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What is Mass
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The Mass number (A), also called atomic mass number or nucleon
number is the total number of protons and neutrons in an atomic
Mass Number (A)
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Carbon atom have 6 protons and 6 neutrons in the centre of
the nucleus, 2 electrons situated in the inner orbital where as
other 4 electrons are outside the orbital.
We can represent carbon atom like
No. of protons(Z)
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Mass spectrometry is an instrumental
technique in which sample is converted to rapidly moving
positive ions by electron bombardment and charged
particles are separated according to their masses.
Mass spectrum is a plot of relative abundance
against the ratio of mass/charge(m/e).
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•To measure relative molecular masses.
• To know the fragmentation of the molecules.
• Comparison of mass spectra with known
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Organic molecules are bombarded with electron
converted into Highly energetic positively charged ions
(Molecular ions or Parent ions)
Further break up into smaller ions
(Fragment ions or Daughter ions)
The formed ions are separated by Deflection in Magnetic
field according to their Mass and Charge
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Loss of electron from a molecule leads to radical cation.
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Electron removed from molecule orbital having lowest
ionization potential (IP).
In general n < π < σ
Compounds Ionization potential
CH4 12.6 eV
C2H4 10.52 eV
CH3NH2 10.3 eV
1 eV = 23 Kcal/mol
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• Inlet system
• Ion source
• Mass Analysers
• Ion Detectors
• Vacuum System
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• SOLIDS SAMPLES with lower vapour pressure
directly inserted into the ionization chamber and volatilization
is controlled by heating the probe.
• LIQUIDS are handled by hypodermic needles
injection through a silicon rubber dam.
• GASES SAMPLES are leaked into the ionisation chamber
directly by the help of mercury manometer.
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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
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.
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Samples are ionised outside the ion source.
This technique include,
1.Electron impact ionization (EIS)
Samples are ionised inside the ion source.
This technique include,
1. Field desorption.(FD)
2. Fast atom bombardment.(FAB)
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Electron impact ionisation Thermally volatile and stable 500 Da
Chemical ionisation Thermally volatile and Stable 500 Da
Electro spray ionisation Polar and Basic 70000 Da
Fast atom bombardment Peptides 7000 Da
Field ionisation Thermally volatile 1000 Da
MALDI Large Biomolecules 3,00,000 Da
Plasma desorption Neutral compounds 500 Da
APCI Thermally liable 1000 Da
SIMS Same as FAB 300-13000 Da
Laser desorption Elemental analysis 500 Da
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• A beam of electrons passes through a gas-phase sample and collides
with neutral analyte molecules (M) to produce a positively
charged ion or a fragment ion.
• Generally electrons with energies of 70 eV are used to form a
• The positive ions are collected in focusing plates and passed to mass
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• The ESI source consists of a very fine needle and a series of
• A sample solution is sprayed into the source chamber to form
•When droplets carry charge exit the capillary end, as the solvent
evaporates, the droplets disappear leaving highly charged
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• Chemical Impact Ionisation between interactions of sample
with large amount of reagent gas.
• Commonly used reagent gases include
• Oxygen and hydrogens are used in Negative ion chemical
ionisation in MS.
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The vaporised sample is introduced into the mass spectrometer with
an excess of a reagent gas (methane) at pressure of about 1 torr.
The excess carrier gas is ionized by electron impact to the primary
ions CHand CH.
These may react with the excess methane to give secondary ions.
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• In this method the molecule pass through sharp metal anode
carrying an electric field of 1010 v m-1
• Electrons are analysed in primary focusing cathode slit.
• ADV :- abundance of molecular ions.
• DISADV :- lower resolution.
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• Useful for nonvolatile and thermolabile compounds.
• Sample is applied to field ion emitter and the solvent allowed to
• Evaporated sample that leads to chemical ionisation or EIS.
• Example:-Nucleotides & Quarternary ammonium compounds.
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• Argon gas ionised by hot filament and focused beam that
bombards the sample.
• Beam impinges the sample, a series of molecular reactions occur
and analyse in MS analyser.
• Ex:-Insulin,Amino glycosides,Phospholipids.
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• Sample is coated with a high energetic fragment Californium 252.
• This fission fragment desorbs positive,negative, and neutral molecules.
• 252cf generates 1012 power at 10,000k, this may ionise the target
• This method involves the interaction of laser beam with sample to
produce both vaporisation and ionisation.
• The vaporised sample passed to mass spectrometers for analysis.
