Mass spectrometry

MASS
SPECTROMETRY
SUBMITTED BY:
PRINCY AGARWAL
M. PHARMA. [QUALITY ASSURANCE]

CONTENTS
 INTRODUCTION
 HISTORY
 PRINCIPLE
 MASS SPECTROMETER
 COMPONENTS OF MASS SPECTROMETER
 ESI [ELECTROSPRAY IONIZATION]
 MALDI [MATRIX ASSISTED LASER SEORPTION IONIZATION]
 FAB [FAST ATOM BOMBARDMENT]
 FIELD DESORPTION
 PLASMA DESORPTION
 APCI [ATMOSPHERIC PRESSURE CHEMICAL IONIZATION]
 APPI [ATMOSPHERIC PRESSURE PHOTOIONIZATION]
 EI [ELECTRON IMPACT IONIZATION]
2

 CI [CHEMICAL IONIZATION]
 FI [FIELD IONIZATION]
 MASS ANALYZERS
 QUADRUPOLE ANALYZER
 MAGNETIC SECTOR ANALYZER
 TIME OF FLIGHT ANALYZER
 QUADRUPOLE ION TRAP ANALYZER
 FTICR ANALYZER
 DETECTORS
 VACUUM SYSTEM
 TYPES OF IONS PRODUCED IN MS
 GENERAL MODES OF FRAGMENTATION
 INTERPRETATION OF MASS SPECTRA 3

INTRODUCTION:
Mass spectrometry is a powerful analytical technique used
to quantify known materials, to identify unknown
compounds within a sample and to elucidate the structure
and chemical properties of different molecules.
It is a microanalytical technique requiring only a few
nanomoles of the sample to obtain characteristic
information pertaining to the structure and molecular
weight of analyte.
This technique basically studies the effect of ionizing energy
on molecules.
Now a day’s mass spectrometry is used in many areas
including pharmaceutical, clinical, geological, biotechnology
and environmental. 4

INTRODUCTION:{cont.}
It depends upon chemical reactions in the gas phase in
which sample molecules are consumed during the
formation of ionic and neutral species.
This will be happened by converting the material to charged
molecules to measure their mass to charge ratio.
Mass spectrometry has both qualitative and quantitative
uses.
 The mass spectrum of each compound is unique and can be
used as a “chemical fingerprint” to characterize the sample.
5

HISTORY:
 Mass spectroscopy was first performed at the Cambridge
university, in 1912 by J.J Thomson (1856-1940) when he
obtained the mass spectra of O2, N2, CO.
 Mass spectroscopy took off in 1930s and advance technology
resulted in the development of double focusing Mass
spectrometers capable of accurate determination.
 The modern techniques of mass spectrometry were devised by
Arthur Jeffrey Dempster and F.W. Aston in 1918 and 1919
respectively.
 In 1989, half of the Nobel Prize in Physics was awarded to Hans
Dehmelt and Wolfgang Paul for the development of the ion trap
technique in the 1950s and 1960s.
 In 2002, the Nobel Prize in Chemistry was awarded to John
Bennett Fenn for the development of electrospray ionization
(ESI).
6

HISTORY: {cont.}
 1934: First double focusing magnetic analyzer was invented by
Johnson and Neil.
 1966: Munson and Field described Chemical Ionization.
 1968: Electrospray Ionization was invented by Dole, Mack and
friends.
 1985: F. Hillenkamp, M. Karas and co-workers describe and
coined the term Matrix Assisted Laser Desorption Ionization
(MALDI).
 1989: W. Paul discovered the Ion Trap Technique. 7

 MASS SPECTROMETER:
It is an instrument in which the substances in gaseous or vapor
state is bombarded with a beam of electrons, to form positively
charged ions (cations) which are further sorted according to their
mass to charge ratio to record their masses and relative
abundances.
 MASS SPECTRUM:
 It is a sorted collection of the masses of all the charged
molecular fragments produced, the relative abundance of each
is the characteristics of every compound.
 The mass spectrum can give detail information about
composition of an organic compound and the position of
functional groups and is also used for the determination of
molecular weight. 8

PRINCIPLE:
In mass spectrometry, organic molecules are bombarded
with a beam of energetic electrons (70 eV) in gaseous
state under pressure between 10-7 to 10-5 mm of Hg,
using tungsten or rhenium filament. Molecules are
broken up into cations and many other fragments.
9

These cations (molecular or parent ion) are formed due
to loss of an electron usually from n or π orbital from a
molecule, which can further break up into smaller ions
(fragment ions or daughter ions).
All these ions are accelerated by an electric field, sorted
out according to their mass to charge ratio by deflection
in variable magnetic field and recorded. The output is
known as mass spectrum.
Each line upon the mass spectrum indicates the
presence of atoms or molecules of a particular mass.
The most intense peak in the spectrum is taken as the
base peak. Its intensity is taken as 100 and other peaks
are compared with it.
10

11
BASIC PRINCIPLE OF MASS SPECTROMETRY

 Mass spectra is used in two general ways:
1) To prove the identity of two compounds.
2) To establish the structure of a new a compound.
 The mass spectrum of a compound helps to establish
the structure of a new compound in several different
ways:
1) It can give the exact molecular mass.
2) It can give a molecular formula or it can reveal the
presence of certain structural units in a molecule.
12

MASS SPECTROMETER:
A mass spectrometer is an instrument which:
 Generates a beam of positively charged ions from the sample under
investigation.
 Produce ions from the sample in the ionization source.
 Separate these ions according to their mass-to-charge ratio in the
mass analyzer.
 Eventually, fragment the selected ions and analyze the fragments in a
second analyzer.
 Detect the ions emerging from the last analyzer and measure their
abundance with the detector that converts the ions into electrical
signals.
 Process the signals from the detector that are transmitted to the
computer and control the instrument using feedback.
13

COMPONENTS OF A MASS SPECTROMETER:
The essential components of a mass spectrometer consist of:
 A sample inlet
 An ionization source
 A mass analyzer
 An ion detector
 Vacuum system
14

15
INLET SYSTEM
FOR SOLIDS, FOR LIQUIDS, FOR GASES
IONIZATION MECHANISMS:
PROTONATION, DEPROTONATION,
CATIONIZATION, ELECTRON EJECTION,
ELECTRON CAPTURE
IONIZATION SOURCE
ESI, MALDI, FAB, FD, PD, APCI, APPI, EI, CI,
FI.
MASS ANALYZER
QUADRUPOLE, MAGNETIC
SECTORS, TIME OF FLIGHT, QIT,
FTICR
DETECTOR
ELECTRON MULTIPLIER, FARADAY
CUP, PHOTOMULTIPLIER
CONVERSION DYNODE, ARRAY
MASS
SPECTROMETER

16
FIGURE REPRESENTING INSTRUMENTATION OF A MASS
SPECTROMETER

INLET SYSTEM:
 The selection of a sample inlet depends upon the sample and the
sample matrix.
 Most ionization techniques are designed for gas phase molecules so
the inlet must transfer the analyte into the source as a gas phase
molecule.
 If the analyte is sufficiently volatile and thermally stable, a variety of
inlets are available.
 Gases and samples with high vapor pressure are leaked directly into
the source region by the help of mercury manometer.
 Liquids and solids are usually heated to increase the vapor pressure
for analysis.
 Liquid samples are handled by hypodermic needles injection through a
silicon rubber dam.
 If the analyte is thermally labile (it decomposes at high temperatures)
or if it does not have a sufficient vapor pressure, the sample must be
directly ionized from the condensed phase.
17

SAMPLE INTRODUCTION METHODS:
1. Direct Vapor Inlet:
 The simplest sample introduction method.
 The gas phase analyte is introduced directly into the source region of the
mass spectrometer through a needle valve. Pump out lines are usually
included to remove air from the sample.
 This inlet works well for gases, liquids, or solids with a high vapor pressure.
 It only works for some samples.
2. Gas Chromatography:
 Most common technique for introducing samples into a mass spectrometer.
 Complex mixtures are routinely separated by gas chromatography and mass
spectrometry is used to identify and quantitate the individual components.
 The most significant characteristics of the inlets are the amount of GC
carrier gas that enters the mass spectrometer and the amount of analyte
that enters the mass spectrometer.
 Ideally all the analyte and none of the GC carrier gas would enter the source
region.
 The most common GC/MS interface now uses a capillary GC column 18

