Mass Spectroscopy

Presented By
F. Y. M. Pharm
Pharmaceutics
❖ Contents:
• Principle
• Theory
• Instrumentation and working of MS
• Different types of ionization Introduction
• Electron impact
• Chemical field
• FAB
• MALDI
• APCI
• ESI
• APPI
• Analyzers of quadrupole
• Time of flight
• Mass fragmentation and it's rules
• Meta stable ions and isotopic peaks
• Applications of MS
INTRODUCTION
 Mass Spectroscopy
• Mass spectroscopy is one of the primary
spectroscopic methods for molecular analysis
available to organic chemistry.

• 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.

• It is not concerned with non- destructive interaction

between molecules and electromagnetic radiation.
• It involves the production and separation of ionised molecules
and their ionic decompositon product and finally the
measurement of the relative abundance of different ions
produced. It is, thus a destructive technique in that the sample is
consumed during analysis.

• In most cases, the nascent molecular ion of the analyte

produced fragment ions by cleavage of the bond and the
resulting fragmentation pattern constitutes the mass spectrum.

• Thus, the mass spectrum of each compound is unique and can

be used as a "chemical fingerprint" to characterize the sample.
 Basic Principle
• Mass spectroscopy is the most accurate method for
determining the molecular mass of the compound and its
elemental composition.

• In this technique, molecules are bombarded with a beam of

energetic electrons.

• The molecules are ionised and broken up into many

fragments, some of which are positive ions.

• Each kind of ion has a particular ratio of mass to charge,

i.e. m/e ratio(value). For most ions, the charge is one and
thus, m/e ratio is simply the molecular mass of the ion.
• 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.
 Basic Theory
• Mass spectroscopy deals with the examination of the
characteristics fragments(ions) arising from the
breakdown of organic molecules.

• A mass spectrum is the plot of relative abundance of

ions against their mass/charge ratio.

• The basic aspect of organic mass spectrometry consist

of bombarding the vapour of an organic compound
with a beam of energetic electron accelarated from a
filament to an energy of 70 eV to form positively
charged ions (molecular ions).
• The additional energy of the elecrons is dissipated in
breaking the bonds in the molecular ion, which
undergoes fragmentation to yield several neutral or
positively charged species.

• This fragmentation may result in the formation of an

even-electron ion and radical.

• The various positive ions, thus formed, can be

accelerated and deflected by magnetic or electric
fields.

• The deflection of ions, however, depends on its mass,

charge and velocity
• Though organic mass spectrometry is routinely used
along with IR, NMR and UV for structure
detemination, its basic theory is different from the
others.

• In mass spectrometry no characteristic selective

absorption of radiation is involved as in the case of the
other three methods, secondly, in the mass
spectrometry, the compound undergoes irriversible
chemical changes unlike in the others, where the
changes are reversible physical changes.

• The mass spectral reactions are much more drastic

than usual chemical reactions.
INSTRUMENTATION OF
MASS SPECTROMETER
​Components of mass spectroscopy
1. Sample inlet systems
• Permits introduction of sample in to the ion source.
• Mass spectrometer should have a vapour sample and it
should enter the ionisation chamber at constant rate.

• The sample is converted into gaseous state in the inlet system.

• The rate at which sample is introduced into ionization

chamber must remain constant so that to achieve relative
abundance of different species in mass spectrum can be
determined.
1. Gas samples:
Transfer of sample from small containers like
glass bulb, of known volume( about 3ml)
coupled to a mercury manometer.
2. Liquid samples:
Hypodermic needle injection through a silicone rubber
dam. The low pressure in reservoir draws in the liquid
and vaporizes it instantly.

3. Solids or Non-volatile liquids:

Directly introduced on silica or platinum probe and then
volatilized by gentle heating.
2. Ion Sources:
Chemical ionization: In this method ionization results
from ion molecule chemical interaction.
The interaction process involves large amount of reagent
gas and a small amount of sample.

The reagent ion and sample molecule may react via any
of the several modes like proton exchange.

Reagent gas: Argon, helium and nitrogen show

fragmentation patterns with increased sensitivity.
3. Mass analyzers and ion separator:

▪ After passing through the electrostatic slits, ions enter

in to the analyzers.
▪ Separate the ions according to their mass by charge
ratio.

Different types of mass analyzers are:

1. Magnetic deflection or sector mass analyzer.
2.Double focus mass analyzer .
3.Quadrupole mass analyzer.
4. Time of flight mass analyzer.
1. Magnetic deflection or sector mass analyzer:

▪ In this type, the homogeneous beam of ions from the

slit can be focus by a magnetic field in a shape of a
sector.
▪ Gas phase molecules are ionized by a beam of high
energy electrons.
▪ Electrons may be ejected from molecules (ionization)
or bonds in molecules may rapture (fragmentation)
▪ Ions are then accelerated in a field (sector) at a
voltage V.
▪ Basis of separation by m/z.
3.Quadrupole mass analyzer:

• The Quadrupole consists of four parallel rods.

