spectroscopy-Mass spectroscopy-peinciple,applications

1
Mass spectroscopy
University College of Pharmaceutical Sciences, Kakatiya University,
Warangal, Telangana

2
 Mass spectrum- a graph of the number of ions
detected as a function of their m/z ratio.
University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana

Principle
3
 To measure relative molecular masses.
 To know the fragmentation of the molecules.
 Comparision of mass spectra with known
compounds.
University College of Pharmaceutical Sciences, Kakatiya University, Warangal,
Telangana

Organic molecules are bombarded with
electron
Converted into highly energetic positively charged
ions(Molecular ions or parent ions)
Further break up into smaller ions( fragment ions or
Daughter ions)
The formed ions are separated by Deflection in
magnetic field according to their Mass and Charge
Mass spectrum
4 University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana

5
Loss of electron from a molecule leads to radical cation.
M e-_ M.+ +e_
radical cation
70eV
Fragments
Cations radicals neutral molecules radical cations
Molecular
ion
15eV
Telangana

Instrumentation
6
 Inlet system
 Ion source
Ionization methods
 Mass analyser
 Ion detector
 Data system

Inlet system
7
 Solid samples with lower vapour pressure
 directly inserted into the ionization chamber and volatilization
is controlled by heating the probe.
 Liquids are handled by hypodermic needles
 Injection through a silicon rubber dam
 Gases are leaked into the ionization chamber
 directly by the help of mercury manometer

Ion source
8
 The minimum energy required to ionise an atom or a molecule is called
Ionization potential.
 The common technique used for the production of ion in mass spectrometer
is by the bombardment of electrons. The bombarding electrons are produced
from an electrically heated tungsten filament.
 Operating pressure 10-6mm.
AB (g)+e AB*+e
AB++2e (most propable)
AB +n + (n+1).e (least propable)
A++B-+e
A++B+2e
AB- (electron capture-less propable)

Ionization methods
9
 Electron ionization (EI)
 Chemical Ionization (CI)
 Field Desorption (FD)
 Fast Atom bombardment(FAB)
 Electrospray Ionization (ESI)
 Matrix Assisted Laser Desorption/Ionization (MALDI)

10
Electron ionization (EI)
 In ES-MS, a beam of high- energy electrons is emitted from a filament
that is heated to several thousand degree Celsius.
 These high- energy electrons strike the stream of molecules that has been
admitted from the sample inlet system.
 The electron- molecule collision strips an electron from the molecule,
creating a cation.
 A repeller plate, which carries a positive electrical potential, directs the
newly created ions toward a series of accelerating plates.
 A large potential difference, ranging from 1 to 10 kilovolts (kV), applied
across these accelerating plates produces a beam of rapidly traveling
positive ions.
 One or more focusing slits direct the ions into a uniform beam.
 The energy required to remove an electron from an atom or molecule is its
ionization potential or ionization energy.

11
Electron ionization (EI)

12
Chemical Ionization(CI)
 In CI-MS, the sample molecules are combined with a stream of ionized
reagent gas that is present in great excess relative to the sample.
 When the sample molecules collide with the pre ionized reagent gas, some of
the sample molecules are ionized by various mechanisms, including proton
transfer, and adduct formation.
 Common ionizing reagents for CI-MS include methane, ammonia, isobutane
and methanol.
 The vaporized sample is introduced into the mass spectrometer with an
excess of a reagent gas ( methane) at pressure of about 1 torr.
 The excess carrier gas is ionized by electron impact to the primary ions
 These may react with the excess methane to give secondary ions.

CH4
.+
+ CH4
CH5
+
and CH3.
CH3
+
+ CH4
C2H5
+
and H2
CH4 + C2H5
+
C3H5
+
+ H2
CH3
+
+ M [M
+
H]+ + CH4
C2H5
+
+ M [M
+
H]+ + C2H4
13
These react with excess methane to give secondary ions.
The secondary ions react with the sample(M)

Field Desorption(FD)
15
 Stable molecular ions are obtained from a sample of low
volatility, which is placed on the anode of a pair of electrodes,
between which there is an intense electric field.
 Desorption occurs, and molecular and quasimolecular ions are
produced with insufficient internal energy for extensive
fragmentation.
 Usually master peak is represented by [M+H]+ ion.
 Synthetic polymers with molecular weights on the order of
10,000Da have been analyzed, but there is a much lower
molecular weight limit for polar biopolymers; here FAB
procedure and others are more superior.

