Mass spectrometry is a technique that uses the deflection of charged particles by a magnetic field to determine the relative masses of molecular ions and fragments. It provides a great deal of information from small samples and can be used to determine molecular mass, structure, and purity. Various ionization sources like electron ionization, chemical ionization, fast atom bombardment, and matrix-assisted laser desorption/ionization are used to vaporize and ionize samples for analysis in mass analyzers such as quadrupoles, ion traps, and time-of-flight instruments. Mass spectra provide the abundance of ions as a function of their mass-to-charge ratio and can reveal molecular structure through characteristic fragmentation patterns.

1. Mass Spectrometry
1

2. Mass Spectrometry
Technique that utilizes the degree of deflection of
charged particles by a magnetic field to find the relative
masses of molecular ions and fragments
a powerful method because it provides a great deal of
information and can be conducted on tiny samples
2

3. Applications
Determining molecular mass
Molecular formula (HRMS)
Confirmation of elemental composition
Finding out the structure of an unknown substance
Verifying the purity of a known substance
Providing data on isotopic abundance
3

4. Reaction monitoring
Crude reaction mixture
Stability studies
Quick product identification (TLC spot)
Selective detector for GC/HPLC
MS provides molecular weight information about
each chromatographic peak
4

5. • Resolution: A measure of how well a mass
spectrometer separates ions of different
mass.
– low resolution: Refers to instruments capable
of separating only ions that differ in nominal
mass; that is ions that differ by at least 1 or
more atomic mass units.
– high resolution: Refers to instruments
capable of separating ions that differ in mass
by as little as 0.0001 atomic mass unit.
Low and High Resolution MS
5

6. • A molecule with mass of 44 could be C3H8,
C2H4O, CO2, or CN2H4
• If a more exact mass is 44.029, pick the
correct structure from the table:
C3H8 C2H4O CO2 CN2H4
44.06260 44.02620 43.98983 44.03740
High Resolution MS
6

7. – 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.
C2 H4 O2
C3 H8 O
60.02112
60.05754
60
60
Molecular
Formula
Nominal
Mass
Precise
Mass
Resolution
– High resolution MS can replace elemental analysis for
chemical formula confirmation 7

8. Mass Spectrometry
Doesn’t involve the absorption of any type of light
Technique involves
 Creating gas phase ions from the analyte atoms or
molecules
 Separating the ions according to their mass-to-charge
ratio (m/z)
 Measuring the abundance of the ions
8
we don’t get the sample back; a destructive method

9. Steps to Get Mass Spectrum
A compound is vaporized and ionized by
bombardment with a beam of high-energy electrons
The electron beam ionizes the molecule by causing
it to eject an electron.
When the electron beam ionizes the molecule, the
species formed is called a radical cation, symbolized as
M+•.
9

10. The radical cation M+• is also called the molecular ion
or parent ion; its mass = molecular weight of M
Because M+• is unstable it decomposes to form
fragments of radicals and cations that have a lower
molecular weight than M+•
The mass spectrometer measures the mass of these
cations.
10

11. The mass spectrum is a plot of the amount of each cation
(relative abundance) versus its mass to charge ratio (m/z)
Since z is almost always +1, m/z actually measures the mass
(m) of the individual ions
11

12. EI,
CI,
ESI,
FAB and
MALDI
12
Quadrupole
Ion Trap
Time of Flight

13. 13
The m/q ratio of the ions that reach the detector can be varied by changing either the
magnetic field (B) or the applied voltage of the ion optics (V)

14. By varying the voltage or magnetic field of the
magnetic-sector analyzer ,
the individual ion beams are separated spatially and
each has a unique radius of curvature according to its
mass/charge ratio.
14

15. IONIZATION SOURCES
15

16. EI: Electron Ionization/Electron Impact
Heated Incandescent
Tungsten/Rhenium Filament
Accel!
e
Vaporized
Molecules
70 eV
Ions To Mass
Analyzer
16
 How EI works:
 Electrons are emitted from a
filament made of tungsten
cathode
 They are accelerated
towards anode by a
potential of 70 eV
 The electrons and
molecules cross (usually at
a right angle) and collide
 The ions are primarily
singly-charged, positive
ions, that are extracted by a
small potential through a slit

