NMR SPECTROSCOPY

Definition
NMR spectroscopy is the study of spin changes at nuclei of atoms
when it absorb electromagnetic radiation (in the radio wave region of 4
to 900MHz) under influence of external magnetic field. It is used to
determine the structure, dynamics, reaction state and chemical
environment of the molecule present the sample.
Types of NMR
1. Proton NMR – 1H NMR
2. Carbon NMR – 13C NMR
Type of nuclei produce NMR spectra
Nuclei with odd mass no(Fermions) only give NMR spectra (e.g
1H, 13C, 19F, 35Cl ) because they have asymmetrical charge

distribution and spin quantum no is ½.

Nuclei with even mass no(Bosons) do not give NMR spectra (e.g
12C, 16O, 14N etc) because of symmetrical charge distribution and

spin quantum no is integral value (0 for carbon and oxygen and 1

for Nitrogen)
Number of signal produce by protons
The number of NMR signal
produce will be depend upon
presence of how many chemically and
magnetically different type of protons
present in the nucleus. Magnetically
and chemically similar type protons
are called as equivalent protons and
we may get only one peak for
equivalent protons e.g Ethane
contains 6 hydrogens and all are
equivalent protons and so we may get
single peak for 6 hydrogns.
Spinning nucleus
The nucleus of hydrogen (proton) behave like spinning bar magnet because
it has both electric charge and mechanical spin. Any spinning charged body
will generate a magnetic field.

Effect of an external magnetic field

The proton will respond to the influence of external magnetic field and tend
to align with field. The proton can adopt two orientation with respect to
external field, either aligned with the field i.e parallel or oppose to the field
i.e anti parallel. Hence external magnetic field applied only force the
protons into two possible orientations .
Energy transition
When no external field applied, nuclei will not spin and spin no is
0. But when external field applied the nuclei will have 2 spin state
namely Ground state (α spin state) and excited state (ß spin state). When
we supply energy in the form of electromagnetic radiation, nuclei in the
ground state will move to excited state and it noted as NMR signal and
this process is called as flipping of proton. After some time it loses extra
energy and come to the ground state as relaxation process.
Precessional motion

Proton being a spinning

magnet, it will move
characteristic way under the
influence of external magnet. It
is defined as wobbling motion
of spinning proton around the
axis of applied magnetic field
and it is due to interaction of
spin & gravitational force.
Precessional frequency
The rate of precession of the magnetic moment of the proton around
the external magnetic field is called as Precessional frequency.
The precessional frequency of the nucleus is directly proportional to
strength of external field and also depends on nature of magnet. The
precessional frequency of different atom will differ.
According to Larmor precession theory, ω = ϒB0
Where ω is Larmor precession frequency , ϒ-gyromagnetic ratio,
B0- Applied magnetic field. (ϒ= ω/B0 ) ϒ value is constant.
But ω = 2πv , 2πv = ϒB0 , v = ϒ/2π B0 , Therefore v α B0
 Criteria required for NMR signal
1. Nuclei should have spin quantum no I = ½
Because the no of spin state can be calculated by following formula
m= 2(I) + 1. m- Magnetic Moment no --> It is no of spin States of a Nuclei
in Magnetic field. Hence nuclei with spin no ½ is ideal for producing
2 spin states.

2. External magnetic field should apply

3. Energy should be supplied in the form of electromagnetic radiations
(radio wave)
Additional principle for 13C NMR spectroscopy
Carbon with mass no of 12 is not producing NMR signal and
spectra due to symmetrical charge distribution as well as spin
quantum no is 0. But Carbon with mass no of 13 i.e isotope of
carbon will produce NMR spectroscopy since its spin quantum no
is ½ and asymmetrical charge distribution.
13C behave like small magnet and spin the resonance and it can
align with (parallel) or oppose (antiparallel) with applied
external magnetic field. When the external energy is supplied in
the form of radio frequency wave, it will excited from ground state
to excited state like proton NMR and noted as NMR signal.
When compare to 1H NMR, 13C NMR producing less intense NMR
signal than proton NMR because isotopic abundance of 1H is
99.99% whereas for 13C is only 1.11% (only 1 % is carbon
isotope remaining carbon with mass no 12).
Chemical shift
Chemical shift is defined as difference between absorption
position of sample proton and absorption position of reference
standard (TMS). The unit of chemical shift is noted as ppm or Hertz
and symbol denoted as δ (delta). It can calculated by the following
formula
δ = frequency of sample proton – frequency of reference standard
/ frequency of reference standard x 106
Measurement of Chemical shift
The usual scale of 1H NMR spectra is 0 to 10 ppm and usual
scale of 13C ranges from 0 to 220ppm.
Chemical shift value ranges from 0 to 10 ppm for most of the
compounds.
Reference standard used and its characteristics
The reference standard used is TMS (Tetra Methyl Silane) in
0.5% concentration and it is added in the sample and so it is
called as internal reference. TMS has 12 protons which are
shielded by highly electropositive nature silicon at centre.
Characteristics of reference standard
1. Chemical inertness
2. Magnetically neutral
3. Produce single sharp peak
4. Miscible with wide range of solvents.
5. Should have volatility for easy recovering from sample
Deshielding effect
A nucleus whose chemical shift has been increased due to
removal of electron density, magnetic induction or other effects is
called as deshielding effect.
Explanation of deshielding effect
When a proton present outside the magnetic field close to
electronegative atom (chlorine) and it withdraw electron from sample
proton and so less electron density and so less applied magnetic field
is required for excitation of nuclei and chemical shift occurs in
downfield.
Factors affecting chemical shift
1. Electronegativity
2. Van der walls deshielding
3. Anisotropic effects
4. Hydrogen bonding
5. Concentration, solvent and temperature effect