16
Field Desorption(FD)

Fast Atom Bombardment (FAB)
17
 Polar molecules such as peptides, with molecular weights up to 10,000 Da can be
analyzed by a “soft” ionization techniques called fast atom bombardment (FAB).The
bombarding beam consists of xenon (or argon) atoms of high translational energy
(Xe). This beam produced by first ionizing xenon atoms with electrons to give xenon
radical cations:
Xe Xe.+ +2e
The radical cations are accelerated to 6-10 keV to give radical cations of high
translational energy (Xe).+which are then passed through xenon. During this passage,
the charged high energy xenon obtains electrons from the xenon atoms to become
high energy atoms(Xe), and the Xe.+ ions are removed by an electric field.
Xe.+ Xe.+
Xe.+ +Xe Xe+ Xe.+
The compound of interest is dissolved in a high boiling viscous solvent such as
glycerol; a drop is placed on a thin metal sheet, and the compound is ionized by the
high energy xenon atoms(Xe).Ionization by translational energy minimizes the
amount of vibrational excitation, and this results in less destruction of the ionized
molecules.The polar solvent promotes ionization and allows diffusion of fresh
sample to the surface.

18
Fast Atom Bombardment (FAB)

Electrospray Ionization (ESI)
19
 ESI involves placing an ionizing voltage-several kilovolts-
across the nebulizer needle attached to the outlet from a high
performance liquid chromatograph (HPLC).
 This technique is used for water- soluble bio molecules-
proteins, peptides and carbohydrates in particular.
 The result is a spectrum whose major peaks consist of the
molecular ion with a different number of charges attached.
 A molecular ion of, for example, about 10,000 Da with a
charge (z) of 10 would behave in a mass spectrometer as
though its mass were about 1000 daltons. Its mass, therefore,
can be determined with a spectrometer of modest resolution-
and cost.

20
Electrospray Ionization (ESI)

21

22
Professor John B. Fenn

Atomic pressure ionization(API)
23
 It is a simple variation of ESI.
 It is applied to the outlet of an HPLC unit attached to the inlet of
the mass spectrometer.
 These variations have in common the formation of a very fine
spray (nebulization) from which the solvent can be quickly
removed.
 The small particles are then ionized by a corona discharge at
atmospheric pressure and swept by the continuous flow of the
particles and a small electric potential that moves the positively
charged particles through a small orifice into the evacuated mass
spectrometer.

24
Atomic pressure ionization(API)
Warangal, Telangana

Matrix Assisted Laser desorption/Ioniztion
(MALDI)
25
 It is mainly used for large bio molecules.
 The sample in a matrix is dispersed on a surface, and is desorbed and
ionized by the energy of a laser beam.
 The matrix serves the same purpose as it does in the FAB procedure.
 This procedure is recently used in several variations to determine the
molecular weight of large protein molecules- up to several hundred kDa.
 The combination of a pulsed laser beam and a time –of flight mass
spectrometer.
 Used in peptide sequencing.
 Matrix selection is critical and depends on the wavelength of the laser beam
and on the nature of the sample. Such as polar compounds as carboxylic
acids (e.g. nicotinic acid), urea and glycerol.
Warangal, Telangana

26
Matrix Assisted Laser desorption/Ionization
(MALDI)

27
Ionizing agents in Mass spectrum
Name and Acronym Ionizing agents Compounds Mass range
Electron Impact (EI) Energetic
electrons
Thermally volatile and
stable
500Da
Chemical Ionization (CI) Reagent gaseous Thermally volatile and
stable
500Da
Fast atom bombardment (FAB) Energetic atomic
beam
Peptides 7000Da
Field Ionization (FI) High –potential
electrode
Thermally volatile 1000Da
Field Desorption (FD) High –potential
electrode
Biopolymers 10000Da
Electospray ionization (ESI) High electrode
field
Polar and basic 7000Da
Matrix –assisted Laser
desorption ionization(MALDI)
Laser beam Laser bio molecules 3,00,000Da
Atomic Pressure Ionization
(API)
Energetic atomic
beam
Thermally liable 1000Da

28
Mass analyser
Dempster’s kinetic energy
1/2.mV2=eV --------------(1)
Where ,
v=velocity of the ions after acceleration
V=potential applied
From Newton’s second law of motion
H eV=mv2/r --------------(2)
Squaring both sides
H2e2v2=m2v4/r2
H2e2=m2v2/r2 --------------(3)
But ½ mv2= eV
mV2=2eV
Putting the value of mv2 in (3)
H2e2=m,.2eV/r2 or H2e= 2mV/r2
Or m/e= H2r2/2V