• Appl:-used for elemental analysis. 31-Aug-14 Solairajan 30
MALDI is a LIMS method of vaporizing and ionizing and sample
molecules are dispersed in a solid matrix such as nicotinic acid.
A UV laser pulse ablates the matrix which carries some of the
large molecules into the gas phase in an ionized form so they can be
extracted into a mass spectrometer.
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Atmospheric pressure chemical ionisation (APCI) is an
analogous ionisation method to chemical ionisation(CI).
Corona discharge is used to ionize the analyte in the
atmospheric pressure region.
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Secondary ion mass spectrometry (SIMS) is based on the
observation that charged particles (Secondary Ions) are ejected
from a sample surface when bombarded by a primary beam of
Primary beam species useful in SIMS include Cs+, O2+, O ,
Ar+, and Ga+
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An ion, after leaving ion source, the ions are separated
according to their m/e ratio.
In this area, the ions are accelerated by both electrostatic
Magnetic sector mass analysers
Double focussing analysers
Quadrupole mass analysers
Time of Flight analysers (TOF)
Ion trap analyser
Ion cyclotron analyser
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H Magnetic field
R Radius of the curvature
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It contains two analysers namely
•Magnetic sector analyser.
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The quadrupole consists of two pairs of parallel rods with applied
DC and RF voltages.
Ions are scanned by varying the DC/Rf quadrupole voltages.
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TOF analyzer – ions are accelerated through a flight tube and
the time of fight to the detector is measured.
Typical flight times are 1 to 50μs.
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The quadrupole ion trap typically consists of a ring
electrode and two hyperbolic end cap electrodes.
As the radio frequency voltage is increased, the orbits of
heavier ions become stabilised,and passed into the detector.
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Fourier transform-ICR mass spectrometry, is a type of mass
analyzer for determining the mass-to-charge ratio (m/z) of ions
based on the cyclotron frequency of the ions in a fixed magnetic
The ions are trapped in a Penning trap(a magnetic field with
electric trapping plates) where they are excited to a larger
cyclotron radius by an oscillating electric field perpendicular to the
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The excitation also results in the ions moving in phase (in a packet).
The signal is detected as an image current on a pair of plates which the
packet of ions passes close to as they cyclotron. The resulting signal is
called a free induction decay (FID), transient or interferogram that
The useful signal is extracted from this data by performing a Fourier
transform to give a mass spectrum.
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The basic principle is that the incident ion strikes the dynode
surface which emits electrons and induces a current which is
amplified and recorded.
The dynode electrode is made of a secondary emitting
material like CsSb, GaP or BeO.
It is ideally suited to isotope analysis.
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Electron multipliers are the most common especially when
positive and negative ions need to be detected on the same
Dynodes made up of copper-beryllium which transduces the
initial ion current ,and electron emitted by first dynode are focused
magnetically from dynode to the next.
Final cascade current is amplified more than million times.
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The dynode consists of a substance( a scintillator) which emits
The emitted light is detected by photo multiplier tube and is
converted into electric current.
These detectors are useful in studies on metastable ions
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All mass spectrometers need a vacuum to allow ions to
reach the detector without colliding with other gaseous molecules
or atoms. If such collisions did occur, the instrument would suffer
from reduced resolution and sensitivity.
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TYPES OF PEAKS IN MS
•Molecular ion peak
•Fragment ions peak
•Rearrangement ions peak
•Metastable ion peaks
•Negative ion peak
Molecular ion Peak:-
When a sample is bombarded with electrons of 9 to 15 eV
energy, the molecular ion is produced, by loss of single electron.
+ 2 e
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Fragment ions Peak:-
when an energy is given further more upto 70 eV,
fragment ions produced, it have lower mass number.
Rearrangement ion Peak:-
Recombination of fragment ion is known as
Metastable ion Peak:-
The ions resulting from the decomposition between the
source region and magnetic analyser are called as Meta stable
ions.These appear as broad peaks called Metastable ion Peaks.
Ions may exist with 2 or 3 charges instead of usual
single charge.The peaks due to these charged ions are known as
Multicharged ion peaks.
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The largest peak in the mass spectrum corresponding
to the most abundant ion or most intense peak in the spectrum is
called as Base Peak.
Negative ion Peak:-
Negative ions are formed from electron bombardment
of sample. These results due to the capture of electron by a molecule
during collision of molecules
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Fragmentation is a type of chemical dissociation.
Fragmentation takes place by a process of heterolysis or homolysis.