Electronegativity
The proton is said to be deshielded if it is attached with an
electronegative atom/group. Greater the electronegativity of atom
greater is the deshielding caused to the protons and greater will be
the chemical shift value.
Electronegative atoms like halogens, oxygen and Nitrogen deshield the
protons. As the electronegativity value increases, the deshielding and
chemical shift value also increase.
Deshielding is directly proportional to no of electronegative atoms and if more
no of electronegative atoms then more deshielding
Compound Chemical Shift
CH3H 0.23 ppm
CH3Cl 3.05 ppm
CH2Cl2 5.30 ppm
CHCl3 7.27 ppm
Deshielding effect decreases with increasing distance from the electronegative
atom
e.g if Bromine attach to the proton directly(-CH2Br) then more deshielding
and more chemical shift value (3.30ppm)but if the distance between
deshielded proton and bromine increases (-CH2-CH2-CH2-Br)then less
deshielding and less chemical shift value (1.25ppm).
Van der walls deshielding

In overcrowded molecule, it is possible that some proton may

be occupying in sterically hindered position. The electron cloud of a
bulkier group (hindered group) will tend to repel the electron cloud
surrounding the proton and so such proton will be deshielded and
chemical shift value will be higher. E.g Methane ( 2.4 ppm) ,propane
(15.5ppm).

2) Cyclohexonol with one methyl group (3.34ppm) ,with two methyl

group (3.65ppm)
Anisotropic effects
Anisotropic effect is also called as space effect. In a
compound containing double or triple bond , circulation of π
electrons in nearby nuclei generate an induced magnetic field
which can either oppose or reinforce the location of proton or
space occupied by the proton. This type of process where
magnetic field is different at different area is called as
anisotropic effect.(non uniformity of magnetic field)
The occurrence of shielding or deshielding can be determined by
the location of proton in the space and this effect is called as
space effect. This can be explained by following illustration
Illustration to explain anisotropic effect
a) Alkene
In alkene if we apply external magnetic field in
the upward direction, due to π electrons in the
double bond, magnetic field is developed and
magnetic wave is surrounded in carbon– carbon
double bond as well in side chain of carbon.
Magnetic wave going upward (same direction of
applied magnetic field) in the proton region
(Deshielding region) and magnetic wave going
downward (opposite direction of applied
magnetic field) in the carbon–carbon double
bond region (Shielding region)
Conclusion
In the case of alkene, the protons are
deshielded due to anisotropic effect with
chemical shift value of 4.6 to 6.5 ppm
b) Carbonyl compounds
In carbonyl compound like aldehydes, π
electrons of double bond develop own
magnetic field which is opposite direction
to the applied magnetic field and the
protons in the region will be shielded,
whereas due to electronegative atom
oxygen, electron density in the
surrounding of double bond will be
reduce and induced magnetic field is
same direction that of applied magnetic
field . Hence in this region deshielding
occurs and the protons are deshielded
with chemical shift value of 9.5 – 10 ppm.
C) Aromatic protons (Benzene)
In aromatic compound π electron cloud in the above and below the plane
and in presence of applied magnetic field they develop magnetic wave. The
magnetic wave within the ring in downward direction (opposite to applied
magnetic field) and magnetic wave in the outside ring is in upward direction
(same direction that of applied magnetic field).
 The aromatic protons (hydrogen) present in the outside the ring
i.e in the deshielding region and so aromatic protons deshielded with
high chemical shift value than alkene protons because strong
deshielding effect (6.5 to 8.0 ppm)

D) Alkyenes (Acetylene)
In the case of alkynes carbon – carbon triple bond in sp
hybridisation and so more electronegative than alkenes and so
theoretically it should more deshielded with more chemical shift
value, but its chemical shift value is less (2.5 ppm) than alkenes (4.5 -
6.5ppm). This is because of magnetic anisotropic effect.
Reason for less chemical shift value for alkynes

In the applied magnetic field, the π electron in the triple bond produce
induced magnetic field and the molecule is oriented in verticular direction
associated with magnetic wave in two different direction i.e magnetic wave
around triple bond is downward direction (opposite to applied field) and so
proton attached to carbon –carbon triple bond present in this shielding
region get shielded with less chemical shift value (2.5ppm).
Hydrogen bonding
In hydrogen bonding, hydrogen atom attached with electronegative atom
and so less electron density around hydrogen atom. Therefore deshielding occurs
in such protons and chemical shift value depend upon strength of hydrogen
bonding.
In general if hydrogen bonding increases, more deshielding occurs and more
chemical shift value.
Effect of concentration, solvent and temperature
Chemical shift value independent of concentration and temperature except
hydrogen bonding condition.
In hydrogen bonding condition, if concentration increases then deshielding will
increase and if concentration decreases then dehsielding will decrease.
In hydrogen bonding condition, if temperature increases then deshielding will
decrease (due to decreasing of hydrogen bonding) and if temperature decreases
then deshielding will increase (due to increasing of hydrogen bonding).
In general NMR signal will differ depend upon the solvent used and so NMR
spectra recorded in deuterated solvent.
Spin-Spin coupling
n+1 rule in spin-spin coupling
The multiplicity of signal is
calculated by n+1 rule.
This is one of the rule to predict
the splitting of proton signals.
This is considered by nearby
hydrogen nuclei. n= no of
protons in nearby nueli.
If zero H atom as neighbour
then n+1 =0+1 =1 (singlet)
One H atom as neighbour then
1+1= 2 (doublet)
Two H atom as neighbour then
2+1=3 (triplet)
Different type of spin-spin coupling
1) Geminal coupling 2) Vicinal coupling 3) Long range coupling
Geminal coupling
In which two coupled proton are attached with same carbon
and they are separated by two bond and the protons are
sterochemically different (non equivalent protons) e.g
Methane diol.
Vicinal coupling
In which, two interactive protons are attached with two
different carbon and separated by three bond. E.g 2,3
dibromobutane.
Long range coupling
In this type, the interacting protons are attached with
different carbons and separated by more than three bonds
(including one double bond)
Possible no of spin orientations in
multiplets
The number of possible spin orientations
will depend upon the neighbouring non
equivalent protons. This can be explained
by following example 1.1,2 tricholoro
ethane.

