Mass spectrometry-Fragmentation

pattern in Aromatic compounds

Annamalai University

By
Dr.R.Rajalakshmi
Associate professor
Department of Chemistry
Annamalai University
 UV spectrum
4.000

1
O
3.000

O
O
Abs.

2.000

1.000

-0.100
2

200.00 250.00 300.00 350.00 400.00

nm.
IR Spectrum
H NMR
1
13
C NMR
 Introduction
 Foundation of mass spectroscopy was laid
in 1898, when Wilhelm Wien, a German
physicist, discovered that beams of charged
particles could be deflected by a
magnetic field.
The first mass spectrometer was developed
in 1912 by J.J. Thompson.
Modern techniques of mass spectrometry
were devised by Arthur Jeffrey Dempster and
F.W. Aston in 1918 and 1919 respectively.
 The Nobel prize for physics(1989) was shared
by Dehmalt and W.Paul for the development of
ion trap technique
In 2002 the nobel prize for chemistry was
awarded to Jhon Bennat Fenn for the
development electron spray ionisation source
Mass spectrometry is the most accurate
method for determining the molecular mass of
the compound and its element composition.
 Types of Mass Spectrometry

Accelerator Mass Spectrometry(AMS)

Gas Chromatography-Mass Spectrometry
Liquid Chromatography-Mass Spectrometry
Inductively Coupled Plasma –Mass
Spectrometry(ICP-MS)
Isotope Ratio Mass Spectrometry(IRMS)
Ion Mobility Spectrometry- Mass Spectrometry
MALDI-TOF
SELDI-TOF
Tandem Mass Spectrometry
Thermal Ionization-Mass Spectrometry(TIMS)
Spark source Mass Spectrometry(SSMS)
Types of ionization techniques

Volatile samples

 Electron Ionization
 Chemical Ionization
Non-volatile samples
 Fast Atom Bombardment(FAB)
 Secondary ion Mass Spectroscopy(SIMS)
 Thermos pray
 Matrix Assisted Laser Desorption Ionization (MALDI)
 Electron spray Ionization (ESI)
 Atmospheric Pressure Chemical Ionization(APCI)
 Electron Ionization (EI)
Electron ionization is one of the most
important ionization sources

 for the -small, hydrophobic, thermally stable

molecules

EI usually generates numerous fragment ions,

it is a “hard” ionization source.
Figure EI mass spectrometry.
 Chemical Ionization (CI)

• CI is applied to samples similar to those analyzed by

EI and is primarily used to enhance the abundance of
the molecular ion.
• Chemical ionization uses gas phase ion-molecule
reactions within the vacuum of the mass spectrometer
to produce ions from the sample molecule.
• The chemical ionization process is initiated with a
reagent gas such as methane, isobutane, or ammonia,
which is ionized by electron impact.
 - -
Reagent(R) + e R + 2e
R + RH RH + R
RH + Analyte(A) AH + R
Fast Atom Bombardment
(FAB) -
• It was done by bombarding a sample in a vacuum
with a beam of atoms, typically Ar or Xe, accelerated
to Kilovolt energies.
• The sample was typically mixed in a matrix.
• The two most common matrixes were glycerol and 3-
Nitro-benzoic acid.
Two of the most common matrices used with
FAB are m-nitrobenzyl alcohol and glycerol
 Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS)

• MALDI-MS was first introduced in 1988 by Tanaka, Karas, and

Hillenkamp.
• It has since become a widespread analytical tool for peptides,
proteins, and most other biomolecules (oligonucleotides,
carbohydrates, natural products, and lipids).
• The efficient and directed energy transfer during a matrix-assisted
laser-induced desorption event provides high ion yields of the
intact analyte
• for the measurement of compounds with sub-picomole sensitivity.
• In addition, the utility of MALDI for the analysis of heterogeneous
samples makes it very attractive for the mass analysis of complex
biological samples such as proteolytic digests.
 Electron spray Ionization

• ESI is a method routinely used with peptides,

proteins, carbohydrates, small
oligonucleotides, synthetic polymers, and
lipids .
• ESI produces gaseous ionized molecules
directly from a liquid solution.
• It operates by creating a fine spray of highly
charged droplets in the presence of an electric
field.
 Atmospheric Pressure Chemical
Ionization (APCI)

APCI has also become an important ionization

source
 because it generates ions directly from
solution
capable of analyzing relatively non polar
compounds.
The ionization originates from the solvent
being excited/ionized from the corona
discharge.
 solvent ions are present at atmospheric
pressure conditions
chemical ionization of analyte molecules is very
efficient; at atmospheric pressure,
 analyte molecules collide with the reagent ions
frequently.
Proton transfer (for protonation MH+
reactions) occurs in the positive mode
 Molecular ion or Parent ion
Molecule is bombarded with electrons in high vacuum in Mass
spectrometer, it is converted into positive ions by loss of an
electron.
These ions are called as Molecular or Parent ions.