3. Liquid Chromatography:
 LC inlets are used to introduce thermally labile compounds not easily
separated by gas chromatography.
 These inlets are used for temperature sensitive compounds.
 The sample is ionized directly from the condensed phase.
4. Direct Insertion Probe:
 The Direct Insertion Probe (DIP) is widely used to introduce low vapor
pressure liquids and solids into the mass spectrometer.
 This is important for analyzing temperature sensitive compounds.
 Although the direct insertion probe is more cumbersome than the
direct vapor inlet, it is useful for a wider range of samples.
5. Direct Ionization of Sample:
 Some compounds either decompose when heated or have no
significant vapor pressure and can be introduced by direct ionization
from the condensed phase.
 These are used for LC-MS, glow discharge MS, FAB and laser ablation.19

IONIZATION METHODS
Ionization method refers to the mechanism of ionization while the
ionization source is the mechanical device that allows ionization to occur.
The different ionization methods are as follows:
1. Protonation
 Protonation is a method of ionization by which a proton is added to a
molecule, producing a net charge of 1+ for every proton added.
 E.g.: More basic residues of the molecule, such as amines; Peptides.
 Can be achieved through MALDI, ESI and APCI.
2. De-protonation
 De-protonation is an ionization method by which the net charge of 1-
is achieved through the removal of a proton from a molecule.
 E.g.: Acidic species including phenols; carboxylic and sulfonic acids.
 Commonly achieved via MALDI, ESI and APCI.
20

3. Cationization
 It produces a charged complex by non-covalently adding a positively charged
cation adduct (e.g. alkali, ammonium) to a neutral molecule.
 E.g.: Carbohydrates are best examples, with Na+ as a common cation adduct.
 Mainly achieved by MALDI, ESI and APCI.
4. Transfer of a Charged Molecule to the Gas Phase
 The transfer of compounds already charged in solution is achieved through
desorption or ejection of the charged species from the condensed phase into the
gaseous phase.
 Commonly achieved through MALDI or ESI.
5. Electron Ejection
 Ionization is achieved through the ejection of an electron to produce a 1+ net
charge, often forming radical cations. It generates significantly fragmented
ions.
 E.g.: Non-polar compounds with low molecular weights.
 Most commonly achieved with electron ionization (EI) sources.
6. Electron Capture
 With the electron capture ionization method, a net charge of 1- is achieved with
the absorption or capture of and electron.
 E.g.: Molecules with high electron affinity, such as halogenated compounds.
21

ION SOURCE
 Ionization of the organic compound is the primary step in obtaining
the mass spectrum. The minimum energy required to ionize the
sample or organic molecule is called as its ionization potential.
 The ion source is the part of the mass spectrometer that ionizes the
material under analysis (the analyte).
 The ions are then transported by magnetic or electric fields to the
mass analyzer.
 Molecular ions are formed when energy of the electron beam
reaches to 10-15 eV.
 Fragmentation of the ion reaches only at higher bombardment
energies at 70 eV.
 Function
1. Produces ion without mass discrimination of the sample.
2. Accelerates ions into the mass analyzer. 22

23
CLASSIFICATION OF ION SOURCES
1. Desorption Sources. a. Electrospray Ionization (ESI).
b. Matrix assisted laser desorption Ionization (MALDI).
c. Fast Atom Bombardment (FAB).
d. Field Desorption (FD).
e. Plasma desorption (PD).
2. Atmospheric
pressure ionization.
a. Atmospheric pressure chemical ionization (APCI)
b. Atmospheric pressure photoionization (APPI)
3. Gas Phase Sources. a. Electron Impact Ionization (EI).
b. Chemical Ionization (CI).
c. Field Ionizations (FI).

ESI (Electrospray Ionization)
 It is a soft ionization technique.
 It is typically used to determine the molecular weights of proteins, peptides
and other biological macromolecules.
 It provides a sensitive, robust and reliable tool for studying.
Advantages
a. Has ability to handle samples with large masses.
b. One of the softest ionization methods available and has the ability to analyze
biological samples with non-covalent interactions.
c. Good sensitivity and therefore, useful in accurate quantitative and qualitative
measurements.
Disadvantages
a. Cannot analyze mixtures very well & when forced to do so, results are
unreliable.
b. Apparatus is very difficult to clean and has a tendency to become overly
contaminated with residues from previous experiments.
c. The multiple charges that are attached to the molecular ions can make for
confusing spectral data.
d. Prior separation by chromatography is required.
24

ESI - Principle
 ESI uses electrical energy to assist the transfer of ions from solution into
the gaseous phase.
 ESI works on the principle of Soft ionization.
 Soft ionization is a useful technique when considering biological molecules
of large molecular mass, because in this process macromolecule is ionized
into small droplets, which are then desolvated into even smaller droplets,
which creates molecules with attached protons.
Applications
a. Protein identification and characterization.
b. Studying non covalent interaction.
c. Probing molecular dynamics
d. Monitoring chemical reactions and studying reactive intermediates.
e. Chemical imaging.
f. Identification and quantification of hemoglobin variants.
g. Screening for inborn errors of metabolism. 25

26
Figure Representing
ELECTROSPRAY IONIZATION MECHANISM

MALDI (Matrix Assisted Laser Desorption Ionization)
 MALDI is also based on “soft ionization” methods where ion formation does not lead
to a significant loss of sample integrity.
 Consequently, the high throughput and speed associated with complete automation
has made MALDI-TOF mass spectrometer an obvious choice for proteomics work on
large-scale.
Advantages
a. Gentle Ionization technique
b. High molecular weight analyte can be ionized
c. Molecule need not be volatile
d. Wide array of matrices
e. Produces singly charged ions thus interpretation becomes easy.
f. Prior separation by chromatography is not required.
Disadvantages
a. MALDI matrix cluster ions obscure low m/z species (<600)
b. Analyte must have very low vapor pressure
c. Pulsed nature of source limits compatibility with many mass analyzers
d. Coupling MALDI with chromatography can be difficult
e. Analytes that absorb the laser can be problematic.
27

MALDI – Principle
The sample for analysis by MALDI MS is prepared by mixing or coating
with solution of an energy-absorbent, organic compound called matrix.
When the matrix crystallizes on drying, the sample entrapped within the
matrix also co-crystallizes. The sample within the matrix is ionized in an
automated mode with a laser beam. Desorption and ionization with the
laser beam generates singly protonated ions from analytes in the sample.
Matrix and Sample Preparation
 The matrix consists of crystallized molecules, of which the three most
commonly used are - 3, 5-dimethoxy-4- hydroxycinnamic acid (sinapinic
acid), α-cyano-4- hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid
(DHB).
 A solution of one of these molecules is made, often in a mixture of highly
purified water and an organic solvent such as acetonitrile (ACN) or ethanol.
 A counter ion source such as Trifluoroacetic acid (TFA) is usually added to
generate the [M+H] ions.
 A good example of a matrix solution would be 20 mg/ml sinapinic acid in
ACN: water: TFA (50:50:0.1).
28

29
 The matrix performs two important functions:
1. It absorbs photon energy from the laser beam and transfers it into
excitation energy of the solid system,
2. It serves as a solvent for the analyte, so that the intermolecular forces
are reduced and aggregation of the analyte molecules is held to a
minimum.
 Some desirable characteristics of a typical MALDI matrix are:
a. A strong light absorption property at the wavelength of the laser flux.
b. The ability to form micro-crystals with the sample.
c. A low sublimation temperature, which facilitates the formation of an
instantaneous high-pressure plume of matrix-sample material during the
laser pulse duration.
d. The participation in some kind of a photochemical reaction so that the
sample molecules can be ionized with high yields.