In this example , two proton namely CH,

CH2 and doublet will produce by CH
protons on CH2 and triplet will produce by
CH2 protons on CH protons.
The possible spin orientation for doublet
will be 1:1 intensity ratio (one parallel
and one antiparallel spin states when
compare to applied field)
The possible spin orientation for triplet will be 1:2:1 intensity
ratio (Both coupling protons are parallel or antiparallel or one
proton will be parallel and another will be antiparallel spin
states to applied field)
signal height equal

Center signal is two time higher

than first and third signal

2nd and third signal are

three time higher than 1st
and 4th signal
Intensity of multiplet signals (Pascal triangle)
The intensity of NMR signal in multiplet can be explained by
Pascal triangle concept.
APPLICATIONS OF SPIN-SPIN COUPLING

1. Spin-spin coupling shows the information regarding

neighbouring protons and based on no and intensity of signal,
we can find information of neighbouring protons present in the
nucleus (No of protons present)
2. We can able to find out whether equivalent protons or non
equivalent protons present as neighbouring proton, because
only non equivalent proton will produce splitting of signal and
equivalent protons donot produce any splitting in the NMR
signal.
e.g No splitting occurs in 1,2 dichloroethane (No splitting
between 2 equivalent CH2 Protons)
3. From the nature of NMR signal splitting occurs, we can able to
find out ratio of intensity of signal in triplet and quartet.
Coupling constant
The distance between the centers of two adjacent peaks in a multiplet is
constant and this constant is called as coupling constant. It is denoted as
J and unit is Hertz or cps (cycles per second). The coupling constant
value is independent of applied external magnetic field and depends on
the structure of molecule.
Features of coupling constant
1) Coupling constant is a measure of how strongly the nucleus affected
by spin states of neighbour proton.

2) Coupling constant value is same for different signals of one

multiplet (J value of 1st and 2nd signal as well as 2nd and 3rd signal)
3) Coupling constant value of Geminal, vicinal and long range
coupling can be expressed by symbol J with superscription value
2,3 and 4 respectively i.e 2J,3J and 4J. The superscripted number
indicate no bonds separated the coupled protons.
4) Coupling constant value in geminal coupling varies with
bond angle e.g J value 0 at 1250 and 20 cps at 1050. J value
varies with dihedral angle in vicinal coupling e.g J value for
vicinal protons arranged in Gauche conformation (dihedral
angle 0 to 1200) is 2-4 cps whereas J value for Anti
conformation (dihedral angle 1800) is 5-12 cps.
5) Coupling constant value differ for
different type of carbon-carbon
linkage (chain length).

6) Coupling constant value also

depend upon presence of double
bond or presence of cis-trans isomer.
Coupling constant value of trans
isomer is higher than cis isomer.
7)Coupling constant value of aromatic protons differ for ortho (8-10HZ),
meta (1-3 HZ) and para (0-1 HZ) position.
8) The coupling constant value increases with the decrease of bond
length.(J value for Geminal coupling is greater than Vicinal coupling)
9) Coupling constants can be either Positive or Negative.
Coupling constant value is positive if the energy of A
(original proton) is lower when X (coupled proton) has
opposite spin as A.
Coupling constant value is negative if the energy of A is lower
when X has the same spin as A.
10) The major factors affecting coupling constants are dihedral
angles, substituents, hybridization and ring strain.
Applications of coupling constant
1.Coupling constants are a measure of the effectiveness of
spin-spin coupling and have been very useful in getting exact
information from Proton NMR spectra of complex molecular
structures.
2. Coupling constant used to distinguish cis and trans isomer
(coupling constant of trans isomer is higher than cis isomer)
3. Coupling constant value used to find out nature of coupled
proton, nature of bond involved and spatial relations between
protons.
Instrumentation of NMR

RF Receiver Coil
NMR spectrophotometer consist of following components
1)Sample holder
2) Magnet
3) A radio- frequency generator
4) A sweep system
5) A RF detector and recorder

Sample Holder
1) Sample holder in NMR is tube shaped and so it is called as
sample tube.
2) It must be transparent to RF radiation , chemically inert and
durable.
3) It is made up of Glass or Pyrex material.
4) They are 6-7 inch long and 3-10 mm diameter with plastic cap.
5) This sample tube used to measure NMR spectra of bulk
samples and solution (0.4ml liquid/solution).
6) This sample tube is present in sample probe which is located
at intersection of applied magnetic field.
7) Sample probe holds sample tube in a magnetic field by single
coil or more no coils (RF coil) and it may be rotate along its axis
for exciting the nuclear spins and receiving & detecting the NMR
signal.
 Magnet
There are three type of magnets
1) Conventional magnet (30 to 6o MHZ)
2) Permanent magnet (60 to 100 MHZ )
3) Large capacity magnet (470 MHZ ) (Mainly used in Modern NMR
spectrometer)
There are two parts of Large capacity magnet
a) Super conducting magnet b) Shim coils
Super conducting magnet
It is an electromagnet made up of superconducting wire.
1) It consists of main field coil made of superconducting Niobium-tin
(Nb3Sn) or Niobium-titanium (NbTi) wire.
2) The superconducting state can be maintained by immersing the wire in
a bath of liquid helium.
3) These coils adjusted with every sample placed in the probe to
compensate sample composition, volume and temperature.
 Advantages of superconducting magnet
1. Strongest Magnet
2. Stable and homogenous field
3. Low running cost.
Shim coils
1. A shim coil is a device used to adjust the homogeneity of a magnetic
field.
2. It is use to correct minor inhomogeneities (due to magnet design,
sample in the probe, variations in the thickness of sample tube) in the
applied magnetic field.
3. For that purpose, surround the sample holder with set of shim coils,
which produce a tiny magnetic field with particular spatial profile to
cancel out the inhomogeneities in the main magnetic field.
Sweep system
Sweep system includes sweep generator and sweep coils present
near by magnet. The main role of sweep generator is altering
magnetic field strength with the help of sweep coils.
There are two type sweep generation methods :
1) Field sweeping 2) Frequency sweeping
Field sweeping
The resonance energy obtained by changing applied magnetic field and
holding RF field constant.
Frequency sweeping
The resonance energy obtained by changing applied RF field value and
holding applied magnetic field constant.
Sweep coils
It is very difficult to vary the magnetic field strength directly in the
magnet and so variation of magnetic field can be done with the help of
two sweep coils (Helmholtz coils)
Radio-frequency generator
1) Radio Frequency Generator is also known as Radio frequency
Transmitter.
2) Radio frequency oscillator is used to generate radio frequency
radiation and it irradiates the sample molecules.
3) Due to applied radio frequency, nuclei gets energy and moves
to excited state. The radio frequency coil surrounding the sample
produce resonance signals.
RF receiver and Amplifier
The Radio frequency receiver coil receive the resonance
signal (NMR signal) and the receiver coil will amplify the
weak NMR signal and amplified signal reaches the detector.
Detector (RF Detector)
The detector will receive the amplified NMR signal and it convert into
electrical signal and stored in the memory.
Recorder
The detected NMR signal are stored in the computer and we can
retrieve the data .