17.  Referred to as hard ionization source due to the high
energy EI source
 Ions are accelerated into the mass analyzer by an
accelerating voltage of ~ 104 V
 Both negative and positive ions are formed by EI
 Negative ions form from molecules containing acid
groups or electronegative atoms
17

18.  Collision between ions and molecules may also result in ions
with higher m/z values than the molecular ion
An example is the (M+1) peak
 Reaction between analyte molecule and H+ to form MH+ or
(M+H)+ in which charge equals a+1
 Low pressure in the ionization source minimizes reaction
between ions and molecules
18

19. Advantages of Electron Ionization
• inexpensive, versatile and reproducible
• fragmentation gives structural information
• large databases if EI spectra exist and are
searchable
Disadvantages of Electron Ionization
• fragmentation at expense of molecular ion
• sample must be relatively volatile
19

20. Chemical Ionization (CI)
 A large excess of reagent gas (1000 – 10000 times) is
introduced into the ionization region
 Pressures in source are typically higher than EI
 Electrons are allowed to bombard the gas-sample
mixture
Examples of reagent gas
- Methane, ammonia, isobutane
20

21.  Reagent gases are much more likely ionized by the
electrons than the sample due to large excess
 Sample molecules are subsequently ionized by
collision with ionized reagent gas molecules
 Considered as soft ionization source
 Less fragmentation and molecular ion is much more
abundant
 Combination of CI and EI spectra provide good
interpretation
21
CI cont’d

22. Chemical Ionization (CI)
- For methane as reagent gas
electrons
with
n
interactio
upon
formed
are
CH
and
CH 3
4









 3
5
4
4 CH
CH
CH
CH
2
5
2
4
3 H
H
C
CH
CH 

 

Proton transfer occurs when sample molecules collide with


5
2
5 H
C
and
CH
22

23. Chemical Ionization (CI)
4
5 CH
MH
CH
M 

 

The following may occur if analyte is a saturated HC
4
2
5
2 H
C
MH
H
C
M 

 

2
4
5 H
CH
H)
-
(M
CH
M 


 

6
2
5
2 H
C
H)
-
(M
H
C
M 

 

29)
(M
m/z
with
)
H
C
(M
H
C
M 5
2
5
2 



 

23

24. 24

25. Fast Atom Bombardment (FAB)
 material to be analyzed is mixed with a non-volatile
chemical protection environment called a matrix
 This is bombarded under vacuum with a high energy (4
– 10 keV) beam of atoms, which forms ions
 atoms are typically an inert gas (Ar or Xe)
 common matrix include glycerol, thioglycerol, 3-
nitrobenzyl alcohol, 18-Crown-6 ether, 2-
nitrophenyloctyl ether, sulfolane, diethanolamine, and
triethanolamine.
25

26. 26
Fast Atom Bombardment

27. Advantages of FAB
Parent Ion
High Mass Compounds (10,000 amu)
Thermally Labile Compounds
27

28. laser desorption . . .
28

29. MALDI
Combined with a MS detector, MALDI became
an indispensable tool in analysis of
biomolecules and organic macromolecules
MALDI involves
incorporation of the analyte into a matrix,
absorption/desorption of laser radiation, and
 then ionization of the analyte
29

30. Formation of Matrix-Analyte
The analyte incorporation in to a suitable matrix is
the first step of the MALDI process, and is an
important feature of the MALDI method
A typical sample preparation involves using 10-6 M
solution of the analyte mixed with 0.1 M solution of
the matrix.
The solvents are then evaporated in a vacuum of the
MS, and the matrix crystallizes with the analyte
incorporated.
30

31. MALDI Matrix
 The matrix must meet the following properties and
requirements:
Be able to embed and isolate analytes (e.g. by co-
crystallization)
Be soluble in solvents compatible with analyte
Be vacuum stable
Absorb the laser wavelength
Cause co-desorption of the analyte upon laser
irradiation
Promote analyte ionization
31

32. 32
Some examples of matrix in MALDI

33. MALDI
• The mechanism remains
uncertain
• It may involve absorption
of light by the matrix
• Transfer of this energy to
the analyte
– which then ionizes
into the gas phase as a
result of the relatively
large amount of
energy absorbed.
– To accelerate the
resulting ions into a
flight-tube in the mass
spectrometer they are
subjected to a high
electrical field.
33