COMPOUND OTHER NAMES SOLVENT
WAVELENGTH
(nm)
APPLICATIONS
2,5-dihydroxy
benzoic acid
DHB, Gentisic
acid
Acetonitrile, water,
methanol, acetone,
chloroform
337, 355, 266
Peptides,
nucleotides,
oligosaccharides,
oligonucleotides
3,5-dimethoxy-4-
hydroxycinnamic
acid
Sinapic acid,
sinapinic acid;
SA
acetone, chloroform
337, 355, 266
Peptides, proteins,
lipids
α-cyano-4-
hydroxycinnamic
acid
CHCA
ethanol, acetone
337, 355
Peptides, lipids,
nucleotides
Picolinic acid PA Ethanol 266 Oligonucleotides
3-hydroxy picolinic
acid
HPA Ethanol 337, 355 Oligonucleotides
30
MALDI Matrix List

32
Applications of MALDI
a. Proteomics To identify, verify and quantitate: metabolites,
recombinant proteins, proteins isolated from natural
source, peptides and their amino acid sequences.
b. Pharmaceutical
Analysis
i. Bioavailability studies
ii. Drug metabolism studies, pharmacokinetics
iii. Characterization of potential drugs
iv. Drug degradation product analysis
v. Screening of drug candidates
vi. Identifying drug targets
c. Microbiology i. It is used for the identification of microorganisms.
ii. Species diagnosis by this procedure is much faster,
more accurate and cheaper than other procedures
based on biochemical tests.
d. Forensic Analysis and
Environmental Analysis
i. Pesticides on food
ii. Soil and groundwater contamination.

FAB (Fast Atom Bombardment)
 FAB is an ionization technique used in mass spectrometry in which a beam of
high energy atoms strikes a surface to create ions.
 It was developed by Michael Barber at the University of Manchester.
 When a beam of high energy ions is used instead of atoms (as in secondary ion
mass spectrometry), the method is known as liquid secondary ion mass
spectrometry (LSIMS).
1. Advantages
a. FAB is extensively used for the ionization of high molecular weight (>5000 D)
samples of biological origin.
b. Extensively used for obtaining mass spectra of salts depending upon the
nature of its cation and anion.
c. The FAB spectra usually provide relatively abundant molecular or quasi-
molecular ions and also show some structurally important fragment ions.
2. Disadvantages
a. The matrix also forms ions on bombardment, in addition to those formed by
the sample which complicates the spectrum.
b. FAB samples the surface rather than the bulk concentration of the solute
present and hence limits quantitative measurement.
33

Principle
 The analyte is dissolved in a viscous liquid, typically glycerol (matrix material) and
ionization is achieved by bombardment of the sample matrix (a metal plate coated
with viscous solution of the sample) by a beam of fast moving neutral atoms.
 The bombarding atoms are usually rare gases, either xenon or argon.
 Common matrices include glycerol, thioglycerol, 3-nitrobenzyl alcohol (3- NBA),
triethanolamine etc.
 This technique is similar to secondary ion mass spectrometry and plasma desorption
mass spectrometry.
 In order to achieve a very high kinetic energy, the atoms of the gas are first ionized
and these ions are then passed through an electric field.
 After acceleration, the fast moving ions enter into a chamber containing further gas
atoms and collision of ions and atoms leads to charge exchange.
Xe•+ (fast) + Xe (thermal) Xe (fast) + Xe+• (thermal)
 The fast atoms formed in this process retain the original kinetic energy of the fast
ions and proceed towards the analyzer.
34

Matrices
 One of the crucial characteristics of FAB is using liquid matrix.
 Due to the high vacuum condition, usual solvent for chemistry laboratory such
as water and other common organic solvent is precluded for FAB.
 Solvent with high boiling point called matrix is necessary to be employed.
 Following table shows examples of matrix.
35
FAB MATRIX LIST
Matrix Observed ions (m/z)
Glycerol 93
Thioglycerol 109
3-Nitrobenzyl alcohol (3-NOBA) 154
n-Octyl-3-nitrophenylether (NOP) 252
Triethanolamine 150
Diethanolamine 106

Applications
a. Elucidation of the amino acid sequence of the oligopeptide efrapeptin D.
This is a potent inhibitor of mitochondrial ATPase activity.
b. The separation and MS analysis of peptides arising from protein
enzymatic digestion.
36
FAB IONIZATION MECHANISM

Field desorption
Introduction
In field desorption method, a multi-tipped emitter (made up of tungsten wire with
carbon or silicon whiskers grown on its surface) similar to that used in FI is used.
Advantages
○ Works well for small organic molecules, low molecular weight polymers and
petrochemical fractions.
Disadvantages
 Sensitive to alkali metal contamination.
 Sample must be soluble in a solvent.
 Not suitable for thermally unstable and non volatile samples.
 Structural information is not obtained as very little fragmentation occurs.
37

Construction & Working
 The electrode is mounted on a probe that can be removed from the sample
compartment and coated with the solution of the sample.
 The sample solution is deposited on the tip of the emitter whiskers either by
 dipping the emitter into analyte solution or
 using a micro-syringe.
 The probe is then reinserted into the sample compartment which is similar
to CI or EI unit.
 Then the sample is ionized by applying a high voltage to the emitter.
 NOTE: In some cases it is necessary to heat the emitter by passing a current
through the wire to evaporate the sample.
 Ionization takes place by quantum mechanical tunneling mechanism, which
involves transfer of ions from the sample molecule to the anode (emitter).
 This results in formation of positive ions which are radical ions (M+) and
cations attached species such as (M+Na)+.
 (M+Na)+ are produced during desorption by attachment of trace alkali metal
ions present in analyte.
38

39
FIELD DESORPTION IONIZATION SOURCE

PLASMA DESORPTION
• Plasma desorption produces molecular ions from the samples coated on a thin
foil when a highly energetic fission fragments from the Californium-252 “blast
through” from the opposite side of the foil.
• The fission of Californium-252 nucleus is highly exothermic and the energy
released is carried away by a wide range of fission fragments which are heavy
atomic ion pairs.
• Ion pair fission fragments depart in opposite directions.
• Each fission of this radio active nucleus gives rise to two fragments traveling in
opposite directions (because necessity of momentum conversation).
• A typical pair of fission fragments is 142Ba18+ and 106TC22+, with kinetic energies
roughly 79 and 104 MeV respectively.
• When such a high energy fission fragments passes through the sample foil,
extremely rapid localized heating occurs, producing a temperature in the range
of 10000K.
• Consequently, the molecules in this plasma zone are desorbed, with the
production of both positive and negative ions.
• These ions are then accelerated out of the source in to the analyzer system. 40

41
He or N2
GLOW
DISCHARGE
SAMPLE
MS INLET
PLASMA DESORPTION IONIZATION MECHANISM

Atmospheric Pressure Chemical Ionization (APCI)
 APCI is an ionization method used in mass spectrometry (commonly LC-MS) which
utilizes gas-phase ion-molecule reactions at atmospheric pressure.
 It is an ionization method that is similar to chemical ionization (commonly used in
GC-MS) where corona discharges on a solvent spray produce primary ions.
 APCI is mainly used with polar and relatively non-polar compounds with a
molecular weight of less than 1500 Da, generally giving mono-charged ions.
Advantages
a. Multiple charging is typically not observed as the ionization process is more
energetic than ESI.
b. Electron transfer or proton loss, ([M-H]-) occurs in the negative mode.
c. Proton transfer (for protonation MH+ reactions) occurs in the positive mode
d. At atmospheric pressure analyte molecules collide with the reagent ions
frequently and hence ionization is very efficient.
Limitations
a. Very sensitive to contaminants such as alkali metals or basic compounds.
b. Relatively low ion currents.
c. Relatively complex hardware compared to other ion sources.
42

PRINCIPLE
 In APCI, the sample is typically dissolved in a solvent and pumped through a
capillary inside an uncharged quartz tube. At the end of the capillary, but still
within the tube, the sample is converted into an aerosol and then vaporized
with the help of nitrogen gas and by heating to very high temperature (~350-
550 °C).
 The gaseous solvent (S) and sample (M) are then ionized by a corona
discharge, in which a highly charged electrode creates an electric field strong
enough to ionize nearby molecules.
 A potential of several kilovolts applied to the electrode typically remove an
electron from a neutral molecule, without depositing enough internal energy
to cause fragmentation.
 The corona discharge may directly ionize an analyte molecule to form a radical
cation (M+●):
M + e- → M+● + 2e-
S + e- → S+● + 2e-
 Frequent collisions between the ions and molecules can transfer charge from
an ion to another neutral. Collision of an ionized solvent ion with an analyte
molecule can create a direct charge transfer to form a radical cation analyte
ion:
S+● + M → M+● + S
To be Cont.
43