29
The mass spectrum can be obtained either by
(i) Changing H at constant V
or
(ii) Changing V at constant H

mass analyzers
30
 Double –focusing mass analyzer
 Quadrapole mass analyzer
 Quadrapole Ion storage (Ion trap)
 Time-of- flight mass analyzer
Warangal, Telangana

Double –focusing mass analyzer
31
 The introduction of an electrostatic field after the
magnetic field permits high resolution so the mass of a
particle can be obtained to four decimal places.
 Ions generated in the source are accelerated toward the
analyzer.
 The magnetic field provides directional focusing.
 The path of the positive ion is again curved by the
electric field applied perpendicular to the flight path of
the ions.
 This double focusing provides resolution as high as
60,000.

32
Double –focusing mass analyzer

Quadrapole mass analyzer
33
 This mass filter uses four voltage- carrying rods.
 Ions entering from one end travel with constant velocity in the direction
parallel to the poles (z direction), but acquire complex oscillations in the x
and y directions by application of both a direct current (dc) voltage
(Vdc)and a radiofrequency (rf) voltage (V rf) to the poles.
 There is a “stable oscillation” that allows a particular ion to pass from one
end of the quadrapole to the other without striking the poles; this
oscillation is dependent on the m/z ratio of an ion.
 Therefore, ions of only a single m/z value will traverse the entire length of
the filter at a given set of conditions.
 All other ions will have unstable oscillations and will strike the poles and
be lost.
 Mass scanning is carried out by varying each of rf and dc frequencies
keeping their ratios constant.

34
Quadrapole mass analyzer

35

+
+
+
- -
36

+
+
+
- -
37

+
+
+
- -
Splat
38

+
-
-
+ +
39

+
-
-
+ +
40

Quadrapole Ion storage (Ion trap)
41
 The ion storage trap is a spherical configuration of the linear quadrapole
mass filter.
 The operations are differ in that the linear filter passes the sorted ions
directly through to the director, whereas the ion trap retains the unsorted
ions temporarily within the trap. They are then released to the director
sequentially by scanning the electric field.
 These instruments are compact (bench top), relatively inexpensive,
convenient to use, and very sensitive. They also provide an inexpensive
method to carry out GC/MS/MS experiments.
 In general, the quadrapole instruments do not achieve the mass range
and the high resolution of the sector instruments.
 However, the mass range and resolution are adequate for unit-resolution
mass spectrometry, and the rapid scan and sensitivity make them
especially suitable for use with capillary gas chromatography.

42
Quadrapole Ion storage (Ion trap)

Time-of- flight mass analyzer
43
 In the time-of-flight (TOF) mass spectrometers, all singly
charged particles subjected to a potential difference V attain
the same translational energy in electron volts (eV).
 Thus lighter particles have the shorter TOF over a given
distance.
 The accelerated particles are passed into a field –free region
where they are separated in time by their m/z values and
collected.
 Since arrival times between successive ions can be less than
10-7s, fast electronics are necessary for adequate resolution.
 Time-of –flight devices are used with sophisticated ionizing
methods(FAB, laser desorption, and plasma desorption)

44
Time-of- flight mass analyzer

Detectors
45
 Faraday cup
 Electron Multiplier
 Photomultiplier

46
Faraday cup detector
 The basic principle is that the incident ion strikes the dynode surface
which emits electrons and induces a current which is amplified and
recorded.
 The dynode electrode is made of a secondary emitting material like
CsSb,GaP or BeO.
 It is ideally suited to isotope analysis

Electron multipliers
47
 Electron multipliers are the most common especially when
positive and negative ions need to be detected on the same
instrument.
 Dynodes made up of copper-beryllium which transduces the initial
ion current and electron emitted by first dynode are focused
magnetically from dynode to the next.
 Final cascade current is amplified more than million times.

48
Warangal, Telangana

Photomultiliers
49
 The dynode consists of a substance ( a scintillator) which emits
photons (light).
 The emitted light is detected by photo multiplier tube and is
converted into electric current.
 These detectors are useful in studies on metastable ions.