34.  produce gaseous ionized molecules from a liquid
solution by creating a fine spray of droplets in the
presence of a strong electric field
 one of the most important techniques for analyzing
biomolecules, such as polypeptides, proteins having
MW of 100,000 Da or more
 Generates positive (M+nH)n
+ and negative (M - nH)n
-
ions and almost no fragmentation. Generates multiple
charged ions.
 Easily coupled to HPLC
Electrospray ionization (ESI)
34

35. 35
Assignment!
Mass Analyzers

36. The Nature of Mass Spectra
• Molecular ion - The ion obtained by the loss of one electron
from the molecule (M+)
• Base peak - The most intense peak in the MS, assigned 100%
intensity
• Fragment ions - Lighter cations formed by the decomposition
of the molecular ion. These often correspond to stable
carbcations.
36

37. base peak, m/z 43
37
M+
fragment ions

38. Isotopes
 Mass spectrometers are capable of separating and detecting individual
ions even those that only differ by a single atomic mass unit
 As a result molecules containing different isotopes can be
distinguished
 This is most apparent when atoms such as bromine or chlorine are present
(79Br : 81Br, intensity 1:1 and 35Cl : 37Cl, intensity 3:1) where peaks at "M"
and "M+2" are obtained
 The intensity ratios in the isotope patterns are due to the natural
abundance of the isotopes
 "M+1" peaks are seen due to the presence of 13C in the sample.
38

39. Bromomethane
39

40. 1-Bromopropane
40

41. 2-Chloropropane
41
m/z 43

42. Getting the Formula from the Mass Spectrum
The Nitrogen Rule
if m/z for M is odd, then the molecular formula must have an odd
number of nitrogens.
If m/z for M is even, then the molecular formula must have an
even number of nitrogens (this includes 0).
For 1-bromopropane, m/z for M=122. The even number is in
accordance with the even number of nitrogens in the formula
(zero).
42

43. 43
The Hydrogen Rule
the maximum number of hydrogens in the molecular formula
is 2C+N+2.
C = # of carbons,
N = # of nitrogens
EX: For CH3CH2CH2Br, there are three carbons, so the max #
of hydrogens is 2(3)+2=8

44. The “Rule of 13” as an aid to guessing a molecular
Formula
Take the Weight of ion, divide by 13
This answer is N, for (CH)N and any numerical remainder is added as H
e.g.; 92. when 92/13 = 7 with remainder = 1; C7H8 weighs 92. This is our
candidate formula.
Can evaluate other alternative candidate formulas possessing heteroatoms.
For each member of the list below, replace the indicated number of CHs in the
above answer
Hetero
substitution
CH
replacement
Hetero
substitution
CH replacement
O CH4 P C2H7
N CH2 S C2H8
O+N C2H6 O+S C4
F CH7 I C10H7
Si C2H4 Cl, Br (use isotopes) 44

45. Degree of unsaturation (One Double Bond Equivalent) is one pi bond or one ring
A triple bond counts as 2 DBE
Having 4 DBE indicates the possibility of a benzene ring. The formula for DBE is
the following:
Important Note: DBE can never be negative and fractional
EX: For CH3CH2CH2Br, the DBE equals 3-(8/2)+(0/2)+1=0. (No pi bonds, no rings.)
45

46. 46

47. Molecular ion
 Three facts:
 The peak must correspond to the highest mass ion on
the spectrum excluding the isotopic peaks
 The ion must have an odd number of electrons –
usually a radical cation
 The ion must be able to form the other fragments on
the spectrum by loss of logical neutral fragments
47

48. Fragmentation
 The time between ionization and detection in most mass
spectrometer is 10-5 sec.
– If a particular ionized molecule can “hold together”
for greater than 10-5 sec. a M+ ion is observed
– If a particular ionized molecule fragments in less
than this time, the fragments will be observed
48

49. Fragmentation – Chemistry of Ions
• One bond s-cleavages:
a. cleavage of C-C
b. cleavage of C-heteroatom (i-cleavage)
C C C C
+
C Z C Z
+
49