• The opposing rods have the same polarity while
adjacent rods have opposite polarity.
• Each rod is applied with a DC and RF (Oscillating
radio frequency) voltage.
• Ions are scanned by varying the DC/RF quadrupole
voltages.
• Only ions with the selected mass to charge ratio will
have the correct oscillatory pathway in the RF field.
4. Time of flight mass analyzer:

• As the name indicates the mass spectrum is obtained

depending upon the time it took for the ion to reach the
detector from accelerating chamber.
• The ions are accelerated and accelerated particles
entering the tube have same kinetic energies.
• The lighter particles arrives at the collector earlier than
heavier particles.

t = L/v ---time of flight equation.

t - time taken for the ion to pass through a drift tube.

v - velocity of the charged particle.
L - length of the tube.
4. Detectors:

▪ Ions are amplified and recorded.

▪ Measure one m/z value at a time (single channel
detectors)
▪ Multiple detectors are used for multiple ion
detection
▪ High resolution magnetic sector instruments use
multiple detectors (called Multicollectors)
Various detectors used are-

I. Faraday cup collector.

II. Electron multiplier.

III. Ion sensitive photographic plates.

IV. Array detector.

I. Faraday cup collector:
➤ Simple and effective means of monitoring ion
current.
➤ When sufficient amount of sample is available,
the measurement can be done by collecting them in
single Faraday cup and scanning for different mass
by charge values.
Advantages:
Current as low as 105A may be detected.
Disadvantage:
It needs high impedance amplifier, which limits the
speed at which spectrum can be scanned.
II. Electron multiplier:
➤ When the amount of sample available is small,
the ion current produced is such a low current,
which cannot be measured directly with a Faraday
cup and further amplification is ca out by the
secondary electron multiplier.
III. Ion sensitive photographic plates:
➤Here the ions directly exposed to a photographic
film.

➤ This type of detectors used in mass analyzers.

Which disperse ions according to their mass to charge
ratio.

➤ The ion sensitive photographic plate integrate the

ions and record them.

➤ This detector provides greater resolution and

highest sensitivity.
IV. Array Detectors:

➤ Used in Time of Flight Mass instruments

➤ Employs a focal plane camera (FPC) consisting of an

array of 31 Faraday Cup

➤ Up to 15 m/z values can be measured simultaneously

➤ Exhibits improved precision compared with single

channel detectors.
5. The vacuum system:
➤ Whole instrument is kept under high vacuum condition
10-6 to 10-7 torr.

Types of pump systems are used –

1. Oil diffusion pumps

2. Mercury diffusion pump

6. Data handling:
➤ Data are digitalized and collected on magnetic tape or
stored in a memory of computer.

7. Recorders:
➤ Multichannel ultraviolet recorder.

➤Pens, tape recorders, oscillators other data collecting

systems are used as recording devices.
Working of Mass
Spectrophotometry
Ionization Types
Electron Impact
• Electron Impact Ionization (EI) is one of the most
common ionization techniques used in mass
spectrometry (MS), especially for analyzing small,
volatile, and thermally stable molecules.
• It provides highly detailed fragmentation patterns that
help identify molecular structures.
• Ionization Energy: Typically 70 eV electrons bombard
the sample.
• Produces extensive fragmentation, useful for
structural analysis
 Working Principle of EI-MS
Step-by-step process:
1.Sample Vaporization → The sample is converted
into gas phase.
2.Electron Beam Interaction → High-energy electrons
collide with sample molecules.
3.Ionization Reaction : M+e−→M+⋅+2e−
where M is the analyte molecule being ionized, e− is the
electron and M+• is the resulting molecular ion.
1.Fragmentation → Unstable molecular ions break
into smaller ions.
2.Mass Analyzer & Detection → Ions are separated
based on m/z ratio and detected.
• In an EI ion source, electrons are produced
through thermionic emission by heating a wire
filament that has electric current running through it.
The kinetic energy of the bombarding electrons
should have higher energy than the ionization
energy of the sample molecule.
• The electrons are accelerated to 70 eV in the region
between the filament and the entrance to the ion
source block. The sample under investigation which
contains the neutral molecules is introduced to the ion
source in a perpendicular orientation to the electron
beam.
• Close passage of highly energetic electrons in
low pressure (ca. 10−5 to 10−6 torr) causes
large fluctuations in the electric field around
the neutral molecules and induces ionization
and fragmentation. The fragmentation in
electron ionization can be described using
Born Oppenheimer potential curves as in the
diagram.
• •The red arrow shows the electron impact
energy which is enough to remove an electron
from the analyte and form a molecular ion
from non- dissociative results.
• Due to the higher energy supplied by 70 eV electrons
other than the molecular ion, several other bond
dissociation reactions can be seen as dissociative
results, shown by the blue arrow in the diagram.
• These ions are known as second-generation product
ions. The radical cation products are then directed
towards the mass analyzer by a repeller electrode.
• The ionization process often follows predictable
cleavage reactions that give rise to fragment ions
which, following detection and signal processing,
convey structural information about the analyte.
❑ Mass Spectrum Interpretation
•Molecular Ion Peak (M⁺·): Represents the intact
molecule.
•Base Peak: The most abundant fragment ion.
•Fragment Ions: Help deduce molecular structure.
•Example mass spectrum of a simple molecule (e.g.,
benzene, ethanol).
 Advantages:
High sensitivity and reproducibility
Extensive spectral libraries for easy identification
Provides rich fragmentation patterns for structural
analysis
Disadvantages:
Extensive fragmentation may lose the molecular ion
peak
Not suitable for large or thermally unstable
molecules
❑ Applications