Types of Fragmentation:-
• Collision induced dissociation(CID)
• Electron capture dissociation(ECD)
• Electron transfer dissociation(ETD)
• Electron detachment dissociation(EDD)
• Photo dissociation
Infrared multiphoton dissociation(IRMPD)
Blackbody infrared radiative dissociation(BIRD)
• Surface induced dissociation(SID)
• Charge remote fragmentation
• Higher energy C-trap dissociation(HCD)
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Collision Induced Dissociation
Molecular ions are accelerated by electrical potential to high
kinetic energy and then allowed to collide with neutral molecules like
He,N or Ar.
Collision between these molecules leads to bond breakage and
formation of fragment ions.
These fragment ions are analysed by mass spectrometer.
Example:- Triple quadrupole spectrometer produces CID
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SORI-CID:-(Sustained Off-Resonance Irradiation Collision-Induced
It is one of CID technique used in Fourier transform ion
cyclotron resonance mass spectrometry.
In this method accelerating ions in cyclotron motion and
increasing the pressure resulting collisions produce CID fragments.
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Electron Capture Dissociation:-
It is a method of fragmenting gas phase ions for tandem mass
spectrometric analysis (structural elucidation).
ECD involves the direct introduction of low energy electrons to
trapped gas phase ions.
Electron-capture dissociation typically involves a multiply
protonated molecule M interacting with a free electron to form an odd-electron
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Electron Transfer Dissociation:-
ETD induces fragmentation of cations by transferring electrons to them.
Example:-peptides or proteins.
Electron Detachment Dissociation:-
EDD is a method for fragmenting anionic species in mass spectrometry.
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Photodissociation is a chemical reaction in which a chemical compound is
broken down by photons.
Absorption of multiple infra red photons by a molecule and leads to
Long interaction of molecule with radiation field like carbon dioxide laser.
It is a technique used in mass spectrometry to fragment molecular ions in the
gas phase by collision of an ion with a surface under high vacuum.
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Charge Remote Fragmentation:-
It is a type of covalent bond breaking that occurs in a gas
phase ion in which the cleaved bond is not adjacent to the location of the
This fragmentation can be observed using tandem mass
Higher-energy C-trap dissociation:-(HCD)
It is a fragmentation technique, used
for peptide modification analysis.
Immonium ions generated via HCD pinpoint modifications such
as phospho tyrosine.
An added octopole collision cell facilitates de novo sequencing.
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Fragmentation of the Molecular ion
Fragmentation of a molecular ion, M, produces a
radical and a cation.
-Only the cation is detected by MS.
(a radical cation)
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Description of Fragmentation process:-
Fragmentation of the odd electron molecular ion (M
occur by Homolytic or Heterolytic cleavage of a single bond.
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Fragmentation rules:- (9 rules)
The height of the M
peak decreases with increasing degree
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The height of the M
Peak decreases with
increasing molecular weight.
Example:- Fatty molecules, steroids.
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The cleavage is favored at alkyl substituted carbons
leads to formation of a carbocation.
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Double bonds, cyclic structures and aromatic rings
and increase the probability of its appearance.
Molecular ion peak
& Base peak
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Double bonds favor allylic cleavage to
give the resonance stabilized cation.
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Saturated rings tend to lose alkyl side chains at the α bond (see
Unsaturated rings can undergo a Retro-Diels-Alder reaction
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Alkyl substituted aromatic compounds are cleaved
preferably at the β bond to the ring, giving the resonance stabilized
benzyl ion (or) tropyllium ion.
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C-C bonds next to hetero atom are frequently
cleaved, leaving the charge on the hetero atom (resonance
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Cleavage is often associated with elimination of small
stable, neutral molecules, such as
CO,olefins,water,ammonia,H2S,HCN,ketene or alcohols (often with
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Mclafferty arrangement can occur in
ketones,aldehydes,carboxylic acids, and esters.
In this rearrangement a radical center in molecular ion derived
from a lone pair or pi bond, removes hydrogen from the Gamma
position(γ), a pi bond is formed between the β and γ position, and
the bond between the α and β positions is broken.
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Alpha cleavage in mass spectrometry is a
characteristic fragmentation of the molecular ion derived
from carbonyl compounds, in which the bond linking the carbonyl
carbon to the atom occupying an alpha position breaks.
It is an expected pathway for carbonyl
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Beta cleavage in mass spectrometry is
a characteristic fragmentation of the molecular ion derived
from some organic compounds, most
notably alcohols, ethers, and amines, in which the bond
connecting alpha- and beta-carbons break.