Type of NMR instruments

1.Constant magnetic field instrument (varying RF)
2.Constant RF transmitter instrument (varying magnetic field)
Working of NMR spectrometer
1) Sample is taken in the sample holder and sample holder is
surrounded by RF coil.
2) Magnet is present near to sample holder and magnetic field is
applied to the sample holder (Field strength can be vary with the
help of sweep coils) and the magnetic field (North south
position) is perpendicular to the sample holder. (Due to applied
magnetic field nuclei present in α spin state with same direction
of applied magnetic field(parallel))
3) Radio frequency transmitter produces the electromagnetic
radiation and it will come through the coil nearby the sample
holder and go through the sample.
4) The electromagnetic radiation is perpendicular to the applied
magnetic field. Due to absorption of suitable RF radiation, nuclei will
move to excited state (ß spin state with anti parallel orientation) which
causes resonance in the nuclei. Then nuclei go to relaxation process and
excess energy released in the form of radio frequency radiation and it is
measured as NMR signal.
5)These NMR signal are received by receiver coil (RF Coil) and it reaches
the amplifier. Then amplifier amplify the signal and it reaches detector.
6) The detector will detect the signal and it recorded in the recorder as
NMR signal.
NMR relaxation process
When we switch off the radio frequency wave, the excited nuclei will looses its
excess energy in non radiative manner by transition from excited state to
ground state. This process is known as NMR relaxation.
Types of relaxation process
1. Spin lattice or longitudinal relaxation(T1)
2. Spin-spin relaxation or transverse relaxation(T2)
Spin lattice type
Spin lattice type
1. In this process, excited nuclei releases its energy and that energy absorbed by the
solvent molecule or lattice molecule (vibrating with same frequency of emitted
radiation) present nearby the nuclei. This process is called as spin lattice relaxation
and denoted as T1.
2. Spin lattice relaxation time(T1) is the measure of average life time of nuclei in the
higher energy state. The energy is transfer to the components of lattice as additional
translational, vibrational and rotational energy. Hence total energy of system
remains same.
3. T1is strongly affected by mobility of lattice. In crystalline lattice mobilities are low
and so T1is large.
4. By enhancing the probability of magnetic fluctuations of proper magnitude, T1
become short. An efficient relaxation process involves short time and results in
broadening of absorption peaks. Smaller the time of excited state, greater is the line
width.
5. This type of relaxation process is not effective in solids.
Spin-spin relaxation

It is due to mutual exchange of spins by two precessing nuclei which

close to each other (neighbouring nuclei). It is also known as transverse
relaxation time T2.

Mechanism
1. In this process, nuclei exchange spins with neighbouring nuclei by
interaction of their magnetic moments (neighbouring nuclei have
identical precessional frequency and differ in magnetic quantum states)
2. The excited nuclei transfer its excess energy to low energy spin state
(Ground state). Nuclei in the low energy spin state absorbing excess
energy and move to excited state (higher energy spin state). Hence there
is no net loss of energy and T2 value for crystalline solids or viscous
liquids is smaller.
3. The greater spin state changes produce more magnetic field
fluxation and so more radio frequency energy absorbed. Hence line
broading will occur.

4. Local magnetic field variation is low in liquid and gases, whereas

in solid such interaction is more and it lead to produce very broad
absorption bonds.
NMR spectroscopy Application
Important Applications
1. Structural determination
2. Qualitative analysis (detection of functional group)
3. Quantitative analysis
4. Determination of molecular weight
5.Determination of hydrogen bonding
6. Study of isomerism in organic chemistry
7. Determination of no of protons from multiplicity of peaks
Qualitative Analysis
NMR spectroscopy used to detect certain functional group
present in the molecule from the characteristic chemical shift
value of various protons associated with molecule.
Quantitative analysis
It is used to estimate the amount of hydrogen or fluorine present in
the unknown compound and the results are better than normal
method of elemental analysis.
The amount of hydrogen can be calculated by comparing peak
area of standard spectrum and unknown compound spectrum by
using following formula
%H = %Hs(A/As)(Ws/W)
A= integral area of unkown
As= integral area of standard
W= weight of unknown Ws= weight of standard
Determination of molecular weight
Molecular weight is the atomic weight of atoms present in the
molecule. The molecular weight of unknown compound
calculated by formula:
M = IsnWMs/InsWs
Is=integrated intensity of standard
n = no of protons in the integrated peak of unknown
W= weight of unknown
I = integrated intensity of the unknown
ns= no of protons in the integrated peak of stanard
Ws= weight of standard
Determination of Hydrogen bonding
It is very use to study hydrogen bonding in metal chelates and in
organic compounds.
Chemical shift value of hydrogen bonded nucleus will be more
than normal condition of nucleus. As the concentration of
hydrogen bonding increases , the chemical shift value also
increases (4-5ppm) and in dilute solution the chemical shift value
decreases(0.5 to 1ppm).
Study of isomerism in organic chemistry
It is used to study Geometrical isomerism occurs in organic
compounds. It is used to differentiate the cis and trans isomer
and in general trans isomer have higher chemical shift value than
cis isomer.
Determination of no of protons from multiplicity of peaks
No of adjacent protons can be find out from the multiplicity of
peaks i.e if one adjacent proton is present then doublet will
recorded and if two adjacent proton is present then triplet will
recorded.
Other applications
1) Detection of Aromaticity
Protons attached to benzoyl ,poly nuclear and hetero cyclic compounds
will be deshielded due to circulation π electrons and so proton signal
will appear in down field than benzene. From that aromatic charter of
compound can be identified.
2) Determination of keto-enol tautomerism
NMR spectroscopy is very useful in studying keto- enol tautomerism.
This can be explained with following example acetyl acetone.
It exhibit keto - enol tautomerism
NMR spectrum of acetyl acetone shows signals for following
equivalent protons