 Alternatively, collision of solvent ions with a neutral analyte molecule may
result in abstraction of a hydrogen atom from the molecule. The resulting
ionized solvent can then ionize the analyte via proton transfer:
S+● + S → [S+H]+ + S[-H]
[S+H]+ + M → [M+H]+ + S
 The resulting analyte ions (M+● or [M+H]+) are then injected into the mass
spectrometer for detection.
Applications
a. APCI is suitable for the analysis of organic compounds with medium - high
polarity.
b. Since positive ionization is dependent on protonation, molecules containing
basic functional groups such as amino, amide esters, aldehyde/ketone and
hydroxyl can be analyzed.
c. Negative ionization depends upon deprotonation, molecules containing acidic
functional groups are analyzed by this method.
d. Can be used as LC/MS interface.
e. In the analysis of pesticides.
f. Analysis of triazines, phenylureas, carbamates and organophosphorus
compounds.
g. In the determination of Vit. D3 in poultry feed supplements.
44

APPI (Atmospheric Pressure Photoionization)
 It has recently become an important ionization source because it
generates ions directly from solution with relatively low background
and is capable of analyzing relatively nonpolar compounds.
 Similar to APCI, the liquid effluent of APPI is introduced directly into the
ionization source.
 The primary difference between APCI and APPI is that the APPI
vaporized sample passes through ultra-violet light (a typical krypton
light source emits at 10.0 eV and 10.6 eV).
 Often, APPI is much more sensitive than ESI or APCI and has been
shown to have higher signal-to-noise ratios because of lower
background ionization.
 Lower background signal is largely due to high ionization potential of
standard solvents such as methanol and water (IP 10.85 and 12.62 eV,
respectively) which are not ionized by the krypton lamp.
Disadvantages
a. It can generate background ions from solvents.
b. It requires vaporization temperatures ranging from 350-500° C, which
can cause thermal degradation.
46

Principle
 In APPI technique samples are ionized by using UV light.
 Molecules interact with photon beam of UV light with vapors of nebulizer liquid
solution.
 Analyte molecules (A) absorb a photon (hν) and become an electronically
excited molecule.
 If the ionization energy (IE) of analyte molecules is lower than the energy of
photon, then the analyte molecule releases energetic electron and become the
radical cation.
Applications
a. It has the capability to ionize compounds with a wide range of polarities while
being remarkably tolerant of matrix components of HPLC additives.
b. APPI has been proved to be a valuable tool for analytes which are poorly
ionized or not ionized by ESI and APCI. In particular
c. APPI was shown to be able to detect steroid hormones down to several ng/L
and had been proven to have much higher sensitivity than ESI.
d. Results indicate that APPI using toluene as dopant provides exceptional
ionization capabilities for a broad range of compounds, in particular for hormones
and sterols compared to APCI and HESI.
47

ELECTRON IMPACT IONIZATION:
 It is the most widely used and highly developed method. It is also known as
Electron bombardment or Electron Ionization.
 Electron impact ionization source consists of a ionizing chamber which is
maintained at a pressure of 0.005 torr and temperature of 200 ± 0.25 degrees.
 Electron gun is located perpendicular to chamber.
 Electrons are emitted from a glowing filament (tungsten or rhenium) by thermionic
emission and accelerated by a potential of 70 V applied between the filament and
anode.
 These electrons are drawn in the ionization chamber through positively charged
slits.
 The number of electrons is controlled by filament temperature and energy.
 The sample is brought to a temperature high enough to produce molecular vapors.
 The gaseous Neutral molecules then pass through the molecular leaks and enter
the ionization chamber.
 The gaseous sample and the electrons collide at right angles in the chamber and
ions are formed by exchange of energy during these collisions between electron
beam and sample molecules.
49

 The positive ions formed in the chamber are drawn out by a small potential
difference (usually 5eV) between the large repeller plate (positively charged)
and first accelerating plate (negatively charged).
 Strong electrostatic field (400 – 4000 V) applied between the first and second
accelerating plates accelerates the ions according to their masses (m1, m2, m3
etc) to their final velocities.
50
Where,
M = Analyte molecule
e- = Electrons
M.+ = Molecular ions

 The ions emerge from the final accelerating slit as a collimated ribbon of ions. The
energy and velocity of ions are given by :-
zV = ½ (m1v1) = ½ (m2v2) = ½ (m3v3)
Where,
z = charge of the ion
V = accelerating potential
v = velocity of ion
Advantages
 Gives molecular mass and also the fragmentation pattern of the sample.
 Extensive fragmentation and consequent large number of peaks gives structural
information.
 Gives reproducible mass spectra.
 Can be used as GC/MS interface.
Disadvantages
 Sample must be thermally stable and volatile.
 A small amount of sample is ionized (1 in 1000 molecules).
 Unstable molecular ion fragments are formed so readily that are absent from mass
spectrum.
51

CHEMICAL IONIZATION
 In chemical ionization the ionization of the analyte is achieved by interaction
of its molecules with ions of a reagent gas in the chamber or source.
 Chemical ionization is carried out in an instrument similar to electron impact
ion source with some modifications such as:-
 Addition of a vacuum pump.
 Narrowing of exit slit to mass analyzer to maintain reagent gas pressure of
about 1 torr in the ionization chamber.
 Providing a gas inlet.
 It is a two part process.
 Step-I Reagent gas is ionized by Electron Impact ionization in the source. The
primary ions of reagent gas react with additional gas to produce stabilized
reagent ions.
 Step-II Reagent ions interact with sample molecules to form molecular ions. In
this technique the sample is diluted with a large excess of reagent gas. Gases
commonly used as reagent are low molecular weight compounds such as
Methane, tertiary Isobutane, Ammonia, Nitrous oxide, oxygen and hydrogen
etc.
 Depending upon the type of ions formed CI is categorized as:-
 Positive Chemical Ionization.
 Negative Chemical Ionization.
52

 Positive Chemical Ionization
 In this technique positive ions of the sample are produced. In positive chemical
ionization gasses such as Methane, Ammonia, Isobutane etc are used
 For example Ammonia is used as reagent gas. First ammonia radical cations are
generated by electron impact and this react with neutral ammonia to form
ammonium cation (reactive species of ammonia CI).
NH3 NH3
.+ + 2 e-
NH3
.+ NH4
+ + NH2
 NH4
+ reacts with the sample molecules by proton transfer or Adduct formation
to produce sample ions
M + NH4
+ [M + H]+ + NH3 Proton transfer
M + NH4
+ [M + NH4]+ Adduct formation
 When Methane is used as Reagent gas. Methane is ionized by electron impact:
CH4 + e- CH4
+ + 2e-
 Primary ions react with additional reagent gas molecules to produce stabilized
reagent ions:
CH4
+ + CH4 CH5
+ + CH3
CH3
+ + CH4 C2H5
+ + H2
53

 The reagent ions then react with the sample molecules to ionize the sample
molecules:
CH5+ + MH CH4 + MH2
+ (Proton transfer)
CH3
+ + MH CH4 + M+ (hydride abstraction)
CH4
+ + MH CH4 + MH+ (Charge transfer)
Negative Chemical Ionization
 Negative chemical ionization is counterpart of Positive chemical ionization.
Negative ions of the sample are formed and oxygen and Hydrogen are used as
reagent gases. This method is used for ionization of highly electronegative
samples. The negative ions are formed by following reactions:-
 Resonance electron capture M + e- M-
 Dissociative electron capture RCl + e- R + Cl-
H2O + e- H + OH-
 The ion molecule reaction occurring between negative ion formed in the chamber
source and the sample molecule include:-
 Charge transfer.
 Hydride transfer.
 Anion- Molecule adduct formation.
54