Types of peaks
50
 Molecular ion peak
 Fragment ion peak
 Rearrangement ion peak
 Metastable ion peak
 Multicharged ion peak
 Base peak
 Negative ion peak

51
 Molecular ion peak:
When a sample is bombarded with electrons of 9 to 15eV
energy, the molecular ion is produced, by loss of single electron.
M M+ + 2e_
 Fragment ions peak:
When an energy is given further more upto 70 eV, fragment ions
produced, it have lower mass number.
 Rearrangement ion peak:
Recombination of fragment ion is known as rearrangement
peaks
 Metastable ion peaks:
The ions resulting from the decomposition between the source
region and magnetic analyser are called as Metastable ions. These
appear as broad called metastable ion peaks.

52
 Multicharged ions:
Ions may exist with 2 or 3 charges instead of usual single charge.
The peaks due to these charged ions are known as Multicharged ion
peaks.
 Base peak:
The largest peak in the mass spectrum corresponding to the most
abundant ion or most intense peak in the spectrum is called as base
peak.
 Negative ion peak:
Negative ions are formed from electron bombardment of sample.
These results due to the capture of electron by a molecule during
collision of molecules.

Mass interpretation
53
 Fragmentation rules
 Mc Lafferty rearrangement
 Alpha cleavage
 Beta cleavage
 Nitrogen rule
 Retro diel’s alder reaction
 IHD

Fragmentation rules
55
 Rule 1:
The relative height of the molecular ion peak is greatest for the
straight –chain compound and decreases as the degree of
branching increases.

56
 Rule2:
The relative height of the molecular ion peak usually
decreases with increasing molecular weight in a homologous
series.
Fatty esters appear to be an exception.

57
Rule 3:
Cleavage is favored at alkyl- substituted carbon atoms; the
more likely is cleavage. This is a concequence of the increased
stability of a tertiary carbocation over a secondary which in turn
is more stable than a primary.
Cation stability order:
Generally, the largest substituent at a branch is eliminated
most readily as a radical, presumably because a long-chain
radical can achieve some stability by delocalization of the lone
electron.

58
Rule 4:
Double bonds, cyclic structures, and especially aromatic (or
hetero aromatic) rings stabilize the molecular ion and thus increase the
probability of its appearance.

59
 Rule 5:
Double bonds favors allylic cleavage and give the
resonance-stabilized allylic carbocation. This rule does not
hold for simple alkenes because of the ready migration of the
double bond, but it does hold for cycloalkenes.

60
 Rule 6:
Saturated rings tend to lose alkyl side chains at the α bond.
This is merely a special case of branching (rule 3). The
positive charge tends to stay with the ring fragment.
Unsaturated rings can undergo a retro-Diels –Alder reaction

61
 Rule 7:
In alkyl- substituted aromatic compounds, cleavage is
very probable at the bond β to the ring, giving the resonance-
stabilized benzyl ion or, more likely, the tropolium ion.
Eg. Mass spectra of n-butyl benzene

62
 Rule 8:
The C-C bonds next to a heteroatom are frequently
cleaved, leaving the charge on the fragment containing the
heteroatom whose nonbonding electrons provide resonance
stabilization.

63
 Rule 10:
Cleavage is often associated with elimination of small, stable,
neutral molecules, such as carbon monoxide, olefins, water,
ammonia, hydrogen sulfide, hydrogen cyanide, mercaptans,
ketenes, or alcohols, often with rearrangement.
Eg. McLafferty rearrangement

McLafferty rearrangement
64
 McLafferty arrangement can occur in ketones, aldehydes,
carboxylic acids and esters.
 In this rearrangement a radical center in molecular ion derived
from a lone pair or pi bond, removes hydrogen from the
Gamma position (y), a pi bond is formed between the β and y
position, and the bond between the α and β positions is
broken.

65
C
H3
O
R
C
H2
OH
+.
+ C
H2 CH2
C
H3
CH2
C
H3
CH2
+.
+ C
H2 CH2
CH3
O
CH2
+
O
H
CH2
CH3
]+.
Warangal, Telangana

66
Double Mc Latterfty Rearrangement
C
H3
O
R
C
H2
OH
+.
+ C
H2 CH2
C
H3
CH2
C
H3
CH2
+.
+ C
H2 CH2
CH3
O
CH2
+
O
H
CH2
CH3
]+.

Αlpha cleavage: Radical-site-initiated
cleavage
67
 Alpha cleavage in mass spectrometry is a characteristic
fragmentation of the molecular ion derived from carbonyl
compounds, in which the bond linking the carbonyl carbon to
the atom occupying an alpha position breaks.
 It is an expected pathway for carbonyl compounds, ethers,
halides, alcohols and amines.
 Write the reaction

Charge- site- initiated cleavage:
Inductive cleavage
68
 Inductive cleavage involves the attraction of an
electron pair by an electronegative heteroatom that
ends up as a radical or as a closed-shell neutral
molecule. While α-cleavage is a fragmentation of
OE+ only, inductive cleavage can operate on OE+
or an EE+.