A distinct two proton singlet appear for ketonic form and four
signals appear for eight protons in the enolic form.
3) Materials science
NMR spectroscopy in solid state can be used to investigate new
materials like glasses, ceramics, polymers and super
conductors. It is also used to investigate reactions taking place
in catalytic surfaces.
Mass spetctrometry
Definition
Mass spectrometry is one of the spectroscopic technique used
to determine molecular weight of the compound by measuring
mass-to-charge ratio (m/z) value of one or more molecule
present in a sample.
Principle
Introduction of principle
Mass spectra are also called as Positive ion spectra or line
spectra. In Mass spectroscopy No electromagnetic radiation is
used for excitation and only electrons are used for
bombardment of molecule to convert neutral molecule to
positive charged ion. There is no ground state and excited state
in mass spectroscopy.
2) All these ions are accelerated and focused as a single beam by
passing through accelerating slits in the electrical strength of
8000V. Positive ions gain certain kinetic velocity (1/2mv2=eV
where m-mass, v-velocity, e-charge, V-Voltage applied) in the
acceleration chamber.
3) Then these ions are sorted out according to their mass to charge
ratio by deflection in variable magnetic field and the ions with low
m/z value is more deflected than high m/z value.

4) Then the ions are reaches the detector and depend upon the
number of ions, electric current is produced and it is recorded. The
output is known as mass spectrum.

5) Mass spectrum is constructed by plotting Relative abundance Vs

m/z value. Each line on the mass spectrum indicates the presence of
atoms or molecules of particular mass. The tallest peak is known as
base peak and it is known as Molecular Ion Peak, which posses 100%
abundance. The corresponding m/z value is noted as molecular
weight of parent compound and other smaller peaks are due to
fragmented ions.
Instrumentation
Instrumentation
The mass spectrometer consists of following
components
1) Sample inlet or sample handling system
2) Ion source or ion chamber
3) Ion Accelerator
4) Ion separator or mass analyzer
5) Ion collector, Detector and Recorder
Sample inlet system
1) Mass spectrometer requires vapor sample and so sample should
convert into gaseous state in the inlet system (by heating) before
enter into ionization chamber.
2) Different type of inlet system used for different sample.
3) Sample from gas bulb is transfer into metering reservoir (pressure
of 30 to 50 torr) and then expand into expansion reservoir (pressure
of 10-3 to 10-1torr) of three-liter capacity.
4) Liquid samples are handled by hypodermic needle through silicon
rubber dam. Then liquid sample withdrawn by reservoir and convert
into vapor immediately.
5) From the sample reservoir, the gaseous samples are leaked into
ionization chamber through pinhole in the diameter of 0.013 to
0.050mm.
6) The sample required is 1µmole.
Ion Chamber (Electron Impact Ionization)
1) From the inlet system, the sample is introduce into ionization
chamber, where sample molecules are bombarded by beam of electrons
(70ev) from glowing filament(tungsten) and molecule ionize into
molecular ion with the loss of one electron. (M + e-  M++2e-) This
electron is collected in ion collector.
2) The ionization potential require for ionization of Molecule to positive
ion is only 10eV and so excess potential (70eV) supply by the filament
use to bombard the M+ ion to produce fragmented positive ions like m1+
and m2+.
3) In addition to Electron Impact Ionization method, three different
Ionization methods available as follows:
i) Chemical Ionization
ii) MALDI (Matrix Assisted Laser Desorption Ionization)
iii) FAB (Fast Atom Bombardment)
Ion Accelerator
1) The electrostatic accelerator system is used to accelerate
the velocity of positive ions M+,m1+& m2+.
2) The accelerator system consist of 3 set of electrodes. First
set of electrode with positive potential(Anode) and other 2
set of electrodes with negative potential(Cathode) in the
increasing potential (8000to 10,000V) .
3) The first set of electrodes repel the positive ions into
right side and other 2 set of electrodes(cathodes)will
attract the positive ions. Hence all positive ions gain certain
kinetic velocity (1/2mv2=eV) in the acceleration chamber.
Mass Analyzer(Magnetic field)
1) The accelerated positive ions reaches the magnetic field area (mass
analyser) and these positive ions require magnetic field to cross this
curvature area.(radius of curvature is fixed and ions with different m/z
value will move based on applied magnetic field value)
2) The magnetic field deflect the ions based on their mass and charge.
Mass of Ion – lighter ion (low molecular weight) deflect more than
heavier ion (high molecular weight)
Charge of Ion – Ions with 2 positive charge is more deflected than ion
with one positive charge.
3) Hence out of 3 positive ions, M+ ion having high molecular weight will
reach detector first (less deflected) and m2+ & m1+
ions will reach the detector one by one later due to less molecular weight
(more deflected).
Other mass analyzer available as follows:
a) Time of Flight (TOF) Analyzer b)Quadrupole Analyzer
Detector
Detector will convert beam of ions into current signal
and amount of current generated based on ion abundance.
In mass spectrometer, three type of detectors namely Faraday
cup collector, electron multiplier tube and photographic plate.
Faraday cup collector
They are based on measurement of direct charge current
produced when an ion hits surface. It consists of insulated
conductor directly connected to an electrometer amplifier.
Advantage
Its cup shape suppressor and guard electrode reduces
the chances of escape of secondary electrons liberated by ion
impact.
Electron multiplier
It is operated similar manner that of photomultiplier
tube. It consists of the primary cathode optimized for the
detection of ions. The sensitivity of this detector is 1000
times that of Faraday cup.