Advantages
 Used for high molecular weight compounds.
 Used for samples which undergo rapid fragmentation in EI.
Limitations
 Not suitable for thermally unstable and non-volatile samples.
 Relative less sensitive then EI ionization.
 Samples must be diluted with large excess of reagent gas to prevent primary
interaction between the electrons and sample molecules.
55
A SCHEMATIC OF CHEMICAL IONIZATION

FIELD IONIZATION
 FI is used to produce ions from volatile compounds that do not give
molecular ions by EI. It produces molecular ions with little or no
fragmentation.
 Application of very strong electric field induces emission of electrons.
 Sample molecules in vapor phase is brought between two closely spaced
electrodes in the presence of high electric field (107 - 108 V/cm) it
experiences electrostatic force.
 If the metal surface (anode) has proper geometry (a sharp tip, cluster of
tips or a thin wire) and is under vacuum (10-6 torr) this force is sufficient
to remove electrons from the sample molecule without imparting much
excess energy.
 The electric field is produced by applying high voltage (20 KV) to these
specially formed emitters (made up of thin tungsten wire).
 In order to achieve high potential gradients necessary to effect
ionization, the anode is activated by growing carbon micro-needles or
whiskers.
 As concentration of sample molecules is high at the anode ion-molecule
reactions often occur which results in formation of protonated species
(M+H)+. Thus both M+ and (M+H) + is observed in FI spectrum.
56

Advantages
 As fragmentation is less, abundance of molecular ions (M+) is enhanced, hence
this method is useful for relative molecular mass and empirical formula
determination.
Disadvantages
 Not suitable for thermally unstable and non volatile samples.
 Sensitivity is less than EI ion source.
 No structural information is produced as very little fragmentation occurs
57
FIELD IONIZATION MECHANISM

MASS ANALYZERS
 With the advent of ionization sources that can vaporize and ionize molecules,
it has become necessary to improve mass analyzer performance with respect
to speed, accuracy, and resolution.
 More specifically, quadrupoles, quadrupole ion traps, time-of-flight (TOF),
time-of-flight reflectron, and ion cyclotron resonance (ICR) mass analyzers
have undergone numerous modifications/improvements over the past
decade in order to be interfaced with MALDI and ESI.
 The first mass analyzers, made in the early 1900’s, used magnetic fields to
separate ions according to their radius of curvature through the magnetic
field.
 The design of modern analyzers has changed significantly in the last few
years, now offering much higher accuracy, increased sensitivity, broader mass
range, and the ability to give structural information.
58

GENERAL PRINCIPLE OF OPERATION OF MASS ANALYZER:
 Once analyte ions are formed in the gas phase, a variety of mass analyzers
are available and used to separate the ions according to their mass-to-
charge ratio (m/z). Mass spectrometers operate with the dynamics of
charged particles in electric and magnetic fields in vacuum described by the
Lorentz force law and Newton’s second law of motion:
F = z(E + v x B) (Lorentz force equation)
F = ma (Newton’s second law of motion)
 Where F is the force applied to the ion, m is the mass of the ion,
a is the acceleration,
q is the ionic charge,
E is the electric field, and
v x B is the vector cross product of ion velocity and magnetic field.
 Combining those equations results in the equation that describes the motion
of charged particles:
(m/z)a = E + v x B
 Two particles with the same physical quantity m/z behave identically, and all
mass spectrometers measure m/z rather than m.
59

SINGLE FOCUSING MAGNETIC DEFLECTION
(DEMPSTER MASS SPECRTOMETER)
 In this spectrometer the positive ions travel in a circular path through
180 degree under a magnetic field H.
Suppose an ion having a charge e is accelerated through a voltage V.
Then the kinetic energy of the ions is expressed as:
Kinetic energy =
1
2
𝑚𝑣2
= eV (1)
Where, v= velocity of the ion after acceleration
V = potential applied
 It may be noted a mass ion travel slowly in a circular path compared
to the lighter fragment.
In a magnetic field H, any ion will experience force Hev.
It produces an acceleration of v2/r in a circular path of radius r.
Magnetic force = Hev
 Counterbalancing centrifugal force
Hev =
𝑚𝑣2
𝑟
60

Hence, form Newton's second low of motion
Hev =
𝑚𝑣2
𝑟
Squaring both sides,
𝐻2 𝑒2 𝑣2 =
𝑚2 𝑣4
𝑟2
𝐻2
𝑒2
=
𝑚2 𝑣2
𝑟2 …. (2)
But
1
2
𝑚𝑣2 = 𝑒𝑉
... mv2 = 2 eV
Putting the value of mv2 in 3rd equation.
𝐻2
𝑒2
=
𝑚. 2𝑒𝑉
𝑟2 or 𝐻2
𝑒 =
2𝑚𝑉
𝑟2
or
𝑚
𝑒
=
𝐻2 𝑟2
2𝑉
Only ions with particular radius will be collected.
 From this equation it is clear that at a given magnetic field strength and
accelerating voltage, the ions of m/v value will follow a circular path of
radius r.
61

 The ions of various m/v values reach the collector, amplified and recorded.
 The mass spectrum can be obtained either by
a) changing H at constant V (Magnetic scanning) or
b) changing V at constant H (Electric Voltage scanning)
Double Focusing Mass Spectrometer
 The limitation in the single focusing instrument is that the resolving power is
limited by initial spread of translational energy of ion leaving the source.
 This problem is overcome by passing the ion through electric field prior to the
magnetic field.
62

PERFORMANCE CHARACTERISTICS OF MASS
ANALYZERS:
 This is the ability of the analyzer to separate different molecular ions, generate
fragment ions from a selected ion, and then mass measure the fragmented ions.
The fragmented ions are used for structural determination of original molecular
ions. The characteristics of these mass analyzers are:
 Resolution describes the ability of a mass analyzer to separate adjacent ions.
 Mass accuracy is the ability of a mass analyzer to assign the mass of an ion
close to its true mass.
 Mass range is usually defined by the lower and upper m/z value observed by a
mass analyzer.
 Sensitivity is the ability of a particular instrument to respond to a given amount
of analyte.
 Scan speed is the rate at which we can acquire a mass spectrum, generally
given in mass units per unit time.
 Tandem mass spectrometry (MS/MS) provides the ability to mass-analyze
sample components sequentially in time or space to improve selectivity of the
analyzer or promote fragmentation and facilitate structural elucidation.
63

TYPES OF ANALYZERS:
 Analyzers are typically described as either continuous or pulsed.
 Continuous analyzers: These analyzers are similar to a filter or
monochromator used for optical spectroscopy. They transmit a single
selected m/z to the detector and the mass spectrum is obtained by scanning
the analyzer so that different mass to charge ratio ions are detected. They
include:
 Quadrupole filters and
 Magnetic sectors.
 Pulsed mass analyzers: These are the other major class of mass analyzer.
These are less common but they have some distinct advantages. These
instruments collect an entire mass spectrum from a single pulse of ions. This
results in a signal to noise advantage similar to Fourier transform or
multichannel spectroscopic techniques. Pulsed analyzers include:
 Time-of-flight,
 Ion cyclotron resonance,
 Quadrupole ion trap mass spectrometers.
64

QUADRUPOLE MASS ANALYZER:
 Quadrupole mass analyzer is one type of mass analyzer used in mass
spectrometry.
 A typical quadrupole mass analyzer consists of four rods with a
hyperbolic cross section that are accurately positioned parallel in a
radial array.
 The quadrupole rods are typically constructed using molybdenum alloys
because of their inherent inertness and lack of activity.
 Very high degrees of accuracy and precision (in the micrometer region)
in rod machining and relative positioning are required to achieve unit
mass accuracy.
 Quadrupole mass spectrometers ~QMSs’ are widely used in both
industry and research for fast accurate analysis of gas and vapors.
 The QMS contains basically three elements;
i) ion source,
ii) mass filter, and
iii) ion detector.
65

