
 RETRO-DIELS ALDER REACTION
SKELETAL REARRANGEMENTS

55. SIMPLE CLEAVAGE
CLEAVAGE OCCURS ONLY ON SINGLE
COVALENT BOND
TWO TYPES
- HOMOLYTIC CLEAVAGE
- HETEROLYTIC CLEAVAGE

56. HOMOLYTIC CLEAVAGE
BREAKAGE OF C-C BONDS
THREE MODES OF CLEAVAGE
MODE I
MODE II
MODE III

57. MODE I
CLEAVAGE OF C-C BOND IN β-POSITION TO SINGLE
BONDED HETERO ATOM
eg:Alcohols,Amines
RO
RO
RO
:
:
Obs. in Mass Spec
Resonance stabilized
neutral
+
+

58. MODE II
CLEAVAGE OF C-C BOND AT α-
POSITION WHERE HETERO ATOM
ATTACHED BY DOUBLE BOND
eg: ketones,esters,amides

59. O O
O
: .
+
++:
+
: :
Obs. in mass spec. Acylium ions
are resonance-stabilized
neutral
Prominent for ketones
CH3C=O+
m/z=43

60. MODE III
CLEAVAGE OF C-C BOND β TO
AROMATIC RING
eg: benzylic cleavage

61. Rearrangements and fragmentations to give good
Carbocations
CH2+
C
H
+ CH2
H C
CH2
CH+
+
Benzylic cation
(stabilized
including
“tropylium” ion
m/z=91
Good cleavage  to aromatic
rings

62. HETEROLYTIC CLEAVAGE
CLEAVAGE OF C-X BOND
CHARGE CARRIED BY CARBON ATOM
ALKYL IODIDES &BROMIDES EASILY
BROKEN

63. CLEAVE  TO HETEROATOMS LIKE
O, N
O
R
: .
+
•
Heterolytic cleavage
R
O:: .
neutral
+
Observed in Mass Spec provided that a
good stabilized carbocation can form
+

64. RETRO-DIELS ALDER
REACTION
MULTICENTRED FRAGMENTATIONS
eg:cyclic olefins
CLEAVAGE OF TWO BONDS
 2 STABLE UNSATURATED FRAGMENTS
 CHARGE CARRIED BY EITHER OF 2
FRAGMENTS
 MORE STABLE ONE PREDOMINATES

65. THE “RETRO -DIELS-ALDER”
CLEAVAGE
+
+
•
+
•
+
+
•
Observed!
Observed!
Cyclohexenes, with favorable 6-
membered transition state. Can include
heteroatoms (N,O, driven by keto-enol
like stability.

66. SKELETAL
REARRANGEMENTS
THREE TYPES
ELIMINATION REACTIONS
ORTHO EFFECT
McLAFFERTY REARRANGEMENT

67. ELIMINATION REACTIONS
ABSTRACTION OF H BY OH ,X OR –
OCH3
ELIMINATION OF H2O,HX,CH3COOH
R-CH2-CH-CH2 R-CH2-CH=CH2
H OCOCH3
+CH3COOH

68. ORTHO EFFECT
OCCURS IN O-SUBSTITUTED
AROMATIC& CIS OLEFINS
H-ATOM IS IN CLOSE PROXIMITY TO
ELIMINATE A NEUTRAL MOLECULE

69. McLAFFERTY REARRANGEMENT
RADICAL CATIONS LOCALIZED ON
CARBONYL GROUP GIVE β
CLEAVAGE
NEEDS H ATOM ON γ-sp3 CARBON
OCCURS IN ALDEHYDES
,KETONES,ESTERS

70. O
+
H
R2
R1
•
O
+
H
•
OH
R1 R2
••
•
+
Loss of
neutral
alkene
The new radical
cation is stabilized
by resonance

71. IMPORTANT EXAMPLE OF
MCLAFFERTY
REARRANGEMENT
OH
OH
m/z = 60
+•
Seen for primary carboxylic acids

72. Nitroaromatics
N
+
O•
O
O
+
Loss of
•N=O
Loss of CO
m/z= 93
(this can
form from
lots of
different
origins)
CH+
Aromatic!
m/z=65
Good test for aryloxy

73. Common Mass Spec Fragments
m/z lost Moiety Compounds Exhibiting Loss
1 H aldehydes
15 CH3 branched sites
16 O sulfoxides, nitro compounds
16 NH2 amides, aromatic amines
17 OH acids
18 H2O alcohols, aldehydes, ketones, ethers

74. Common Mass Spec Fragments
m/z lost Moiety Compounds Exhibiting Loss
26 CN alkylcyanides
29 C2H5 or CHO alcohols
31 OCH3 or CH2OH methyl esters, alcohols
35 Cl halide-containing
45 OC2H5 or COOH ethyl esters or carboxylic acids

75. FRAGMENTATION RULES
Peak height of [M]+.↑ for straight chain
compounds
Peak height ↓ as branching increases
Cleavage favoured acco: to stability of
carbocation
30 >20 >10
Aromatic rings & cyclic structures stabilize [M]+.