Photographic plate detector

This detector system is most sensitive than any other
detector, because the photographic plate integrates the ion
signal over period of time. It is processed with
photographic techniques and read with the aid of
densitometer. It is used more effectively in double focusing
spectrometer.
Recorder
It records the relative abundance of each ion having
specific mass/charge ratio to give a mass spectrum.
Vacuum System:

a) A high vacuum is to be maintained in the

instrument.

b) The inlet system is generally maintained at 0.015

torr, the ion source at 10-3 torr and analyszer tube at
10-7 torr or as low as possible.

c) The oil diffusion and mercury diffusion and mercury

diffusion pumps are commonly used in different types
of combinations.
Working of Mass spectrometer
1) Vaporized form of sample (liquid/solution is converted
into vapor by heating coil) is injected into ionization chamber
through pin hole.
2) In ionization chamber, sample molecule is bombarded by
electron in the potential of 70eV (from tungsten filament)
and so molecule looses one electron to produce Molecular
positive ion (M+). For this conversion only 10to15eV potential
is required and excess potential use to ionize the molecular
positive ion to produce fragmented ions m1+&m2+.
3) Now these three positive ions are entered into acceleration
chamber to increase and attain kinetic velocity equal to
1/2mv2=eV. This can be achieved by passing the positive ions
through 3 set of electrodes (one anode and two cathodes).
4) When the ions pass through the anode, it repels the positive ions
into right side and other 2 set of electrodes (cathodes) will attract
the positive ions. Hence all positive ions gain certain kinetic velocity
(1/2mv2=eV) in the acceleration chamber.
5) Then the accelerated ions enter into mass analyzer, where
magnetic field is applied. Now positive ions will be sorted out based
on their m/z value and positive ion with low m/z value (low
molecular weight) will more deflected than positive ion with high
m/z value (M+). Hence M+ ion will first reaches detector followed by
m2+ and m1+.
6) The detector will produce electrical signal based on the no ions
reaches the detector.
7) The electrical signal produce in the detector will be amplified
and amplified signals recorded in the recorder as mass spectrum.
Ionization techniques in Mass spectrometry
Introduction
Mass spectrum is significantly dependent upon the
ionization technique. There are two basic parameters
such as Number of peaks and Intensity of molecular ion
peak are important for studying mass spectrum. Hence
one good ionization technique has to produce more no of
peaks and similarly high intense molecular ion peak.
Classification of ionization technique(Based on energy)
1) Hard ionization technique
It requires high energy and it produce increased no of
fragmented molecular ion. E.g Electron Ionization
method.
2) Soft ionization technique
It requires comparatively less energy and comparatively less
no of fragmentation of molecular ion.E.g Chemical ionization
method, Fast Atom bombardment(FAB)method & Matrix
Assisted Laser Desorption Ionization (MALDI) method.

Classification of ionization techniques based on methods

1) Gas phase ionization methods
Sample is vaporized before ionization.
Eg: Electron ionization and chemical ionization methods
2) Desorption methods
Liquid /solid samples are directly converted into gaseous ions.
Eg: Fast Atom bombardment(FAB)method & Matrix Assisted
Laser Desorption Ionization(MALDI)method.
Electron ionization method
1) It is also known as electron bombardment ionization technique. In
this method a high energy(70eV) of beam of electrons passes through
the gaseous sample.
2) The electrons are collided with neutral molecule and remove one
electron from the molecule to produce positive charged ion (M+).
3) The ionization potential require for conversion of M to
M+is only 10-15eV and excess potential use to ionize the
molecular positive ion to produce fragmented ions
m1+&m2+,m3+and etc.
4) The positive ions will accelerate to get kinetic velocity
(1/2mv2=eV)by passing through 3 set of electrodes(one
anode and two cathodes) with high electrode potential
value up to 8000V. Positive ions will be repelled by anode
and attracted by two cathodes to get required kinetic
velocity.
5) This method is suitable for volatile liquid but molecular
ion peak is either weak or absent. Some of ions may
decompose during the process and so no M+ ion peak.
Chemical ionization method
Introduction
It is a gaseous phase ionization technique and it is one of the
important soft ionization technique. In this method
less fragmentation occur and so intense molecular ion peak
will appear. Hence this method is very useful method for
ionization of molecules like alcohol, ether & amino acid (These
molecules are highly fragmented in Electron ionization method
and so molecular ion peaks will not to be detected)
Definition
It is a ionization technique involving formation of
molecular ions by reaction between analyte and reactant
ions of reagent gas (Methane,isobutane &ammonia)
Steps involved in chemical ionization
1)Introduction of carrier gas into ionization chamber
A carrier gas or reagent gas (methane, iso-butane or
ammonia) is introduced into ionization chamber at
slightly higher pressure (1torr)than Electron ionization
method.
2) Formation of primary ions
First carrier gas will be ionized by electron
bombardment in the ionization chamber to produce
primary ions. This can be expressed as follows:
3) Formation of secondary ions
Now these primary ions will react with excess of carrier gas
Methane to produce 3 type of secondary ions.
4) Reaction of analyte molecule with secondary ions
Secondary ions will react with analyte molecule in three
different reaction to produce molecular ions.
Proton transfer reaction
Analyte molecule react with secondary ions to produce
M+1 ion (Molecule with one additional proton) and it is
called as quasi molecular ion
M +CH5+  [M-H]+ +CH4 i.e [M+1]+
M+C2H5+  [M-H]+ +C2H4