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Retro Diels-Alder fragmentation occurs in 3-cyano-cyclohexene,
lets first look at the fragmentation of cyclohexene.
First ionization occurs and electrons from the double bond
transfer to an adjacent carbon and an electron from the bond
between the 3 and 4 carbons transfers to form a second double bond
that is conjugate with the first one.
These rearrangements cleave the molecule between the 3 and 4
carbon and 5 and 6 (where another electron is transferred to form a
double bond between the 4 and 5 carbons). This leaves an olefin and
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The nitrogen rule states, that a molecule that has no or even
number of nitrogen atoms has an even nominal mass, whereas a
molecule that has an odd number of nitrogen atoms has an odd
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The molecular ion appears at m/z 121, indicating an odd number
of nitrogen atoms in the structure.
Odd number of
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In a hydrocarbon where all carbon atoms have only single bonds
and no rings are involved, the compound would have maximum
number of H atoms.
If any of the bonds are replaced with double or triple bonds, there
would be deficiency of H atoms. By calculating the index of
hydrogen deficiency(IHD), we can calculate molecular formula and
how many multiple bonds and rings are involved. IHD is also called
the Degree of Unsaturation.
A double bond and ring each counts as one IHD.
A triple bond counts as two IHD.
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Mass Spectrum of compounds:-
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Fragmentation of Cyclo Hexane:-
= 84 (Molecular ion Peak),
= 56 (Base Peak), (M-28)
= 69 (Fragment ion Peak), (M-15)
= 43 (Fragment ion Peak), (M-41)
= 29 (Daughter ion Peak),
= 15 (Daughter ion Peak).
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Possible Fragmentations are:- Mol.wt-46
C2H5OH+ =46 (Molecular ion peak)
= 31 (Base Peak)
= 27 (Fragment ion Peak)
= 15 (Daughter ion Peak)
Daughter ion Peak
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= 99 (Molecular ion Peak)
= 44 (Base Peak)
= 57 (Fragment ion Peak)
= 29 (Fragment ion Peak)
Base Peak- Mclafferty
Peak- α cleavage
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Gas chromatography–mass spectrometry (GC-MS) is a
method that combines the features of gas-liquid chromatography
and mass spectrometry to identify different substances within a test
Applications of GC-MS include :-
Explosives investigation, and
Identification of unknown samples.
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What is Tandem MS:-
-Uses 2 (or more) mass analyzers in a single instrument.
-One purifies the analyte ion from a mixture using a
-The other analyzes fragments of the analyte ion for
identification and quantification.
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Tandem mass spectrometry, also known
as MS/MS or MS2, involves multiple steps of mass spectrometry
selection, with some form of fragmentation occurring in between
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Components of Tandem Mass
MS-1 Collision cell MS-2
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Applications of Tandem MS
Biotechnology & Pharmaceutical
To determine chemical structure of drugs and drug metabolites.
Detection/quantification of impurities, drugs and their metabolites
in biological fluids and tissues.
Analysis of liquid mixtures
Nutraceuticals/herbal drugs/tracing source of natural products
Clinical testing & Toxicology
Inborn errors of metabolism, cancer, diabetes, various poisons,
drugs of abuse, etc.
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Matrix-assisted laser desorption/ionization (MALDI) is a
soft ionization technique used in mass spectrometry allowing the
analysis of biomolecules (biopolymers such
as DNA, proteins, peptides and sugars) and
large organic molecules (such as polymers, dendrimers and
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MALDI is based on the bombardment of sample molecules
with a laser light to bring about sample ionisation.
The sample is pre-mixed with a highly
absorbing matrix compound for the most consistent and reliable
The matrix transforms the laser energy into excitation
energy for the sample, which leads to sputtering of analyte and
matrix ions from the surface of the mixture.
Most commercially available MALDI mass spectrometers
now have a pulsed nitrogen laser of wavelength 337 nm.
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Applications of MS:-
Elucidation of the structure of the organic and biological molecules.
Determination of molecular mass of peptides, proteins, and
Monitoring gases in patients breath during surgery.
Identification of drugs abuse and metabolites of drugs of abuse in
blood, urine, and saliva.
Analyses of aerosol particles.
Determination of pesticides residues in food.
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Instrumental methods of chemical analysis by willard
Organic spectroscopy by William kemp
Spectroscopic identification of organic compounds by Silverstein
Instrumental analysis by skoog
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