50. Fragmentation – Chemistry of Ions
• One bond s-cleavages: a-cleavage of C-heteroatom
C C Z C C Z
+
C C Z C C Z
+
C C Z C Z
+ C
50

51. Fragmentation – Chemistry of Ions
 Two bond s-cleavages/rearrangements:
a. Elimination of a vicinal H and heteroatom:
b. Retro-Diels-Alder
C C Z Z
+ H
H
C C
+
51

52. Fragmentation – Chemistry of Ions
McLafferty Rearrangement
Abbreviated: H
+
H
52

53. Fragmentation – Chemistry of Ions
 When deducing any fragmentation scheme:
The even-odd electron rule applies: “thermodynamics dictates
that even electron ions cannot cleave to a pair of odd electron
fragments”
The order of carbocation/radical stability is benzyl/3° >
allyl/2° > 1° > methyl > H * the loss of the longest carbon
chain is preferred
Fragment ion stability is more important than fragment radical
stability
53

54. Fragmentation Patterns of Groups
 Alkanes
Apply the stability of carbocations (or radicals)
This is governed by Stevenson’s Rule – the fragment with the
lowest ionization energy will take on the + charge – the other
fragment will still have an unpaired electron
Example: iso-butane
CH3
+
CH3
+
54

55. For straight chain alkanes, a M+ is often observed
Ions observed: clusters of peaks CnH2n+1 apart from the loss of –
CH3, -C2H5, -C3H7, etc.
Fragments lost: ·CH3, ·C2H5, ·C3H7, etc.
In longer chains – peaks at 43 and 57 are the most common
55

56. Mass spectrum of n-heptane
43
M+
57
56

57. Mass spectrum of 2,2-dimethylhexane
57
57
M+ 114

58. Alkenes
Ions observed: clusters of peaks CnH2n-1 apart from -C3H5, -C4H7, -
C5H9 etc. at 41, 55, 69, etc.
Terminal alkenes readily form the allyl carbocation, m/z 41
R
H2
C
+
R
C
H
CH2 H2C C
H
CH2
58

59. (E) 2-Hexene
59

60. cycloalkenes
 Retro-Diels-Alder is significant: observed loss of 28
+
60

61. Mass spectrum of 1-methyl-1-cyclohexene
 Side chains are easily fragmented
M+ 96
81
68
61

62. Alkynes – Fragment Ions
 For terminal alkynes, the loss of terminal hydrogen is observed
(M-1)
 Terminal alkynes form the propargyl cation, m/z 39 (lower
intensity than the allyl cation)
R
H2
C
+
R
C CH H2C C CH
62

63. Mass spectrum of 1-pentyne
M+ 68
H
67
H
39
63

64. Aromatic Hydrocarbons – Fragment Ions
 Very intense molecular ion peaks and little fragmentation of the ring
system are observed
 Where alkyl groups are attached to the ring, a favorable mode of
cleavage is to lose a H-radical to form the C7H7
+ ion (m/z 91)
 This ion is believed to be the tropylium ion; formed from
rearrangement of the benzyl cation
CH2
CH3
75 eV e-
64

65. Toluene
CH3
+
.
m/z = 92
loss of H
. CH2
m/z = 91
+
+
tropylium ion
m+
m-1
65

66. Propylbenzene
CH2CH2CH3
+
.
loss of
CH2CH3
.
CH2
+
+
m/z = 91
m/z = 120
m+
m-29
66

67. Isopropylbenzene
C
H3C CH3
H
+
.
loss of
CH3
.
CHCH3
+ CH3
+
m/z = 105
m/z = 120
m+
m-15
67

68. McLafferty Rearrangements in Alkyl Benzenes


a
CH2
CH2
CHCH3
H
m+ 134
loss of CH
3CH=CH2
.
+
CH2
H
H
+
.
m/e 92
CH2
+
m/e 91
- propyl
.
68

69. Alcohols– Fragment Ions
 The largest alkyl group is usually lost; the mode of cleavage typically
is similar for all alcohols:
primary
secondary
tertiary
OH
H2C
O H
+
O H
+
OH O H
+
OH
m/z
31
59
45
69

70. Alcohols – 2-pentanol
M+ 88
OH
45
70

71. Alcohols– Fragment Ions
 Dehydration (M - 18) is a common mode of fragmentation
 For longer chain alcohols, a McLafferty type rearrangement can
produce water and ethylene (M - 18, M - 28)
O
H
R H O
H
R
H
+
71