Note: proton transfer reaction take place in amine and

alcohol.
Mass spectrum of Ephedrine obtained by Electron ionization method
not showing Molecular ion peak due to decomposition of ions by high
energy of electrons and Mass spectrum of ephedrine by chemical
ionization method showing M+1 peak (Molecular ion + Hydrogen)
with m/z value of 166 (original molecular weight is 165+1)
Hydride transfer reaction
In this reaction analyte molecule react with secondary
ions to produce M-1 ion (Molecule with one proton less).
M + C2H5+ [ M-1]+ + C2H6
Note: Hydride transfer reaction takes place in carboxylic
acid and n-alkane.
Electrophilic addition reaction
In this reaction secondary ions are added with analyte
molecule to produce addition product as [M-CH3]+
M +CH3+  [M-CH3]+ or (M+15)
Conclusion
1) Mass spectrum resulting from chemical ionization
method contains well defined [M+1]+ and [ M-1]+
peaks.
2) [M+1]+ ions do not undergo significant
fragmentations, the spectra are as simple as
compared to spectra obtained in Electron ionization
method and very useful to determine molecular
weight of compound.
Matrix Assisted Laser Desorption Ionization
(MALDI) method
It is one of soft ionization method classified as
desorption ionization method. In this method laser
beam is used to hit the surface of sample matrix to
produce molecular ions.
It is mainly used to determine molecular weight of
peptides, proteins and antibiotics (molecular weight
up to 3,00,000 unit). This method produce less
fragmentation and intense molecular ion peak.
Methodology
Material required
1)Sample
2)Matrix (to protect biomolecule from direct exposure of laser light). It
should be acidic in nature with low molecular weight and should have
polar functional group. It should able to absorb laser beam of light.
e.g Nicotinic acid, urea, picolinic acid, 3-hydroxy picolinic acid & 2,5
dihydroxy benzoic acid.
Steps involved in the process
 Steps involved in the process
1)Sample and matrix is dissolved in the solvent separately
in the concentration of 0.1mg/ml and 10mg/ml
respectively.
2)Then both solution are mixed together and cocrytalized
to get crystal mixture of matrix and analyte.(1:1000 to
1:1,0000)
3) Type of laser beam
4) Laser beam will hit the crystal mixture of matrix and
sample (0.5 -20 ns time)and sample will convert into
gaseous state by the heat energy of laser beam. Further
sample and matrix will convert into ions due to the
translational energy of laser beam.
Two type of ionization process:
1) Protonation : one proton from matrix transfer to the
sample and molecule become M-H ion. (M+H[MH]+
2) Deprotonation :
One proton loss from molecule and molecule become
[M[M-H] -+H+
These ions are called as quasi molecular ions. Then
these ions are desorbed from surface and reaches the
mass analyser (Time of flight mass analyser). Finally the
ions are detected and recorded as mass spectrum. In the
mass spectrum M+1 peak will appear to show molecular
weight of compound (Exact molecular wt +1 )
Application of MALDI Technique
1) Matrix-assisted laser desorption/ionization-time of flight
(MALDI-TOF) mass spectrometry (MS) has become a widely used
technique for the rapid and accurate identification of bacteria,
mycobacteria and certain fungal pathogens in the clinical
microbiology laboratory
2) The MALDI-TOF can be used in profiling and imaging proteins
directly from thin tissue sections and known as MALDI imaging
mass spectrometry (MALDI-IMS). It provides specific
information about the local molecular composition, relative
abundance and spatial distribution of peptides and proteins.
Fast Atom bombardment(FAB)method
It is one of the soft ionization technique classified under
desorption technique . In this method sample is mixed with
matrix and mixture of sample matrix is bombarded by
accelerated neutral atom like xenon. It is use to determine
molecular weight of compounds such as
polypetides,olegonucleotide (M.Wt of 300 to 6000 daltons)
Characteristics of Matrix
1) It should be non volatile.
2) It should be low vapor pressure liquid.
3) It should be electrolyte and inert material.
e.g Glycerol, Thioglycerol, 3-Nitro Benzyl alchol,
Diethnolamine or triethanolamine.
Methodology
1)Sample solution is mixed with matrix to get sample-matrix
mixture.
2) The Xenon or Argon gas is entered into the ionization
chamber and it is bombarded by electrons to convert the xenon
into xenon cation radical.
3)Then xenon cation radical accelerated by 6000
to 10,000 eV and it reaches Neutral atom
production area.