Drug and pharmaceutical analysis

Environmental monitoring (pesticides, pollutants)
Forensic investigations
Organic compound identification
Chemical field / Chemical Ionization
❖ Chemical field / Chemical Ionization:

Introduction :

•Chemical Ionization (CI) is a soft ionization

technique in mass spectrometry.
•It uses reagent gases to generate ionized
species that react with the analyte.
• Results in minimal fragmentation, making
molecular weight determination easier.
Working Principle:

•1. A reagent gas (e.g., methane, isobutane,

ammonia) is introduced.
•2. High-energy electrons ionize the reagent gas,
forming reagent ions.
•3. These reagent ions transfer a charge to the
analyte, forming (MH⁺) or (M⁻).
•4. The ions are analyzed in a mass spectrometer.
Types of Chemical Ionization:

1. Positive Chemical Ionization (PCI):

• Proton transfer reaction (MH⁺ formation).

• Suitable for basic compounds (amines, alcohols,
etc.).
•
2. Negative Chemical Ionization (NCI):

• Electron capture or deprotonation (M⁻ formation).

• Suitable for electron-deficient molecules
(halogenated, acidic compounds).
Comparison: CI vs. EI
Chemical Ionization (CI) Electron Ionization (EI)

 Ionization Type: Soft  Hard

 Fragmentation : Low  High
 Molecular Ion  Weak or absent
Visibility : Strong  Structural analysis
(MH⁺)
 Best For : Molecular
weight determination
Advantages:

Strong molecular ion peak for easy

identification.

Low fragmentation, useful for thermally

unstable compounds.

Positive and negative ionization modes for

selectivity.
Disadvantages:

Requires a reagent gas and additional setup.

Limited to compounds that can undergo

protonation or electron capture.

Less fragmentation may make structural

elucidation harder.
Applications of Chemical Ionization:

• Pharmaceutical Analysis – Identifying drug

molecules and metabolites.

• Environmental Studies – Detecting pesticides and

halogenated pollutants.

• Biochemical Research – Studying lipids, steroids,

and other biomolecules.

• Petroleum Industry – Characterizing hydrocarbons

in crude oil.
Conclusion:

• Chemical Ionization is a useful soft ionization

technique in mass spectrometry.

• Provides strong molecular ion signals with minimal

fragmentation.

• Suitable for pharmaceutical, environmental, and

biochemical applications.

• Choice of reagent gas and ionization mode

determines effectiveness.
FAB
 MALDI

(MATRIX –ASSISTED LASER

DESORPTION/IONIZATION)
INTRODUCTION
 Matrix-assisted laser desorption/ionization
 Soft ionization technique used in mass spectrometry
 Analysis of bio molecules and large organic
molecules
 The ionization is triggered by a laser beam
 It is used to determine the molecular weight of
Peptides, Proteins, Antibodies upto size to 300 kDa
• The sample is dispersed in a large excess of matrix material
which will strongly absorb the incident light.
• The matrix contains chromophore for the laser light and since the
matrix is in a large molar excess it will absorb essentially all of
the laser radiation
• The matrix isolates sample molecules in a chemical environment
which enhances the probability of ionization without
fragmentation
• Short pulses of laser light (UV, 337 nm) focused on to the sample
spot cause the sample and matrix to volatilize
• The ions formed are accelerated by a high voltage supply and
then allowed to drift down a flight tube where they separate
according to mass
• Arrival at the end of the flight tube is detected and recorded by a
high speed recording device
 ❑ Mechanism of MALDI
The mechanism of MALDI Done in three
steps...
1. Formation of a Solid Solution
2. Matrix Excitation
3. Analyte lonization

1. Formation of solid solution

• It is essential for the matrix to be in access thus leading to the

analyte molecules being completely isolated from each other.