72. Ethers– Fragment Ions
 The largest alkyl group is usually lost to a-cleavage; the
mode of cleavage typically is similar to alcohols:
 Cleavage of the C-O bond to give carbocations is
observed where favorable
R
H2
C O R R H2C O R
+
R
H
C O R R CH O R
R
R
+
72

73. Ethers– Fragment Ions
 Aromatic ethers can generate the C6H5O+ ion by loss of
the alkyl group rather than H; this can expel CO as in
the phenolic degradation
O
R
O
R + C O + C5H5
+
73

74. Example MS: ethers – butyl methyl ether
M+ 88
O
45
74

75. Example MS: ethers – anisole
M+ 108
O
93
O
77
75

76. Aldehydes - Fragment Ions
a-cleavage is characteristic and often diagnostic for aldehydes – can
occur on either side of the carbonyl
-cleavage is an additional mode of fragmentation
R H
O
R C O + H
R H
O
H C O
+
R
M-1 peak
m/z 29
H
O
+
R
R
H
O m/z R+
M - 41
can be R-subs.
76

77. Aldehydes - Fragment Ions
d) McLafferty rearrangement is observed if -Hs present
e) Aromatic aldehydes – α-cleavages are more favorable, both to lose H· (M -
1) and HCO· (M – 29)
m/z 44
m/z R+
Remember:
aromatic ring
can be subs.
+
O
H
H
R
O
H
R
C O + H
H
O
O
H
+
O
H
77

78. Ketones - Fragment Ions
a-cleavage can occur on either side of the carbonyl – the
larger alkyl group is lost more often
R R1
O
R C O + R1
R1 is larger than R
M – 15, 29, 43…
m/z 43, 58, 72, etc.
78

79. Ketones - Fragment Ions
 McLafferty rearrangement is observed if -H’s present
 Aromatic ketones – a-cleavages are favorable primarily to lose R· (M – 15,
29…) to form the C6H5CO+ ion, which can lose CO
Remember:
aromatic ring
can be subs.
+
O
R1
H
R
O
H
R
R1
C O + R
O
R
+ C O
m/z 105
m/z 77 79

80. ketones (aromatic) – propiophenone
M+ 134
C O
O
m/z 105
m/z 77
80

81. Esters
Most important a-cleavage reactions involve loss of the
alkoxy- radical to leave the acylium ion
The other a-cleavage (most common with methyl esters,
m/z 59) involves the loss of the alkyl group
R
R1
O
R C O + OR1
O
R
R1
O
R C
O
+
O
O R1
81

82. Esters - Fragment Ions
McLafferty occurs with sufficiently long esters
R1
O
+
O
H
R1
O
O
H
82

83. Esters - Fragment Ions
 One interesting fragmentation is shared by both benzyloxy esters and
aromatic esters that have an ortho-alkyl group
O
O
H
OH
fragmentation
+
CH2
C
O
ketene
O
R
O
C
H2
H
C
HO
R
O
CH2
+
benzyloxy ester
ortho-alkylbenzoate ester
83

84. Esters (benzoic) – methyl ortho-toluate
M+ 150
C
O
CH2
O
O
119
O
O
91
m/z 118
84

85. Carboxylic Acids - Fragment Ions
 Most important a-cleavage reactions involve loss of the hydroxy-
radical to leave the acylium ion
 The other a-cleavage (less common) involves the loss of the alkyl
radical. Although less common, the m/z 45 peak is somewhat
diagnostic for acids.
R
H
O
R C O + OH
O
R
H
O
R C
O
+
O
O H
85

86. Carboxylic Acids - Fragment Ions
McLafferty occurs with sufficiently long acids
aromatic acids degrade by a process similar to esters, loss of the HO·
gives the acylium ion which can lose CO:
H
O
+
O
H
H
O
O
H
m/z 60
H
O
H
O
C
O
+
+ further loss of
CO to m/z 77
86

87. Carboxylic Acids - Fragment Ions
 As with esters, those benzoic acids with an ortho-alkyl group will lose
water to give a ketene radical cation
O
H
O
C
H2
H
C
HO
H
O
CH2
+
ortho-alkylbenzoic acid
87