Βeta cleavage
69
 Beta cleavage in mass spectrometry is a characteristic
fragmentation of the molecular ion derived from some organic
compounds, most notably alcohols, ethers and amines, in
which the bond connecting alpha- and beta- carbons break.

Retro-diels- Alder reactions
70
 Unsaturated six-membered rings can undergo a retro Diels-
Alder fragmentation to produce the radical cation of a diene
and a neutral alkene- the hypothetical precursors to the
cyclohexene derivative if it had been prepared in the forward
direction via the [4pi+2pi] diene + dienophile cycloaddition
known as Diels Alder reaction.
C
H2 CH2
+ C
H2
CH2
-e + +

Two- bond cleavage
71
 In this process, an elimination occurs, and the odd – electron
molecular ion yields an OE+ and an even - electron neutral
fragment N, usually a stable small molecule of some type
:H2O, a hydrogen halide, or an alkene.

Alkanes
72
 The relative height of the parent peak decreases as the
molecular mass increases in the homologous series.
 Groups of peaks in the mass spectrum are observed 14 mass
units apart. The most abundant peaks correspond to CnH2n+1
ion.
 The most intense peaks are due to C3 and C4 ions at m/e 43 and
m/e 57 respectively.
 There is no preferred charge stabilization site to favor any
specific cleavage.
 The peaks corresponding to CnH2n+1 ions are also accompanied
by CnH2n
+ and CnH2n-1
+ ions in much less abundance.

73
Mass spectrum of dodecane
C3H7
+
C4H9+
C2H5
+
C5H11+
C6H13+
C7
C8 C9 C10

Branched chain alkanes
74
 Bond cleavage takes place preferably at the site of branching. Due
to such cleavage, a more stable secondary or tertiary carbonium
ions results.
 Generally, larger substituent at branch is eliminated readily as a
radical. The radical achieves stability by the delocalization of lone
electron.
 The relative abundance of the parent ion is least and is mostly not
observed.
 Great number of fragments result from a branched chain
compound compared to the straight chain compound. It is due to
greater pathways available for cleavage.
 The signals corresponding CnH2n-1
+ ions follow weak signals
which appear 2 units below them.

75
Mass spectrum of 3,3 dimethyl hexane
C2H5+
C3H7+
C4H9+
C5H11+
C6H13+
C7H15+

Alkene
76
 The molecular ion peak in the spectra of unsaturated compounds is
more intense than the corresponding saturated analogues. The reason
is the better resonance stabilization of the charge on the cation
formed by the removal of one of the π electrons. Mono–olefines
contain CnH2n-1
+ ions in their mass spectra.
 The relative abundance of the molecular ion peak decreases with
increasing molecular mass.
 A cyclic olefines also shows group of peaks which are 14 mass unit
apart.
 The general mode of fragmentation induced by a double bond is the
allylic cleavage.
 The CnH2n ions (fragments) formed by McLafferty rearrangement
are more intense.

Cycloalkanes
77
 The relative abundance of the molecular ion of cycloalkane is
more as compared to the corresponding alkane.
 It favors cleavage at the bond connecting the ring to the rest of
the molecule.
 Fragmentation of the ring is usually characterized by the loss
of two carbon atoms as C2H4
+(28 mass units) and C2H5
+(29
mass units).
 The stability of the fragment ion depends upon the size of the
ring.
 Fragment ions are commonly observed by the loss of alkenes
or alkenyl ions. The side chain on the ring breaks and lone or
odd electron remains on the ring.

78
Mass spectrum of cyclohexane

79
Mass spectrum of naphthalene

80
Mass spectrum of P-Xylene

acetylenes
81
 For 1-Butyne and 2-Butyne, the molecular peak is the base
peak.
 The relative abundance of the molecular ion peak decreases as
the molecular mass of the alkyne increases.
 In alkynes, the fragment ions are generally formed by the loss
of alkyl radicals.
 Thus, M+-15, M+-29 etc. peaks are generally noticed in the
mass spectra of alkynes.