• This eases the formation of the homogenous solid solution

required to produce a stable desorption of the analyte.
2. Matrix Excitation

• The laser beam is focussed onto the surface of the matrix-

analyte solid solution.
• The chromophore of the matrix couples with the laser
frequency causing rapid vibrational excitation, bringing
about localised disintegration of the solid solution.
• The clusters ejected from the surface consists of analyte
molecules surrounded by matrix and salt ions.
• The matrix molecules evaporate away from the clusters to
leave the free analyte in the gas-phase.
3. Analyte lonisation

• The photo-excited matrix molecules are stabilised through

proton transfer to the analyte.
• Cation attachment to the analyte is also encouraged during
this process.
• It is in this way that the characteristic [M+X(X= H, Na, K
etc.) analyte ions are formed.
• These ionisation reactions take place in the desorbed matrix-
analyte cloud just above the Surface.
• The ions are then extracted into the mass spectroscopy for
analysis
❑ INSTRUMENT

The MALDI technique combined with a MS detector (MALDI-

MS) became an indispensable tool in analysis of biomolecules
and organic macromolecules.

MALDI involves
incorporation of the
analyte into a matrix,
absorption/desorption
of laser radiation, and
then ionization of the
analyte.
o According to Sigma Aldrich, the matrix must meet
the following properties and requirements :

• Be able to embed and isolate analyte (e.g. by co-

crystallization)
• Be soluble in solvents compatible with analyte
• Be vacuum stable
• Absorb the laser wavelength
• Cause co-desorption of the analyte upon laser
irradiation
• Promote analyte ionization
❑ MALDI MATRIX
• The analyte incorporation in to a suitable matrix is the
first step of the MALDI process, and is an important
feature of the MALDI method.
• A typical sample preparation involves using 10 M
solution of the analyte mixed with 0.1 M solution of the
matrix.
• The solvents are then evaporated in a vacuum of the
MS, and the matrix crystallizes with the analyte
incorporated
 ❑ MALDI LASER

• Numerous gas and solid state lasers have been developed for
use in MALDI.
• Most MALDI devices use a pulsed UV laser N, source at 337
nm.
• Neodymium-yttrium aluminum garnet (Nd:YAG) Emits at 355
nm and gives a longer pulse time
• IR lasers are also used
• The most common IR laser is the erbium doped-yttrium
aluminum garnet (Er:YAG). Emits at 2.94 micrometer.
• It is "softer" than the UV, which is useful for certain
biomolecules.
 MALDI Advantages

• Gentle Ionization technique

• High molecular weight analyte can be ionized
• Molecule need not be volatile
• Sub-picomole sensitivity easy to obtain
• Wide array of matrices
MALDI Disadvantages

• MALDI matrix cluster ions obscure low m/z species (<600)

• Analyte must have very low vapor pressure
• Pulsed nature of source limits compatibility with many mass
analyzers
• Coupling MALDI with chromatography can be difficult
• Analytes that absorb the laser can be problematic
• Fluorescein-labeled peptides
❑ APPLICATIONS OF MALDI
Applications of MALDI mass spectrometry in detection of-

• Synthetic polymers
• Peptides and proteins
• Oligonucleotides
• Oligosaccharides
• Lipids
• Inorganics
• Bacterial identification
Atmospheric Pressure Chemical
Ionization (APCI).
INTRODUCTION

This is an ionization method in which the sample is

ionized using an ion molecule reaction with a reactant
ion. Sample solution is nebulized by the N2 nebulizer
gas to form a spray as it enters the heater(at about
400°C), and both sample and solvent molecules are
vaporized to a gaseous state. The solvent molecules are
ionized by the corona discharge, and stable reactant
ions are formed.
Protein transfer occurs between these reactan
ions and sample molecules (ion molecule
reaction), and the sample molecules either add
or lose protons to become ions. This ion
molecule reaction is known to occur in a
variety of patterns, such as protein shift
reactions, electrophilic addition reactions, etc.
 The APCI process

 ➤The sample is in a flowing stream of a carrier liquid (or

gas) and is nebulized at moderate temperatures.

 ➤This stream is flowed past an ionizer which ionizes the

carrier gas/liquid.

 ➤ The ionized stream acts as the primary reactant ions,

forming secondary ions with the analytes.
The ions are formed at AP in this process,
and are sent into the vaccuum

➤In the vaccuum, a free-jet expansion

occurs to form a Mach disk and strong
adiabatic cooling occurs.