88. 88
Carboxylic acid
O
Ch3(CH2)4CO (small) 99
CH3(CH2)4 71
CH3(CH)3 57
CH3(CH2)2 43
CH3CH2 29
CH3 CH2 CH2 CH2 CH2 C OH
45 CO2H
59(small) CH2CO2H
73 (CH2)2CO2H
87 (CH2)3CO2H

89. Carboxylic acids (aromatic) – p-toluic acid
M+ 136
OH
O
119
OH
O
91
89

90. Amines
 Follow nitrogen rule – odd M+, odd # of nitrogens
 a-cleavage reactions are the most important
fragmentations for amines; for 1° n-aliphatic amines
m/z 30 is diagnostic
R
C
N R
C
N
+
90

91. Example MS: amines, 1° – pentylamine
M+ 87
NH2
30
91

92. Amides
 Follow nitrogen rule – odd M+, odd # of nitrogens;
 a-cleavage reactions afford a specific fragment of m/z
44 for primary amides
 McLafferty observed where -hydrogens are present
R
C
NH2
O
R + O C NH2
m/z 44
O
NH2
H
O
NH2
H
+
92

93. Example MS: amides (aromatic) – benzamide
M+ 121
C
NH2
O
77
C
NH2
O
105
93

94. Benzamide
94

95. Nitriles - Fragment Ions
 Follow nitrogen rule – odd M+, odd # of nitrogens
 Principle degradation is the loss of an H-atom (M – 1) from a-
carbon:
 Loss of HCN observed (M – 27)
 McLafferty observed where -hydrogens are present
H +
R
H2
C C N R C
H
C N
C
N
H
H2C
C
N
H
+
m/z 41
95

96. nitriles – propionitrile
M+ 155
M-1 54
- HCN (m/z 27)
96

97. Halogens - Fragment Ions
 Fluoro- and iodo-compounds do not have appreciable
contribution from isotopes
 Chloro- and bromo-compounds are unique in that they will show
strong M+2 peaks for the contribution of higher isotopes
 For chlorinated compounds, the ratio of M+ to M+2 is about 3:1
 For brominated compounds, the ratio of M+ to M+2 is 1:1
97

98. Halogens - Fragment Ions
 Principle fragmentation mode is to lose halogen atom,
leaving a carbocation
 Loss of HX is the second most common mode of
fragmentation
R + X
R X
R +
C X
C
R
H
H H
H
C
H
CH2 H X
98

99. Example MS: chlorine – 1-chloropropane
M+ 78
m/z 49, 51
43
Cl
H2C Cl
M+2
99

100. Example MS: bromine -p-bromotoluene
M+ 170
M+2
91
Br
100

101. Example MS: iodine – iodobenzene
M+ 204
77
I
101

102. Hyphenated Mass Techniques
m/z
15
29
43
57
85
99 113 142
71
Mass: Detection
Chromatography-Mass Spectroscopy :
Separation + Detection
GC-MS LC-MS CZE-MS
Chromatography: Separation
102
Capillary Zone Electrophoresis

103. GC-MS
Sample
Sample
5890
1.0
DEG/MIN
HEWLETT
PACKARD
HEWLETT
PACKARD
5972A
Mass
Selective
Detector
D C
B
A
A
B
C
D
Gas Chromatograph (GC) Mass Spectrometer
Separation Identification
B
A C
D
A
D
B
C
MS
Gas chromatography-mass spectrometry (GC-MS) is a method that combines
the features of gas-liquid chromatography and mass spectrometry to identify
different substances within a test sample.
103

104. Gas Chromatography-Mass Spectrometry (GC-MS)
 To analyze a urine sample for tetrahydrocannabinol, (THC) the principle
psychoactive component of marijuana, the organic compounds are extracted from
urine, purified, concentrated and injected into the GC-MS
 THC appears as a GC peak, and gives a molecular ion at 314, its molecular weight
104

105. Tandem Mass Spectrometry
Tandem mass spectrometry, also known as MS/MS, involves multiple
steps of mass spectrometry selection, with some form of fragmentation
occurring in between the stages.
105

106. 106
Exercise

107. 107

108. 108

109. 109