Benefits:
 Classical mass spectra.
 Good repeatability.
 Relatively small and cost-effective systems.
 Low-energy collision-induced dissociation (CID) MS/MS spectra leads
to efficient conversion of precursor to product.
Limitations:
 Limited resolution.
 Peak heights are variable as a function of mass discrimination.
 Peak height vs. mass response should be 'tuned'.
 Not compatible for pulsed ionization methods.
 Low-energy collision-induced dissociation (CID) MS/MS rely most
probably on energy, collision gas, pressure, and alternative factors.
Applications:
 Majority of bench top GC/MS and LC/MS systems.
 Triple quadrupole MS/MS systems.
 Sector/quadrupole hybrid MS/MS systems. 66

MAGNETIC SECTOR MASS ANALYZER:
 Sector mass analyzers are the most mature of the MS mass analysis
technologies, having enjoyed widespread use from the 1950s through
to the 1980s.
 Magnetic sectors bend the trajectories of ions accelerated from an ion
source into circular paths; for a fixed accelerating potential, typically
set between 2 and 10 kV, the radii of these paths are determined by
the momentum-to-charge ratios of the ions. In such a manner, the ions
of differing m/z are dispersed in space.
 Magnetic sector instruments are often used in series with an electric
sector, high resolution and tandem mass spectrometry experiments.
 When utilizing a magnetic sector alone, resolutions of only a few
hundred can be obtained, primarily due to limitations associated with
differences in ion velocities. To correct for this, electric sectors can be
placed before or after the magnetic sector and is thus called as double
focusing sector instruments.
68

Benefits:
 Classical mass spectra.
 Very high reproducibility.
 High resolution.
 High sensitivity.
 High dynamic range.
Limitations:
 Not well-suited for pulsed ionization methods (e.g. MALDI).
 Usually larger and higher cost than other mass analyzers.
Applications:
 All organic MS analysis methods.
 Accurate mass measurement.
 Isotope ratio measurements.
 Quantitation.
69

TIME OF FLIGHT ANALYZER:
 A time-of-flight (TOF) mass spectrometer is a non-scanning mass
analyzer that emits pulses of ions (or transients) from the source.
 These ions are accelerated so that they have equal kinetic energy
before entering a field free drift region, also known as the flight tube.
 A time-of-flight (TOF) instrument consists of a pulsed ion source, an
accelerating grid, a field-free flight tube, and a detector.
 Different Modes of Time of Flight Analyzers:
 The mass analyzer used in TOF-MS can be either be a linear flight
tube, applying a bended geometry the flight path to reduce the
influence of neutral particles (Poschenrieder type) or a employ one (V-
type) or more reflectrons (W-type) to enhance the path length within
a given flight tube.
• Linear TOF (high mass range but low mass resolution)
• Reflectron TOF (lower mass range but high mass resolution) 71

Benefits:
 Fastest MS analyzer.
 Well suited for pulsed ionization methods (method of choice for
majority of MALDI mass spectrometer systems).
 High ion transmission.
 MS/MS information from post-source decay.
 Highest practical mass range of all MS analyzers.
Limitations:
 Requires pulsed ionization method or ion beam switching (duty
cycle is a factor).
 Fast digitizers used in TOF can have limited dynamic range.
 Limited precursor-ion selectivity for most MS/MS experiments.
Applications:
 Almost all MALDI systems.
 Very fast GC/MS systems, 72

QUADRUPOLE ION TRAP ANALYZER:
 The Quadrupole ion storage trap mass spectrometer (QUISTOR) is a
recently developed mass analyzer with some special capabilities.
 The quadrupole ion trap is an extraordinary device that functions both
as an ion store in which gaseous ions can be confined for a period of
time, and as a mass spectrometer of large mass range variable mass
resolution, and high sensitivity.
 As a storage device, the quadrupole ion trap confines gaseous ions,
which are either positively or negatively charged and when required
ions of each polarity also.
 A typical (three-dimensional quadrupole) ion trap consists of a
cylindrical ring electrode and two end-cap electrodes. T
 he end-cap electrodes contain holes for the introduction of ions from
an external ion source and for the ejection of ions toward an external
detector.
 A He bath gas (∼1 mbar) is used to stabilize the ion trajectories in the
trap. 74

Benefits:
 High sensitivity,
 Compactness and mechanical simplicity
 Ion/molecule reactions can be studied for mass-selected ions,
 High resolution
 Non-destructive detection is available using Fourier transform techniques.
 Multi-stage mass spectrometry (analogous to FTICR experiments)
Limitations:
 Poor quantitation.
 Very poor dynamic range (which can be compensated for by employing auto-
ranging).
 Collision energy not well-defined in Collision Induced Dissociation [CID] MS/MS.
 Quality of the mass spectrum is influenced by many parameters such as
excitation, trapping, and detection conditions.
 Mass measurement accuracy is relatively poor.
Applications:
 Benchtop GC/MS, LC/MS and MS/MS systems.
 Target compound screening.
 Ion chemistry.
 Non-destructive ion detection.
75

76
QUADRUPOLE ION TRAP ANALYZER

FOURIER-TRANSFORM ION CYCLOTRON
RESONANCE MASS ANALYZER {FTICR-MS/FTMS}
 Fourier transform ion cyclotron resonance mass spectrometry is a
type of mass analyzer (or mass spectrometer) for determining the
mass-to-charge ratio (m/z) of ions based on the cyclotron frequency
of the ions in a fixed magnetic field.
 FT-ICR is the highest performance MS technique available, offering
unrivalled resolution and mass accuracy.
 A FTMS can be considered an ion-trap system, where the ions are
trapped in a magnetic rather than in a quadrupole electric field.
 The Ion Cyclotron Resonance (ICR) mass spectrometer uses a
superconducting magnet to trap ions in a small sample cell. This type
of mass analyzer has extremely high mass resolution and is also
useful for tandem mass spectrometry experiments.
 The FTMS consists of an ion source (in this case an Electrospray ion
source), some ion optics to transfer the ions into the magnetic field
(in this case an RF-Only Quadrupole ion guide), and the Ion Cyclotron
Resonance (ICR) cell or Penning trap.
77

Benefits:
 Highest mass resolution of all mass spectrometers.
 Well-suited for ion chemistry and MS/MS experiments.
 Well-suited to be used with pulsed ionization techniques like MALDI,
 Non-destructive ion detection; ion re-measurement.
 Mass calibration is stable in FTICR systems with superconducting magnet .
Limitations:
 Dynamic range is limited.
 Strict low-pressure requirements demands a mandatory external source for
a number of analytical applications.
 Artifacts like harmonics and sidebands are present in the mass spectra.
 Quality of the mass spectrum is influenced by many parameters such as
excitation, trapping, and detection conditions.
 Generally low-energy Collision Induced Dissociation, therefore the spectrum
depends on collision energy, collision gas, and other parameters.
Applications:
 Ion chemistry.
 High-resolution MALDI and electrospray experiments for high-mass analytes.
 Laser desorption for materials and surface characterization.
78

DETECTORS
 Once the ions are separated by the mass analyzer, they
reach the ion detector, which generates a current signal
from the incident ions.
 The most commonly used detectors in MS are as follows:
Faraday cup
Electron multiplier
Photomultiplier dynode
Charge (or Inductive) Detector
80

FARADAY CUP
 A Faraday cup involves an ion striking the dynode (BeO, GaP, or CsSb)
surface which causes secondary electrons to be ejected.
 This temporary electron emission induces a positive charge on the
detector and therefore a current of electrons flowing toward the
detector.
 This detector is not particularly sensitive, offering limited
amplification of signal, yet it is tolerant of relatively high pressure.
81

ELECTRON MULTIPLIER
 It is the most common means of detecting ions. It is made up of a series (12
to 24) of aluminum oxide (Al2O3) dynodes maintained at ever increasing
potentials.
 Ions strike the first dynode surface causing an emission of electrons. These
electrons are then attracted to the next dynode held at a higher potential
and therefore more secondary electrons are generated.
 Ultimately, as numerous dynodes are involved, a cascade of electrons is
formed that results in an overall current gain on the order of one million or
higher.
 The high energy dynode (HED) uses an accelerating electrostatic field to
increase the velocity of the ions and serves to increase signal intensity and
therefore sensitivity.
82