➤Cooling promotes the stability of

analyte ions
Benefits:

• Excellent LC/MS interface

• Good for less-polar compounds

➤ Compatible with MS/MS methods

Mass range:

➤ Low-moderate Typically less than

2000 D.
Limitations:

➢ Very sensitive to contaminants such as

alkali metals or basic compounds.

➢ Relatively low ion currents.

➢ Relatively complex hardware compared

to other ion sources.
ESI
 APPI
( Atmospheric Pressure
Photoionization)
❖ Introduction
• APPI (Atmospheric Pressure Photoionization) is one
of the ionization techniques used in mass
spectrometry (MS), particularly for analyzing
nonpolar and less polar compounds.
• It operates under atmospheric pressure and is often
used in combination with liquid chromatography
(LC-MS).
• Similar to APCI , the liquid effluent of APPI is
introduced directly into the ionization source.
•The primary difference APCI &APPI is that the APPI
•Vaporized sample pass through the ultra violet light (a
typically krypton light source emit at 10.0eV &10.6eV)

•Often APPI is much more sensitive than ESI OR APCI

❖ PRINCIPAL
• In APPI techniques samples are ionized by using UV
light
• A UV lamp (typically 10 eV–11 eV) is used to ionize
either the analyte directly or a dopant/solvent, which
then transfers the charge to the analyte.
• Molecules interact with photon beam of UV light
with vapor of nebule solutions
• Analyst molecules (AI) absorb Photon (hv) and
became a electrically exited molecules.
• If the ionization energy (IE) of analyte molecule is
lower than the Photon, the energetic molecules release
energetic electrons and became radial
• The ionization occurs at atmospheric pressure,
making it suitable for coupling with liquid
chromatography (LC).
APPI MECHANISM
❖ ADVANTAGES
• Works well for nonpolar and low-polarity
compounds.

• Complementary to ESI (Electrospray Ionization) and

APCI (Atmospheric Pressure Chemical Ionization).

• Suitable for volatile and thermally stable molecules

❖ DISADVANTAGES
• It required vaporization temperature ranging from
350-550°C, which can cause thermal degradation

• Much more expensive than ESI and APCI

• Depends on solvent
❖ Application
1. Pharmaceutical Analysis – Used for detecting and
characterizing low-polarity drug molecules.
2. Petroleum Analysis – Identifies and quantifies
hydrocarbons, including polycyclic aromatic
hydrocarbons (PAHs).
3. Environmental Monitoring – Detects pollutants,
pesticides, and other organic contaminants in
environmental samples.
4. Lipidomic – Analyzes neutral lipids and sterols that
are difficult to ionize with ESI.
5. Metabolomics – Helps in profiling nonpolar
metabolites in biological samples.
6. Food Safety – Identifies food contaminants, including
persistent organic pollutants (POPs).
7. Forensic Science – Used for detecting drugs of abuse
and other illicit substances.
8. Polymer and Material Science – Characterizes
synthetic polymers and their degradation products.
9. Explosives Detection – Identifies trace amounts of
explosive compounds.
10. Cosmetic and Fragrance Industry – Analyzes
volatile and semi-volatile fragrance compounds.
ANALYZERS OF
QUADRAPOLE
QUADRUPOLE MASS ANALYZER
(QMA)

1. The QMA is one type of mass analyser which is

used in mass spectroscopy.
2. It is also called as transmission QMA, quadrupole
mass filter.
3. Consists of four parallel rods that separate ions
based on their mass-to-charge ratio (m/z) using a
combination of DC and RF voltages.
PRINCIPLE:

1.It consists of four metal rods which are parallel to

each other.
2. The opposing rod pair is connected together
electrically.
3.A R.F voltage and a D.C voltage is applied.
4. lons will travel through the quadrupole.
5. Only ions of certain m/z ratio will reach the
detector
Construction:

• It consists of four electrically conducting parallel

rods.
• One diagonally opposite pair of rods is held at
+Udc volts and the other pair at Ude volts.
• A radiofrequency oscillator supplies +Vcosot to
the first pair and -Vcosot to the second pair.
• Ions enter to the quadrupole through a circular
aperture.
WORKING:
• lons from the ion source will make their way to
the quadrupole mass filter and finally reach the
detector.
• The main functions of QMA is to separate the
particles based on m/z ratio
• Now, a mixture of ions comes from the ion source.
• This mixture will have positive, negative, small
and large particles.
• For instance let us consider the pair of positive
electrodes first
• Now let us consider the positive charged particles
are approaching the positive electrode.
• Let us now consider the large size particle first.
• A positive D.C current is applied to the pair of
electrodes.
• When they approach the positive electrode, they
get repelled.
• They just make the way out of the filter.
• An A.C current is applied to the electrode.
• This A.C will have both positive and negative
phase.
• When positive phase exists, it will make the
electrode positive
• Thus ions get repelled.
• But when negative phase exists, it will make the
electrode negative.
• Thus the large particles will get attracted. But when
positive phase again exists, they repel and make
their way out of the filter.
• But the small particles, since they have small mass,
they will be attracted quickly than large particles.
but when positive phase exists, they get repelled. by
the time they get repelled, the negative phase exists
and they get attracted and they finally crash on the
electrode.
• Hence large particles reach the detector first.
• This is called as high pass filter.
• Now let us consider the negative electrode.
• A D.C current of negative is applied to the
electrode.
• Taking again, the positive charged particles into
consideration, we see that they get attracted
towards the electrode and they crash.
• This is not a filter.
• An A.C current is applied to the electrode.
• The large particles will get attracted towards
negative electrode.
• By the time it gets attracted, the electrode will
become positive.
• Now let us consider the negative electrode.
• A D.C current of negative is applied to the
electrode.
• Taking again, the positive charged particles into
consideration, we see that they get attracted
towards the electrode and they crash.
• This is not a filter.
• An A.C current is applied to the electrode.
• The large particles will get attracted towards
negative electrode.
• By the time it gets attracted, the electrode will
become positive.
ADVANTAGES:

1. Compact and reliable.

2. Low cost analytical tool.
3. Determine the molecular weight in many
compounds.
4. To identify the structure of many compounds.
DISADVANTAGES:

1. It is a low resolution mass analyzer.

2. Only one m/z ratio value can be obtained at a
time.
3. Broader m/z ratios values can be obtained only
after long scanning periods.
4. Not suited for pulsed ionisation method.
APPLICATIONS:

1. Compositional analysis of gases and volatile

liquids.
2. Mainly used in residual gas analysis.
3. Used in liquid chromatography- mass
spectrometry and gas chromatography- mass
spectrometry.
4. Used in both qualitative and quantitative
analyses.
Time of flight
 ❑ INTRODUCTION:

• Stephens first described the concept of time of flight in 1946.

• As the name implies, time of flight mass spectrometer

separates ions and measure their m/z based on the time they
take to pass ("fly") from the ion source to the detector.
❑PRINCIPLE:

• Ions with different masses and same kinetic energy

travel when accelerated and the ions with smaller
masses (less m/z value) reaches early to the detector
than the bigger masses.
•Sample Ionization: The sample is ionized using techniques like laser
ablation or electrospray ionization to generate ions (charged particles).
•Acceleration of Ions: Ions are accelerated by an electric field to the same
kinetic energy.
•Ion Flight Tube: The accelerated ions travel through the flight tube,
which is a vacuum chamber with no electric or magnetic fields.
•Detection: Once ions hit the detector, a signal is produced based on the
time it takes to reach the detector.
•Measurement of Time of Flight: The time taken by each ion to travel
through the flight tube is measured.
•Calculation of m/z: Using the time-of-flight data and the known energy
of the ions, their mass-to-charge ratio (m/z) is calculated.
•Mass Spectrum Generation: Finally, a mass spectrum is generated
showing the distribution of the ions based on their m/z values.
 ❑VARIANTS OF LINEAR TOF ANALYSER

1. Ion mirror/ion reflectron / reflectron.

2. Time-lag focussing

o NEED FOR VARIANT TYPE OF TOF ANALYSER

• Ions with very similar m/z ratio may have relatively poor
mass resolutions.
• This is due to the spatial distribution in the ion source and
their proximity to the applied electric field, not all the ions
receive the same kinetic energy.
• This leads to components in mixtures being unresolved from
one another and large errors in molecular
weight measurements.
ION MIRROR/ION REFLECTRON / REFLECTRON

• Construction and working:

• It consists of stack of donut shaped lens connected by a series
of resistors across which voltage is applied.

• These lenses repell the ions entered.

• These ions are reflected down at second flight tube to a

second detector.

• Mass resolution is achieved as ions of different kinetic

energies penetrate the mirror to differing degrees.
TIME-LAG FOCUSSING / PULSED ION EXTRACTION /
DELAYED EXTRACTION

Construction and working:

• In time-lag focusing TOF applying accelerating potential is
delayed.

• Ions from the ion source will have some kinetic energy
and moves with different velocities

• Application of potential gives more energy to the ions

which are away from the detector than closer to it.

• The amplitude is adjusted in such a way that all the ions

reach the detector at the same time.
 ❑ ADVANTAGES OF TOF MASS ANALYSER

• Mass range is unlimited.