M + e → M+° + 2e—

The order of energy required to remove electron is as follows

—
σ electrons > non-conjugated π > conjugated π > non bonding
or lone pair of electrons.
 Fragment ion

• CH2
Br Fragmentation
and Br

m/z=170 m/z=91 m/z=79

 Multi charged ions
 . Some times ions may also exist with two or three charges instead
of usual single charge in the mass spectrum. These are known as
doubly or triply charged ions. They are created as follows:
 M+° + e- → M++ + 3e-
 But under normal operating conditions, most of the ions produced
are single charged. The doubly or triply charged ions are recorded
at a half or one third of the m/e value of the single charged ions.
 Formation of these multiple charged ions is more common in
hetero aromatic compounds. They are also common in inorganic
mass spectrum. Gases such as CO , N2,CO2 and O2 have measurable
peaks corresponding to CO+2,N+2,and O+2
 Metastable Ions:
Fragment of a parent ion will give rise to a new ion
(daughter) plus either a neutral molecule or a radical.
M1+ M2+ + non charged particle
An intermediate situation is possible; M1+ may
decompose to M2 + while being accelerated.
The resultant daughter ion M2 + will not be recorded
at either ; M1 or M2, but at a position M* as a rather
broad, poorly focused peak. Such an ion is called a
metastable ion.
 Significance of Metastable ions
Metastable ions are useful in helping to
establish fragments routes.
Metastable ion peak can also be used to
distinguish between fragmentation Processes,
which occur in few microseconds.
 General rules for fragmentation:
The relative height of the molecular ion peak is
greatest for the straight chain compound and
decreases as the degree of branching increases.
The relative height of the Molecular ion peak
usually decreases with increasing molecular
weight in a homologous series.
Cleavage is favoured at alkyl substituted carbon
atoms; the more substituted, the more likely is
cleavage. CH3+ < RCH2+ < R2CH+ < R3C+
 STEVENSONS RULE

When an ion fragments, the positive charge

will remain on the fragment of lowest
ionization potential.
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.
Double bonds favour allylic cleavage -
resonance stabilized allylic carbonium. eg:
Mass spectrum of 1-butene

Saturated rings tend to lose alkyl side chains

at the α carbon atom. This positive charge
tends to stay with the ring fragment.
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 tropylium ion: Ex: mass spectra of n-butyl benzene.

8. Cleavage is often associated with elimination of small, stable, neutral

molecules such as carbon monoxide, olefins, water, ammonia, hydrogen
sulphide, hydrogen cyanide, mercaptans, ketone, or alcohols, often with
rearrangement.
 Factors influencing Fragmentation
process:
1. Bombardment energies
2. Functional groups
 3. Thermal decomposition
 General modes of fragmentation:

Simple cleavage
 1. Homolytic cleavage
 2. Heterolytic cleavage
 3. Retro Diels-Alder reaction

 Rearrangement reactions accompanied by transfer

of atoms.
 1. Scrambling
 2. Mc Lafferty rearrangement
 3. Elimination
 Homolytic cleavage
Here fragmentation is due electron
redistribution bonds.

R H2C CH2 R' •

R CH2• H2C R'

e-

R CH2- H2C R'

2. Heterolytic cleavage
3. Retro Diels-Alder reaction:
 Rearrangement reactions accompanied by
transfer of atoms:
1. Scrambling:

.
Mc Lafferty rearrangement:
 3. Elimination:
Fragmentation due to loss of small molecule:
 Loss of small stable molecules such as H2O, CO2, CO,C2H4 from molecular ion
during fragmentation. Ex- An alcohol readily looses H2O molecule and shows a
peak 18 mass units less than the peak of molecular ion.

Fragmentation adjacent to the branching point:

 In case of branched alkanes, bond fission takes place adjacent to the
branching point. Hence this leads to the formation of more stable carbocation
Ex: 3-methyl pentane
Fragmentation Pattern in
Aromatic Compounds
 Benzene
+
•
-H+
+ Empty sp2 orbital

M / Z=78 M / Z=77

H • H

C
(C4H3+)
C
H
M / Z=51
•
H
 Alkyl benzene

+
H2C CH3 • CH2

-CH3 -C2H2

M/Z=65
M/Z=91
M/Z=106
 Alkyl halide

+
X •

+
-X-
-C2H2 C4H3+

M/Z=51
 + +
• -( HC CH)
C6H5F C4H3F •

M/Z=96 M/Z=70
 Ethers
H2 +
O C H •

+
•
-CH2O -H+ C4H3+

M/Z=77 M/Z=51
 CH3

O O

• + • + ••
-CH3 -C=O
+

-C2H2

C3H3+
 Carbonyl compounds

O+
O + -X
• CO+
C

X
M/Z=105
 O

OH

R CO+

O
M/Z=105
C

OR

Cl

NH2
 H
-CO RDA
+
CO C4H3+ C

M/Z=51 C
M/Z=105
H
 Phenol

+
O H • O
H H
H

-H • -CO -H •
H
M / Z=65
M / Z=66
M / Z =94
 OH
H H
OH
OH
-C O
-H •

H3C H2C
M / Z=108 M / Z=107 M/Z=79

-H 2

-C 2H 2
C4H3
M/Z=51
 Primary amines

NH NH2 •
H H
•
-H• •
-HCN -H

M/Z=92 M/Z=93 M/Z=66 M/Z=65

 Nitro Benzene
- •
O O
•
N O N O O

-N O
Rearrangement

M / Z=123 M / Z=93
M / Z=123
• C6H5O•
NO2 -C O

NO

M / Z=30
-C2H2

M/Z=65

M / Z=77 M / Z=51
3-(2,4-dichlorobenzylideneamino)-2-(2,4-dichlorophenyl)-2,3-dihydro-1,3-
oxazepine-4,7-dione

• Molecuar mass of the compound = 444

• m/z = 446
4-(2-(5-(4-fluorophenyl)-4,5-dihydro-3-(thiophen-2-yl)pyrazol-1-yl)thiazol-
4-yl)-2H-chromen-2-one

S
N N

O O

Molecuar mass of the compound = 473