Aromatic compounds
82
 The molecular ion peak in aromatic compounds is fairly
abundant as compared to the corresponding alkanes and
alkenes containing the same number of carbon atoms.
 In aromatic compounds, M++1 and M++2 are also noticed. The
reason is fairly large abundance of the molecular ion peak.
 In case of polynuclear hydrocarbons, doubly or triply charged
(M2+,M3+ ions) are possibly formed. Doubly charged
molecular ions(m/2e)appear at integral m/e values.
 If the aromatic ring is substituted by an alkyl group, a
prominent peak is formed at m/e 91.Hence benzyl (C6H5CH2
+)
cation formed rearranges to tropylium cation.(C7H7
+)

83
 The benzyl cation formed rearranges to more stable tropylium cation
which appears at m/e 91. Tropylium cation in turn loses a molecule
of acetylene to form C5H5+ at m/e 65.
 Cleavage of a carbon-carbon bond which is in the β-position to the
aromatic ring is an energetically favoured fragmentation mode.
CH3
CH3
+
+
-(CH3CH2).
Benzyl cation
rearrangement
m/e 91
m/e 65
loss of -C2H2
]+. ]+.
R
+
rearrangement
m/e 91
]+.
M
+
(parent ion)
tropylium cation

Mass spectrum of toluene
85

86
 The molecular ion peak of primary and secondary alcohol is usually
of low abundance. It is not detected in tertiary alcohols.
 The parent ion peak is formed by the removal of one electron from
the lone pairs on the oxygen atom of primary and secondary
alcohols.
 The number of fragmentation modes in alcohols depend upon the
fact whether it is primary, secondary or tertiary alcohol.
 The fragmentation of carbon-carbon bond adjacent to oxygen
atom(α- cleavage) is the preferred fragmentation mode.
Alcohols

87
 The signal at m/e 31 appears in large abundance in the mass
spectrum of methanol and other aliphatic primary alcohols. The
signal corresponds to the formation of oxonium ion ( CH2= OH+)
and is formed by the cleavage of carbon-hydrogen bond in methanol.
 Primary alcohols show M+-18 peaks corresponding to the loss of
water.
C
H3 OH
C
H2 O
+
H
+. -H.
R
OH
C
H2 O
+
H
-RCH2.
+.
M
+
.
(Primary alcohol)
m/e 31
m/e 31

88
 A primary alcohol having a chain of four or more carbon atoms
shows a peak which corresponds to M+-(18-CnH2n). It can be shown
mechanistically as follows:
 Long chain members may show peaks corresponding to successive
loss of H- radicals at M-1,M-2,M-3. It can be represented as shown.
H O
C
H3
C
H3 H
O
H2 +
C
H3
CH3
+
.
+ C
H3
CH2
.
+
C
H3
CH3
]+.
C
H3
CH3
O
H H
C
H3
O
+
H
C
H3
O
H
C
H3
O
+
(M
+
.)
M
+
-1
M
+
-2
M
+
-3

89
 The CH2=OH+ is the most significant peak in the spectra of
primary alcohols.
 Secondary alcohols cleave to give prominent peaks due to R-
CH=OH+ at m/e 45,59,73….Tertiary alcohols fragment to
give prominent peaks due to RR’C=OH+ at m/e 59,73,87….
 In addition to the α- cleavage, primary alcohols also undergo
β-,γ-,δ- cleavage to form peaks at m/e 45,59, 73…

Mass spectrum of1-Butanol
90

Mass spectrum of2-butanol
91

92
 The relative abundance of the parent ion (M+) of aromatic alcohol is
fairly large.
 Some of the fragment modes of benzyl alcohol are loss of one, two or
three hydrogen atoms.
 The fragment ion, (M+-H) further eliminates CHO radical.
 (M+-H) fragment of benzyl alcohol also rearranges to form hydroxy
tropylium ion.
Aromatic alcohols

93
 The –OH group in the benzylic positions fragments in a way which
favors retention on the aryl group.
OH
OH
C
H3
O
H
C
H3
O
+
C6H5
+ C4H3
+
-[C2H2]
-H.
-CO
+
-H.
Hydroxy tropylium ion
-2H.
(M
+
)
m/e 77
m/e 51

94
 Aliphatic ethers show molecular ion peak almost of negligible
abundance. The presence of oxygen atom in ethers can be
known from strong peaks at m/e 31,45, 59 etc. and these peaks
represent RO+ and ROCH2+ fragments.
 The most characteristic fragmentation mode is the loss of one
of the alkyl groups to form an oxonium ion (RO+) or the alkyl
cation.
 The ions so formed is the result of α- cleavage.
Ether