• Excellent sensitivity due to lack of resolving slits.

❑ DISADVANTAGES OF TOF MASS ANALYSER

• Limited use in case of pulsed ionization techniques.

• Mass resolution is usually less than 20000.Fast electronics

are necessary for adequate resolution.
 ❑APPLICATION OF TOF MASS ANALYSER

• Due to its fast scanning capability it is increasingly

being used in LC-MS instrumentation.

• Generally it is used in case of fast analysis or in high

chromatographic resolution techniques.
Mass Fragmentation and its Rules
Content

• Basic concepts
• Fragmentation Process
• McLafferty Rearrangement
Basic Concept

• Mass spectrometry uses high energy electrons to break a

molecule into fragmentation.
• A beam of high-energy electrons breaks the molecule
apart.
• The masses of the fragments and their relative abundance
reveal information about the structure of the molecule.

Separation and analysis of the fragments provides

information about:
1. Molecular weight
2. Structure
Fragmentation Process
• Bombardment of molecules by an electron beam with
energy between 10-15ev usually results in the ionization of
molecules by removal of one electron (Molecular ion
formation)

• When the energy of electron beam is increased between 50-

70ev, these molecular ions acquire a high excitation
resulting in their break down into various fragments. This
process is called "Fragmentation process".
McLafferty Rearrangement
Fragmentation due to rearrangement of Molecular or Parent
ion:
• The cleavage of bonds in Molecular ion is due to the
intramolecular atomic rearrangement. This leads to
fragmentation whose origin cannot be described by simple
cleavage of bonds.
• When fragments are accompanied by bond formation as
well as bond for breaking, a rearrangement process is said
to have occurred.
• Such rearrangement involves the transfer of hydrogen from
one part of the molecular ion to another via, preferably, a
six-membered cyclic transition state.
• This process is favoured energetically because as many
bonds are formed as are broken.
• Compounds containing hydrogen atom at position gamma
to carbonyl group have been found to a relative intense
peak.
• This is probably due to rearrangement and fragmentation
is accompanied by the loss of neutral molecule. This
rearrangement is known as Mc Lafferty rearrangement.
Butanal contains a y- hydrogen atom. The McLafferty ion
formed in this case is shown below:

Similarly, a large number of organic compounds viz. ketones,

amines, alcohols, esters, acids which contain a y-hydrogen
atom forms as a McLafferty rearrange ion,
Thus, the molecular formula of the unknown compound can
be determined from the various fragment ions and also the
parent ion of the mass spectrum.
More example of McLafferty rearrangement's are:
• A double McLafferty rearrangement is also reported in ketones.
• The second hydrogen atom originates exclusively from the y
position.
• A secondary hydrogen is preffered to a primary hydrogen atom
in this process.
The mechanism involves.
i. Ketonisation of the intermediate enol ion by the hydrogen
transfer.
ii. ii. Hydrogen transfer to enolic oxygen rearrangement in 4-
Heptanone.
META Stable Ions
&
Isotopic Peaks
 META Stable Ions

• These ions are typically formed during the ionization

process in mass spectrometry and are not in their most
stable state.
• A metastable ion is Excited state ,unstable and typically
break apart.
• These are ions that fragment after leaving the ion source
but before reaching the detector, resulting in broad, low-
intensity peaks at non-integral mass-to-charge ratios.
• Metastable ions are formed when an ion, with enough
internal energy, dissociates (fragments) during its flight
through the mass spectrometer, but before it's detected.
• Fragment of a parent ion ( Molecular) will give rise to a new
ion (daughter) plus either a neutral molecule or a radical.

M1+ M2+ + Non charged particle

• Consider that M1+ is the parent ion and M2+ is daughter ion.
• If the fragmentation of parent ion occurs in source then called
as fragment ion.

• If the reaction M1+ M2+ takes place in the source,

then M2+ may travel the whole analyzer region and recorded
as M2+ ion.

• If the transition M1+ M2+ occurs after the source exit and
before arrival at the collector, then M2+ is called a metastable
ion.
• The position of metastable ion is given by:
M*= M22/M1
• The resultant daughter ion M2+ will not be recorded as either
M1, but at a position M* (observed metastable ion m/z
value) as a rather broad, poorly focused peak. Such ion
called as metastable ion.
The process involves the following steps:
• An ion is formed, often by methods like electron impact (EI)
or electrospray ionization (ESI).
Ionization and Fragmentation:
•In a mass spectrometer, molecules are first ionized in an ion
source, creating ions (including molecular ions, M+).
•Some of these ions, particularly those with excess internal
energy, can become unstable and undergo fragmentation,
breaking down into smaller ions (fragment ions).
Field-Free Regions:
•After leaving the ion source, ions travel through field-free
regions, where there are no electric or magnetic fields to
influence their motion.
Metastable Ion Formation:
• If an ion with excess internal energy fragments within one of
these field-free regions, the resulting fragment ion is called a
metastable ion.
• Decay occurs infield free region, not at detector.
Detection:
• Metastable ions, because they fragment in flight, appear as
weak, diffuse peaks at non-integer m/e values in the mass
spectrum, distinct from the peaks of precursor ions that
fragment in the source or those that reach the detector intact.
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.
5) 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.
6) Hard to detect
Meta stable ion Peak
 Isotopic Peaks