PHOTOMULTIPLIER CONVERSION DYNODE:
 The photomultiplier conversion dynode detector is not commonly used.
 It is similar to electron multiplier in design where the secondary
electrons strike a phosphorus screen instead of a dynode. The
phosphorus screen releases photons which are detected by the
photomultiplier and are then amplified using the cascading principle.
 One advantage of the conversion dynode is that the photomultiplier
tube is sealed in a vacuum, unexposed to the environment of the mass
spectrometer and thus the possibility of contamination is removed.
83

ARRAY DETECTOR
 An array detector is a group of individual detectors aligned in an array
format.
 The array detector, which spatially detects ions according to their
different m/z, has been typically used on magnetic sector mass analyzers.
 Spatially differentiated ions can be detected simultaneously by an array
detector.
 The primary advantage of this approach is that, over a small mass range,
scanning is not necessary and therefore sensitivity is improved.
84

CHARGE (OR INDUCTIVE) DETECTOR
 Charge detectors simply recognize a moving charged particle (an
ion) through the induction of a current on the plate as the ion
moves past.
 This type of detection is widely used in FTMS to generate an image
current of an ion.
 Detection is independent of ion size and therefore has been used on
particles such as whole viruses.
85

VACUUM SYSTEM:
 In order to work in a predictable and efficient way, mass analyzers require
high levels of vacuum so that the analyte ions under investigation must be
manageable and sensitive to the electrostatic components of the instrument.
 Vacuum technology is used to remove the majority of background (air)
molecules, as the deflection of ions by the spectrometer on a specific
pathway takes place only under the influence of electric, magnetic and/or
radiofrequency fields, which would otherwise result in deviation due to
collision.
 A high level of vacuum within the instrument prevents deviation of the
analyte ion from the specified path and assists the processes of ion movement
and separation within the following ways:
 By providing an adequate mean free path for the analyte ions.
 By facilitating collision free ion trajectories.
 By the reduction of ion-molecular reactions.
 By minimization of background interference. 86

Examples of Vacuum Pumps:
 Rotary pumps
 Foreline pumps
 Turbo-molecular pumps
 Diffusion pumps
87

TYPES OF IONS PRODUCED IN MS
1. Molecular ion (Parent ion)
2. Fragment ions
3. Rearrangement ions
4. Metastable ions
5. Multiple charged ions
6. Isotope ions
7. Negative ions
88

MOLECULAR ION (PARENT ION)
 When a 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-
Where, M – represents the Molecule;
M+°– represents the Molecular or Parent ion
 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.
 Most molecules show a peak for the molecular ion, the stability of
which is usually in the order—
Aromatic > Conjugated acyclic polyenes > Alicyclics > n- hydrocarbons
> ketones > ethers> Branched chain hydrocarbons > Alcohols. 89

Characteristics of Molecular ion
Molecular peak is observed if molecular ion remains intact long
enough (10-6 seconds) to reach the detector.
This peak gives the molecular weight of the compound. The
molecular ion peak is usually the peak of the “highest mass
number.”
The molecular ion M+° has mass, corresponding to the molecular
weight of the compound from which it is generated. Thus the
mass of a Molecular ion M+° is an important parameter in the
identification of the compound.
Significance of Molecular ion
Molecular ion peak gives the molecular weight of the compound.
i.e. m/z of molecular ion = molecular weight of the compound.
Ex: C2H5+ (m/e=29) gives the molecular weight of Ethane. 90

IMPORTANT FEATURES OF PARENT ION PEAK
a) The molecular ion peak in aromatic compounds is relatively
much intense due to the presence of 𝜋- electron system.
b) Unsaturated compounds give more intense peak as compared
to saturated or cyclic molecule.
c) Absence of molecular ion peak means that the compound
under examination is highly branched or tertiary alcohol.
d) Primary and secondary alcohol gives very small molecular ion
peaks.
e) In case of chloro or bromo compounds isotope peaks are also
formed along with the molecular ion peak.
 For chloro compounds: M+ and (M++2) peaks are formed in the
intensity ratio 1:3.
 For bromo compounds: M+ and (M++2) peaks are formed in the
intensity ratio 1:1.
91

FRAGMENT ION
When the energy is given further more upto 70 eV,
fragment ions are produced, which has smaller masses.
Formed by both heterolytic and homolytic cleavage of
bonds by simple cleavage and rearrangement process.
Formation is governed by bond dissociation energy and
steric factors. e.g.: ethyl chloride.
92

REARRANGEMENT IONS:
 Rearrangement ions are the fragments whose origin cannot be
described by simple cleavage of bonds in the parent ion, but are result
of intramolecular atomic rearrangement during fragmentation.
 These are probably due to recombination of fragment ions and known
as rearrangement peaks.
 Ex: Prominent peak in spectrum of diethyl ether occurs at m/e 31. This
is due to the ions CH3O+, which is formed by rearrangement of C2H5O+
ions.
 The ‘McLafferty rearrangement’ is a common example. In case of
carbonyl compound.
93

METASTABLE ION
Consider that M1
+ is the parent ion and m1
+ is daughter ion.
If the reaction M1
+ m1
+ takes place in the source, then m1
+
may travel the whole analyzer region and recorded as m1
+ ion.
If the transition M1
+ m1
+ occurs after the source exit and
before arrival at the collector, then m1
+ is called a metastable
ion.
The position of metastable ion is given by:
𝑚∗
=
𝑚1
2
𝑀1
It is important to remember that m* has a distance below m1 on
mass scale same as that of m1 and M1.
The relative abundance of the metastable peak is often of the
order of 10-2 or less compared to the abundance of parent or
daughter ion.
94

CHARACTERISTICS OF METASTABLE IONS:
1) They do not necessarily occur at the integral m/e values.
2) These are much broader than the normal peaks.
3) These are of relatively low abundance.
4) These have low kinetic energy.
95
METASTABLE PEAKS

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-
M+° + e- → M+++ + 4e-
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. 96

ISOTOPE IONS
 Most elements are mixture of two or more stable isotopes
differing by one or two mass units.
 Chlorine and Bromine have two isotopes (35Cl, 37Cl and 79Br, 81Br)
in the ratios 3:1 and 1:1 respectively.
 Thus in the spectrum of methyl bromide the molecular ion peak
is the doublet consisting of two equally intense peaks one at m/e
94 (CH3
79Br) and the other at m/e 96 (CH3
81Br).
 These isotopic clusters are referred to as isotopic peaks. They are
helpful in determining the presence of such elements in a
molecule. 97

NEGATIVE IONS
 The positive ions predominate in electronic impact ionization
because of greater stability. The Negative ions are not very
useful in structural determinations.
 The formation of Negative ions is very rare but these can be
produced in three ways:
1.AB + e- → A+ + B-
2.AB + e- → AB-
3. AB + e- → A+ + B- + e-
98

99
GENERAL MODES OF FRAGMENTATION
SIMPLE
CLEAVAGE
REARRANGEMENT
REACTIONS ACCOMPANIED
BY TRANSFER OF ATOMS
SKELETAL
REARRANGEMENT
a) Heterolytic
Cleavage
b) Homolytic
Cleavage
c) Retro
Diels-Alder
Reaction
a) H-transfer Reaction
b) Scrambling
c) McLafferty
Rearrangement
d) Elimination
e) Ortho-effect
(Ortho Elimination)
a) Molecular ion
rearrangement with
elimination of a
neutral molecule
b) Rearrangement
leading to the
formation of
carbonium ions
c) Rearrangement
leading to the loss of
electron deficient
species
d) 1,2- alkyl or aryl shift

(A) SIMPLE CLEAVAGE
(i) Heterolytic Cleavage
In this type of cleavage both the electrons of the α bond are
taken over by one of the atoms; the fragments are even electron
cation and a radical with the positive charge residing on the
alkyl group.
It may be noted that the cleavage of C ̶ X (X= O,N,S, Cl) bond is
more difficult than that of a C ̶ C bond.
It can be shown by fragmentation of alkyl halide.
100

(ii) Homolytic Cleavage
Homolytic cleavage reactions are very common and can be classed in the
following types:
(a) MODE I.
 Operates in compounds in which a hetero atom is singly bonded to a
carbon atom.
 Parent ion is formed by the removal of one electron from the hetero-
atom. A new bond is formed with the adjacent atom through the
donation of the unpaired electron and the transfer of an electron from
the adjacent bond.
 Eg: alcohols, ethers, amines
101

(b) MODE II.
 When a hetero atom is attached to a carbon atom by a double bond, α-
cleavage is the preferred fragmentation mode.
 Eg: ketones, esters, amides etc.
 Compounds containing C ═ N or C ═ S groups do not show this type of
fragmentation.
102
(c) MODE III.
Benzylic cleavage is an energetically preferred fragmentation mode. It
involves the cleavage of C ̶ C bond which is β to the aromatic ring.