95
Mass spectrum of Diethyl ether
C
H3
O
CH3
C
H3
O
+
CH3
C
H2 O
+
H
C
H3
O
+
H
m/e 45
m/e 31
m/e 59

Aryl ethers
96
 In case of aromatic ethers, the molecular ion peak is fairly
abundant. Methyl phenyl ethers show two main
fragmentations.
 Primary fission occurs at the bond β- to the ring. Loss of
methyl gives an ion M-15.It further splits to lose carbon
monoxide.
 The fragmentation pattern is shown below:

97
Mass spectrum of anisole
O
CH3
OH
+
-CH3.
]+.
]+
-CO
m/e 93
M
+
.
m/e 108

Aliphatic aldehydes and ketones
98
 The intensity of the molecular ion peak decreases as the alkyl
chain length increases.
 The major fragmentation processes are α and β – cleavage. In
α- cleavage, the bigger group on either side of the carbonyl
group (ketone ) preferably lost.
 In aldehydes and ketones containing γ- hydrogen atom, Mc
Lafferty rearrangement ion is most significant. In an aldehyde,
which is not α- substituted, a peak due to this is formed at m/e
44. it may be base peak.
 The Mc Lafferty rearrangement ion in methyl ketones which
are not α- substituted appears at m/e 58 and is quite abundant.

99
 In lower aldehydes, α-cleavage is prominent with retention of
charge on oxygen.
 In aldehydes, methyl or alkyl radical is preferably lost
compared to hydrogen radical.
C
H3
O
H
+
C
H3
O
+
C
H3
O
+
H
H
O C
H2
H
OH
+
m/e 44
m/e 29
M
+
(m/e 72)
(MR ion) Base peak

Mass spectrum of Pentanal
100

101
 In these compounds, parent ion peak is intense. M+.-1, M+.-28
due to the elimination of CO in benzaldehyde are formed. Peak
at m/e 77 due to C6H5
+ followed by the one at m/e 51 due to
C4H3
+ also result.
 Benzaldehyde
Aromatic aldehydes and ketones
H5C6
O
+
C6H5CHO
+
C6H6
+ C6H5
+
C4H3
+
m/e 51
m/e 77
m/e 78
(M
+
.-1)
(M
+
.) -(C2H2)
-(H)
-(CO)

Warangal, Telangana
102
R
O
O
+
C4H3
+
m/e 51
m/e 77
m/e 105
M
+
(parent ion)
_R.
-CO
_C2H2
]+.
+
In ketones, the loss of larger group is preferably by a
α- cleavage.
Consider the fragmentation of alkyl phenyl ketone:

Mass spectrum of Benzaldenyde
103

Mass spectrum of Benzophenone
104
Telangana

105
 The molecular ion peak in aliphatic acids is less intense as compared
to that of aromatic acids.
 Carboxylic group is directly eliminated by α- cleavage and a signal
is formed at m/e 45.
 If α- carbon atom is not substituted in aliphatic acids containing a γ-
hydrogen atom, a Mc Lafferty rearrangement ion is formed at m/e
60. It is often the base peak.
 In short chain acids, M-OH+. And M-COOH+. Peaks are prominent.
Aliphatic acids
C
H3
OH
O
OH
O
C
H3
O
+
C
H3
CH3
+
m/e 43
m/e 71
m/e 45
(M
+
)
m /e 88
]+
M
-
(OH)
M
+
.-(COOH)

Mass spectrum of Pentanoic acid
106 H
OH
O
C
H2
OH
OH
(M
+
)
(MR ion) (base peak)
m/e 60
H
OH
O
O
OH
C
H2
OH
CH3

Aromatic acids
107
 In aromatic acids, the parent ion parent is intense.
 Some other prominent peaks are M-17+ and M-45+
 If an alkyl group is present or any other hydrogen bearing
group be present ortho to –COOH group, then a signal due to
M-18 (loss of water molecule) is also observed.
 It is called ortho effect.

Esters
108
 The molecular ion peak is weak.
 The fragment ion due to α- cleavage is usually observed.
 In methyl esters, peaks due to R-CO+,R+,CH2O+ and
CH3OCO+(m/e 59) are observed.
 The methyl esters not substituted at the α- carbon atom show
McLafferty rearrangement ion at m/e 74.
 Methyl substitution at α-carbon atom shifts the position of
McLafferty rearrangement peak at m/e 88.
 The molecular ion peak is comparatively more intense. Benzyl
acetate and alkyl acetate eliminate neutral ketene molecule to
form a base peak.