76. Contd..….
Double bonds favours allylic cleavage
Unsaturated rings undergo retro-Diels
Alder reaction
Alkyl substituted aromatics give tropylium
ion
Cleavage releases small,stable,neutral
molecules like CO,H2O etc

77. FACTORS AFFECTING
FRAGMENTATION
THERMAL DECOMPOSITION
BOMBARDMENT ENERGY
RELATIVE RATES OF COMPETING
FRAGMENTATION ROUTE

78. ISOTOPIC ABUNDANCE
3 Classes of Isotopes
 A - only a single isotope
– EX: F, P, I
A+1 - two isotopes with significant relative abundance
differing by 1 mass unit
– EX: H, C, N
A+2 - two isotopes with significant relative abundance
differing by 2 mass units
– EX: Cl, S, O

79. Natural Isotopic Abundance of
Common Elements in Organic
Compounds
Element Isotope Relative
abundan
ce
Isotope Relative
abundan
ce
Isotope Relative
abundan
ce
Carbon 12C 100 13C 1.11
Hydrogen 1H 100 2H .016
Nitrogen 14N 100 15N .38
Oxygen 16O 100 17O .04 18O .20
Sulfur 32S 100 33S .78 34S 4.40
Chlorine 35Cl 100 37Cl 32.5
Bromine 79Br 100 81Br 98.0

80. BASE PEAK
MOST INTENSE PEAK
ASSIGNED A VALUE OF 100%
OTHER PEAKS RELATIVE TO BASE
PEAK

81. INTERPRETATION
OF MASS SPECTRA

82. GENERAL RULES FOR INTERPRETATION
1.EXACT MOLECULAR WEIGHT
2.ISOTOPE EFFECT
3.NITROGEN RULE

83. 1.EXACT MOLECULAR
WEIGHT
IDENTIFICATION OF THE PARENT
PEAK
DETERMINES THE MOLECULAR
WEIGHT
DETERMINES MOLECULAR FORMULA
FROM MOLECULAR WEIGHT

84. 2. ISOTOPE EFFECT
 CARBON 12 HAS AN ISOTOPE, CARBON 13.
12C= 100%,
13C= 1.1%.
 IF A COMPOUND CONTAINS 6 CARBONS, EACH
ATOM HAS A 1.1% ABUNDANCE OF (13)C.
 IF THE MOLECULAR ION PEAK IS 100%, THEN
THE ISOTOPE PEAK (1 MASS UNIT HIGHER)
WOULD BE 6X1.1%=6.6%.

85. LOOK FOR M+2 PEAK
eg:Cl,S,Br
LOOK FOR M+1 PEAK
eg:C,H,N
PEAK INTENSITY RATIO OF
Cl - 1:3
Br - 1:1

86. 3.NITROGEN RULE
COMPOUND CONTAINING C, H, O, AND AN
EVEN NUMBER OF NITROGENS (OR NO
NITROGENS) WILL HAVE AN EVEN
MOLECULAR WEIGHT
C6H7BrN2
 COMPOUND CONTAINING C, H, O, AND AN
ODD NUMBER OF NITROGENS WILL HAVE
AN ODD MOLECULAR WEIGHT
C2H7NS

87. Major Steps in Analysis of Mass
Spectral Data
IDENTIFICATION OF MOLECULAR ION
– BASE PEAK
EXAMINATION OF ISOTOPIC DISTRIBUTION
PATTERN
– DETERMINE ELEMENTAL COMPOSITION
ANALYSIS OF FRAGMENTATION PATTERN
– PROPOSE POSSIBLE STRUCTURES
– COMPARE POSTULATED SPECIES TO
AVAILABLE REFERENCE SPECTRA

88. MASS SPECTRA OF SOME
CHEMICAL CLASSES

89. ALKANES
Molecular ion peaks are present,
possibly with low intensity.
The fragmentation pattern
contains clusters of peaks 14
mass units apart (which
represent loss of (CH2).
Hexane

90. Fragmentation of Alkane:
•These shows regular clusters of peaks separated by
14 (CH2) mass units. The largest peak in each cluster
is normally CnH+
2n+1 fragment occurring at m/z =14n+1
straight and branched-chain alkanes show grossly
similar spectra the major difference being in the
relative intensities of the peaks.
•Normally a largest substituent at a branch is
eliminated as a radical (stability reasons). Cleavage is
also favoured to give a more stable of the
carbocations.