• An isotopic ion in mass spectrometry refers to an ion that

arises from an isotope of an element present in a molecule.
• Isotopes are atoms of the same element with the same
number of protons but a different number of neutrons,
leading to a different atomic mass.
• Many elements have naturally occurring isotopes (atoms of
the same element with different numbers of neutrons).
• Mass spectrometry separates ions based on their mass-to-charge
ratio (m/z).
• The mass spectrometer detects ions with different masses
corresponding to the isotopes of the elements in the molecule.
• These isotopes have slightly different masses, leading to multiple
peaks in the mass spectrum, even for the same molecule.
• These peaks, other than the main molecular ion peak, are called
isotopic peaks.
 Mass Spectrum Peaks: When a molecule is ionized in mass
spectrometry, peaks corresponding to different isotopes of the
elements in the molecule appear in the mass spectrum. These peaks
are typically observed as clusters of peaks near the parent ion.
Isotopic Distribution: The relative intensities of these isotopic
peaks depend on the natural abundance of the isotopes. For
example:
•The ¹³C isotope occurs about 1.1% of the time for each carbon
atom in the molecule, so the intensity of the peak corresponding
to the molecule with ¹³C is much smaller than the peak
corresponding to the molecule with ¹²C.
•Similarly, ²H occurs less frequently than ¹H, so deuterated
molecules show less intensity in the corresponding isotopic peak.
Isotopic Patterns: The appearance of isotopic peaks can help
identify the elements in the molecule. For example:
• A molecule of C₆H₁₂O₆ will show peaks corresponding to ¹²C
and ¹³C and will have a characteristic isotopic pattern based on
the distribution of carbon isotopes.
Use of Isotopes in Mass Spectrometry:
• Isotopic labeling is often used in quantitative analysis and
structural elucidation.
• For instance, deuterium (²H) labeling can help track reactions or
processes, and the pattern of isotopic peaks can provide
structural information.
Significance of Isotopic Peaks:
•Molecular Identification: The presence of isotopic peaks allows
for structural determination and confirmation of molecular
formulas.
•Elemental Analysis: They provide information about the
elements present in the molecule (for example, chlorine or
bromine isotopic patterns).
•Quantitative Analysis: Isotopic peaks can help in quantitative
analysis, particularly when using isotope labeling.
Structural Elucidation Using Isotopic Peaks
1.Presence of Halogens:
• If an M+2 peak is nearly equal in intensity to the molecular ion
peak, bromine is present.
• If the M+2 peak is one-third the intensity of the molecular ion
peak, chlorine is present.
2. Multiple Carbon Atoms:
• The larger the M+1 peak, the more carbon atoms are present in
the molecule.
3.Sulphur or Oxygen Contribution:
• A small M+2 peak (4%) suggests sulphur in the structure.
• Oxygen does not significantly contribute to isotopic peaks but
affects mass calculations.
Determining Molecular Formula
Isotopic peaks help in molecular formula determination in the
following ways:
1. M+1 Peak Analysis (Carbon Count Estimation)
• The intensity of the M+1 peak (one unit higher than the molecular
ion peak) is influenced mainly by the presence of 13C .
2. M+2 Peak Analysis (Heteroatom Identification)
The M+2 peak can indicate elements like oxygen, sulphur, chlorine, or
bromine:
• If M+2 is about 4% of M, sulphur (34S-4.4%) may be present.
• If M+2 is 24-25% of M, chlorine (37Cl-24.2%) is likely present.
• If M+2 is nearly equal to M, bromine ( 79Br and 81Br are about
50:50) is present.
APPLICATION
 Referances

1. Chatwal GR, Anand SK. Instrumental Methods of Chemical

Analysis. 5th ed. Mumbai: Himalaya Publishing House; 2014.

2. Hoffman, Edmond, and Vincent Stroobant. Mass

Spectrometry: Principles and Applications. Great Britain: John
Wiley & Sons Ltd., 2007.

3.Eskandari M, Mousavi S. Current role and potential of triple

quadrupole mass spectrometry in biomedical research. Int J Mass
Spectrom. 2022;116794.
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