109
C
H3
O
CH3
O
]+.
C
H3 O
O
+
C
H3
CH3
OCH3
+
H
CH3
O C
H2
O CH3
OH
m/e 59
(M
+
- molecular ion)
+
m/e 43
m/e 31

Mass spectrum of Diethyl ether
Warangal, Telangana
110

Amides
111
 The molecular ion peak of straight chain monoamides is usually
discernible.
 The McLafferty rearrangement peak in amides is usually the
base peak.
 The M.R. ion appears at m/e 59.Primary amides give a strong
peak at m/e 44 due to H2N-C=-O+
 A moderate peak at m/e 86 results from γ-δ- carbon cleavage ,
possibly accompanied by cyclisation.
 When the N-alkyl groups on C2 are longer and acyl moiety is
shorter than C3, another mode of cleavage predominates. This is
the cleavage of the N-alky group beta to the nitrogen atom, and
cleavage of the C-N bond with migration of α-H atom of the
acyl moiety.

Mass spectrum of Benzamide
Warangal, Telangana
112

Halogen compounds
113
 The molecular ion abundance of a particular alkyl halide increases
as the electro negativity of the halogen substituent decreases.
 The relative abundance of the molecular ion decreases with increase
in chain length and increasing in branching.
 Compounds containing chlorine and bromine show characteristic
isotope peaks. A compound containing one chlorine atom shows
M+2 peak which is one third in intensity of parent peak.
 A mono bromo compound shows (M+2) peak which is of the same
intensity compared to the parent peak.
 In the parent ion, charge resides on the halogen atom.
 Important fragmentation mode is α- cleavage with charge retention
by the halogen containing fragment. Another mode leads to the loss
of halide radical.

Mass spectrum of Ethyl chloride
114

Mass spectrum of Ethyl bromide
115

Mass spectrum of Dichloroethane
116

Mass spectrum of Dibromonitromethane
117

118
 If the molecular ion is formed at the odd mass number, then
the molecule carries an odd number of nitrogen atoms.
 The molecular ion peak in monoamines is formed in very
small abundance and is undetectable in long chain or
branched chain amines.
 For primary amines, the base peak is formed at m/e 30 due to
CH2=NH2
+.It results from the molecular ion by α- cleavage.
 The parent ion may lose an alkene to form a fragment ion at
M+-CnH2n.
 Loss of largest branch from the α- carbon atom is preferred.
Amines

119
 In higher aliphatic alkyl amines, β- cleavage is not very
significant.γ- cleavage is sometimes preferred.
N
R
R
1
R
2
R
4
R
3
N
+
C
H3
C
H3 CH3
CH3
N
C
H3
C
H3 CH3
CH3
-[R
2
]
+.
here R
2
. R
1
, or R

Nitro compounds
122
 The molecular ion peak in aliphatic nitro compounds is
usually absent but it is prominent in aromatic compounds.
 In aliphatic compounds, the signals due to NO+ and NO2
+ are
usually observed.
 In aromatic nitro compounds, the signals for NO+ and NO2
+
are commonly observed.

Mass spectrum of 1-Chloro-3-
nitrobenzene
123

Aliphatic nitriles
124
 The molecular ion peak of aliphatic nitriles is weak or may be
absent.
 A weak but diagnostically useful (M_1) peak if formed by the
loss a α- hydrogen atom to form a stable ion.
 The base peak of straight chain nitriles between C4 and C9 is
m/e 41.This peak is due to the ion resulting from hydrogen
rearrangement from hydrogen rearrangement in a six
membered transition state.
Telangana

READING A MASS SPEC
125
Fragment Due to loss of… Interpretation
M+• -1 -H• Aldehydes, tert. Alcohols, cyclic
amines
M+• -2 Multiple -H• Secondary alcohols
M+• -3 Multiple -H• Primary alcohols
M+• -4 to -13 (doubtful) Consider contaminants
M+• -14 (doubtful) CH2• , N• not good losses
M+• -15 CH3• Available methyl groups, methylesters
M+• -16 O• Peroxides
M+• -17 OH• Alcohols, phenols, RCO2H
M+• -18 H2O alcohols
M+• -19 -F•
M+• -20 -HF
M+• -21 to -25 No peaks expected
M+• -26 HCCH
M+• -27 •HC=CH2 or HCN HCN from pyridine, anilines
M+• -28 CO or CH2=CH2 Check for McLafferty

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