91. ALCOHOL
An alcohol's molecular ion is small
or non-existent.
Cleavage of the C-C bond next to
the oxygen usually occurs.
A loss of H2O may occur as in the
spectra below.
3-Pentanol
C5H12O
MW = 88.15

92. ALDEHYDE
Cleavage of bonds next to
the carboxyl group results in
the loss of hydrogen
(molecular ion less 1) or the
loss of CHO (molecular ion less
29).
3-PHENYL-2-PROPENAL
M.W-132.18

93. Fragmentation of Aldehyde:
The major fragmentation is like that in ketones C-C and
C-H bond cleavage adjacent the C=O bond (α-
cleavage).
Β-cleavage is also observed to give the hydrocarbon
ion R+.
A β, ϒ-bond cleavage occurs by the loss of a radical
and resonance stabilized cation e.g., at m/z = 57 in
butyraldehyde.
McLafferty rearrangement is useful to distinguish
between isomeric aldehydes.

94. 2-Butenoicacid
C4H6O2
MW = 86.09
In short chain acids, peaks due to
the loss of OH (molecular ion less
17) and COOH (molecular ion less
45) are prominent due to cleavage
of bonds next to C=O.
CARBOXYLIC ACID

95. Ethyl acetate
C4H8O2
MW = 88.11
Fragments appear due to
bond cleavage next to C=O
(alkoxy group loss, -OR) and
hydrogen rearrangements.
ESTER

96. Fragmentation of Aliphatic Carboxylic Acids, Esters
and Amides:
These show α-cleavages on either side of C=O group.
McLafferty ion is an intense characteristic peak in all the classes.
In carboxylic acids, α-cleavage by OH+ loss is not facile (except in
aromatic acids). Ions corresponding to COOH+ m/z = 45 or the
loss of radical COOH+ are observed in short chain acids.
Cleavage of a C-C bond – some distance from the functionality
also occur to a significant extent.
Fragmentation in esters at the ether oxygen (α-cleavage) gives
acylium ion RCO+ which by the loss of CO gives the carbocation
particularly when it is secondary or tertiary.
In the amides the C-N bond resists cleavage (except in aryl
amides). Fragmentation of aliphatic amides is by C-C α-cleavage
to yield O=C-NH2, m/z = 44.

97. 1-Bromopropane
C3H7Br
MW = 123.00
HALIDE
1.The presence of chlorine or
bromine atoms is usually
recognizable from isotopic peaks.
2.Iodides and bromides are easily
released

98. 4-Heptanone
C7H14O
MW = 114.19
Major fragmentation peaks
result from cleavage of the C-C
bonds adjacent to the carbonyl.
KETONE

99. Fragmentation of Ketones:
The molecular ion peaks are intense.
The major fragmentation peaks from aliphatic ketones arise due to
loss of alkyl groups attached to the carbonyl group i.e., α-cleavage
to give resonance stabilized acylium ion.
Often the base peak is due to the loss of larger alkyl group.
Further fragmentation of the acylium ions by CO loss may be
facile to give carbocations.
When one of the alkyl chains contains three or more carbon
atoms, the α,β bond cleaves with the six-membered cyclic H atom
shift from the ϒ-carbon to oxygen atom to give a McLafferty ion.
The McLafferty rearrangement is useful to distinguish between
ketones.

100. Fragmentation of Phenols:
Phenols show mass spectra which contract strongly with those of a
aliphatic alcohols:
•Phenols do not undergo α-cleavage or dehydration.
•Keto-enol tautomerism is followed by loss of CO.
•Substituted phenols undergo ring expansion and fragmentation
through substituents.
Fragmentation of Ethers:
C-O cleavage with charge largely retained by the hydrocarbon portion
with formation of an oxy radical.
•α-cleavage i.e., cleavage of the first C-C bond counting from the
functionality to give resonance stabilized product.
•The rearrangement on the product of α-cleavage in which an alkene is
lost.

101. CONCLUSION
MASS SPECTRUM PROVIDES INFORMATION
ABOUT
-MOLECULAR WEIGHT OF ORGANIC
& INORGANIC COMPOUNDS
-MOLECULAR FORMULA OF
COMPOUNDS
-ANALYTICAL TOOL FOR
CHARACTERIZING ORGANIC
MOLECULES

102. THANK YOU