(iii) Retro Diels-Alder Reaction
 This reaction is an example of multicentered fragmentation
which is characteristic of cyclic olefins.
 It involves the cleavage of two bonds of a cyclic system
resulting in the formation of two stable unsaturated fragments
in which two new bonds are formed.
 In simple system, the charge is carried by a diene.
103

(B) REARRANGEMENT REACTIONS ACCOMPANIED BY TRANSFER OF
ATOMS
(i) Hydrogen Transfer Rearrangement Reaction
These involve intramolecular hydrogen transfer rearrangements in
aliphatic hydrocarbons and aromatic compounds. An important
type of hydrogen transfer reaction is scrambling.
SCRAMBLING
 It takes place by the rupture and reformation of C ̶ C bonds and
involves equilibration of identity.
 The formation of tropylium ion from benzyl derivatives is a good
example of hydrogen scrambling.
104

(ii) McLafferty Rearrangement
 It involves the migration of γ-hydrogen atom followed by the cleavage
of a β- bond. The rearrangement leads to the elimination of neutral
molecules from aldehydes, ketones, amines, unsaturated compounds
etc.
 The rearrangement proceeds through a sterically hindered six
membered transition state.
 The structural requirements for this reaction are a side chain of at least
three carbon atoms bearing a γ-hydrogen and a double bond which
could be a carbonyl group or an olefinic double bond or an aromatic
system.
105

(iv) Ortho- Effect
 In suitably substituted aromatic compounds or cis-olefins, the substituent and
a hydrogen can come in close proximity so as to help the elimination of a
neutral molecule. This effect is known as ortho- effect.
 Eg: elimination of methyl alcohol from cis-methylcrotonate.
106
(iii) Elimination
 It operates not only from the molecular ion but also from the fragment ion.
The positive charge remains on the carbon containing fragment.
 The elimination of ketene (CH2 ═ C ═ O) is a characteristic fragmentation
mode of n- alkyl amides and O-acetates of phenols.

(C) SKELETAL REARRANGEMENT
(i) Molecular ion rearrangement with elimination of a neutral molecule
 Induced by radical or charge sites or both may found to have some influence
on rearrangement processes.
 Unsaturated esters and carbonates undergo rearrangements with the loss of
carbon dioxide.
107
(ii) Rearrangement leading to the formation of carbonium ion
It is the rearrangements of even electron fragment ions by the migration of a
nucleophilic species to an electron deficient center which could be a
carbonium ion or other even electron ionic center.

(iii) Rearrangements leading to the loss of an electron deficient species
These are very rare.
108
(iv) 1,2- Alkyl or Aryl Shift
The ring expansion and ring contraction processes in aromatic and
heterocyclic compounds are simple 1,2- alkyl migration whereas
sulphoxides and sulphones undergo 1,2- aryl migration.

INTERPRETATION OF MASS SPECTRA
 Nitrogen rule
 The nitrogen rule states that organic molecules that
contain hydrogen, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur,
and the halogens have an odd nominal mass if they have an odd
number of nitrogen atoms or an even mass if they have an even
number of nitrogen atoms are present. The nitrogen rule is true for
structures in which all of the atoms in the molecule have a number of
covalent bonds equal to their standard valency, counting each sigma
bond and pi bond as a separate covalent bond.
 Rings plus double bonds
 From degree of unsaturation principles, molecules containing only
carbon, hydrogen, halogens, nitrogen, and oxygen follow the formula
 𝑅𝑖𝑛𝑔𝑠 + 𝜋 𝑏𝑜𝑛𝑑𝑠 = 𝑢 = 𝐶 −
𝐻
2
−
𝑋
2
+
𝑁
2
+ 1
Where, C is the number of carbons, H is the number of hydrogen, X is the
number of halogens, and N is the number of nitrogen. 109

 Even electron rule
 The even electron rule states that ions with an even number of
electrons (cations but not radical ions) tend to form even-
electron fragment ions and odd-electron ions (radical ions) form
odd-electron ions or even-electron ions. Even-electron species
tend to fragment to another even-electron cation and a neutral
molecule rather than two odd-electron species.
 OE+•→EE++ R•, OE+•→OE+•+ N
 Stevenson's rules
 The more stable the product cation ion, the more abundant the
corresponding decomposition process . Several theories can be
utilized to predict the fragmentation process, such as the electron
octet rule, the resonance stabilization and hyperconjugation and
so on. 110

 Rule of 13
 The Rule of 13 is a simple procedure for tabulating possible chemical
formula for a given molecular mass. The first step in applying the rule
is to assume that only carbon and hydrogen are present in the
molecule and that the molecule comprises some number of CH "units"
each of which has a nominal mass of 13. If the molecular weight of the
molecule in question is M, the number of possible CH units is n and
𝑀
13
= 𝑛 +
𝑟
13
where r is the remainder. The base formula for the molecule is
𝐶 𝑛 𝐻 𝑛+𝑟
and the degree of unsaturation is
𝑢 =
(𝑛 − 𝑟 + 2)
2
 A negative value of u indicates the presence of heteroatoms in the
molecule and a half-integer value of u indicates the presence of an odd
number of nitrogen atoms.
 On addition of heteroatoms, the molecular formula is adjusted by the
equivalent mass of carbon and hydrogen. For example, adding N
requires removing CH2 and adding O requires removing CH4. 111

SOME EXAMPLES
FOR INTERPRETATION
OF
MASS SPECTRA
112

• 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.
113

114
• Mass spectrum of 2-methylpentane

115
• Alkenes:
– Fragmentation typically forms resonance
stabilized allylic carbocations

116
• Aromatics:
– Fragment at the benzylic carbon, forming a
resonance stabilized benzylic carbocation (which
rearranges to the tropylium ion)
CH
H
CH Br
H
C
H
H
or
M+

117
Aromatics may also have a peak at m/z = 77 for the
benzene ring.
NO2
77
77
M+ = 123

118
• 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+

119
• MS for 1-propanol
CH3CH2CH2OH
H2C OH
M+-18 M+

120
• Amines
– Odd M+ (assuming an odd number of nitrogens
are present)
– a-cleavage dominates forming an iminium ion
CH3CH2 CH2 N
H
CH2 CH2CH2CH3 CH3CH2CH2N CH2
H
m/z =72
iminium ion

121
86
CH3CH2 CH2 N
H
CH2 CH2CH2CH3
72

122
• Ethers
– a-cleavage forming oxonium ion
– Loss of alkyl group forming oxonium ion
– Loss of alkyl group forming a carbocation

123
MS of diethylether (CH3CH2OCH2CH3)
H O CH2
H O CHCH3
CH3CH2O CH2

124
• Aldehydes (RCHO)
– Fragmentation may form acylium ion
– Common fragments:
• M+ - 1 for
• M+ - 29 for
RC O
RC O
R (i.e. RCHO - CHO)

125
MS for hydrocinnamaldehyde
C C C H
H
H
H
H
O
133
105
91
91
105
M+=134

126
• Ketones
– Fragmentation leads to formation of acylium ion:
• Loss of R forming
• Loss of R’ forming
RCR'
O
R'C O
RC O

127
• MS for 2-pentanone
CH3CCH2CH2CH3
O
CH3C O
CH3CH2CH2C O
M+

128
• Esters (RCO2R’)
– Common fragmentation patterns include:
• Loss of OR’
– peak at M+ - OR’
• Loss of R’
– peak at M+ - R’

129
C
O
O CH3
105
77
77
105
M+ = 136
MS for Methyl benzoate

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