4) In this chamber neutral atom of xenon is

present and the accelerated xenon cation radical
react with xenon neutral atom. Now the excess
energy posses by xenon radical transfer to
neutral atom(Xe) and the neutral atom gets
accelerated.
5) Now this accelerated xenon atom hit the surface of sample matrix
(which is directly taken in sample probe) and this accelerated xenon
atom ionize the sample matrix by using their translational energy.
6)During this process, the matrix material like glycerol ionize
to loose one hydrogen and this hydrogen is protanated to
sample to form [M+H]+ ion (quasi molecular ion). Finally this
[M+H] ion will desorbed from the surface and get accelerated to
reach Mass analyzer.
7) In the mass analyzer ,ions are separated based on m/z value
and detected by detector. The signals are amplified to get
spectrum in the recorder and in this spectrum M+1 peak may
appear to show the molecular weight of compound.
(Exact molecular weight +1)
Application of FAB method
1) It is suitable method for the analysis of highly
polar, thermally unstable compounds, especially
peptides and proteins.
2) It provides detailed sample molecular structure
information and widely used in biomedical field.
Mass Analyzer
A mass analyzer is the component of the mass
spectrometer that takes ionized masses and
separates them based on charge to mass
ratios(m/z)and outputs them to the detector
where they are detected.
Types of mass analyzer
1. Magnetic field
2. Time of flight mass analyzer (TOF)
3. Quadrupole mass analyzer
Time of flight mass analyzer
It is one of the mass analyzer use to separate different ions of
sample based in their mass to charge ratio without using
electric/magnetic field. This is achieved by accelerating all
particles at the same electric potential and then measuring
the time taken by each ion to reach the detector.
Principle
The main principle involved in this method is the velocities of
two ions varies depending on the mass of ions provided both
ions are created from same source and both should have same
kinetic energy. In this condition, the velocity of lighter ion is
faster than heavier ion. Hence when these ions travel towards
detector, lighter ion will reach detector first followed by
heavier ion.
This can be expressed by following mathematical equation
Kinetic energy of ion accelerated by electrode potential is
expressed as
eV = 1/2mv2 (eV-applied potential,v- velocity of ions)
but v=l/t (l-path length, t-time)
Hence eV=1/2m(l/t)2or m/e=2V t2/ l2
In this equation length(l) and applied potential V is
constant for all ions and so we can use term k (k= 2V/ l2)
Therefore, m/e =kt2or m/e α t2. It indicate that ions with
different molecular mass will reach detector with different
time and so this method is called as time of flight mass
analyzer.
Methodology
Construction of instrument
1) It consist of ionization chamber, acceleration
chamber, flight tube (Drift path) and finally connected
with detector.
2) In ionization chamber, sample holder attached with
matrix contains sample.
3) In acceleration chamber, One positive electrode and
two negative electrodes with increasing potential are
present.
4) Flight tube ,which is also known as drift path and
ions are travelling through this drift path to reach
detector.
Working
1) Ions (M+,m1+,m2+ )are produced by the attack of laser beam on
matrix (MALDI method)and reaches acceleration chamber.
2) The ions are accelerated by applying electric potential with
same kinetic energy by three set of electrodes.
3) Now all the ions have to travel through the drift path (same
distance)to reach detector. If it is assume that molecular weight of
the ions in the increasing order from m2+,m1+ and M+ then ion with
low molecular weight (m2+) will reach the detector followed by m1+
& M+.
4) Hence the ions are separated by time of flight i.e ion with high
molecular weight take more time to reach detector than ion with
low molecular weight. By this manner the ions are separated in
this method. This analyzer is mainly used in MALDI ionization
method .
Advantages
1) Increased mass accuracy and mass resolution
2) Greater sensitivity
3) Rapid analysis
4) It can analysis sample of unlimited mass
range
5) Moderate cost
Limitation
At high molecular mass, resolution is difficult
because flight time is longer.
Quadrupole mass analyzer
It is one type of the mass analyzer use to
separate the ions using electric field. It consists
of four cylindrical metal rods arranged in a
square parallel to the direction of ion beam
(adjacent rods are of opposite charge.) The rods
are connected to Radio frequency and direct
current generators and it is use to measure m/z
value of ions in a mixture.
Methodology

1) It consists of four cylindrical metal rods and which are attached to

battery. Two positive charge rods are diagonal to each other and
similarly two negative charge rods are diagonal to each other.
2) Combination of RF and DC will generate oscillating electrostatic
field between the rods.
3) Now ions with different m/z value (M+= High
molecular weight, m1+ = moderate molecular
weight, m2+= Low molecular weight) will enter
into the analyzer and separation will be take
place based on their molecular weight.
4) Then radio frequency and DC voltage is applied
to the rods . Depending upon the ratio of RF
amplitude and DC voltage oscillating electrostatic
field will be generated.
5) Now due to this electrostatic field , the ion will oscillate two
way depending upon following two conditions:
i) Condition1: If RF frequency is greater than DC voltage then
large bombardment of smaller particles than larger
particles(distance travel by smaller particles are more) and so
larger particle will hit the detector surface first [M+ will reach
the detector first followed by m1+& m2+] . It is known as High
Pass filtration process.
ii) Condition II: If DC voltage is greater than RF frequency then
larger particles deviate more than smaller particles and so
smaller particles will hit the detector surface first (m2+ will
reach the detector first followed by m1+ & M+ ).This is known as
Low pass filtration process.
6) The ions posses inappropriate m/z value (other than 1-1000) will
undergo an unstable oscillation and they hit the rods without
reaching detector.
7) Hence ions with correct m/z value will undergo stable oscillation
and they will reach the detector to produce signal.
Advantages
1. Classical mass spectra. 2. Good reproducibility
3. Relatively small and low cost systems. 4. Rapid analysis
Limitations
1) Limited reproducibility.
2) Peak height variable based on mass
3) Not suitable for pulse wave ionization method.(MALDI)
Fragmentation
In mass spectrometry, fragmentation is the
dissociation of energetically unstable molecular ions
(M+ion formed by electron bombardment in the ion
chamber) into smaller ions called as fragment ions or
daughter ions. This fragment ion gives information
regarding the structure of the compound.
Importance of Fragmentation
1)Fragmentation of molecule produce large no of ions
with different molecular weight and in mass spectrum
separate peaks will be obtained for each fragment ions.
2) In Mass spectrometry, Molecular ion peak is important
peak to determine molecular weight of compound and
fragment ion peaks also useful to determine molecular
weight and structural information of the unknown
molecule.
3) The frequency and size of fragments are depended on
the structure & bond energy of sample molecule and
unique spectrum will be obtain for particular type of
compound due to unique pattern of fragmentation.
General rules for predicting prominent peaks (Electron
impact ionization method)
1)The existence of molecular ion peak in the spectrum is
dependent on the stability of molecular ion. The order
of decreasing stability of molecular ions is aromatic and
heteroaromatic compounds > conjugated polyenes >
alkenes > cyclo-alkanes > ketones > n-alkanes-ethers >
acids > alcohols > amines. Thus molecular ions are
usually very abundant in compounds contain aromatic
or heteroaromatic ring system.
2) In alkanes, the relative intensity (height) of molecular
ion peak is greatest for straight chain compounds. The
relative intensity of molecular ion peak decreases with
increased degree of branching and also with increasing
molecular weight in homologous series.
3) The fragmentation is favored at more substituted
compound because tertiary carbocation is more stable
than secondary, which is more stable than primary.
4) In alkyl substituted ring compounds,
fragmentation favored at the bond at ß position in
the ring to produce resonance stabilized benzyl ion

5) Saturated rings containing side chain, lose the

side chain at α bond and formation of ring fragment
cation
6) The cleavage of C-X bond is more difficult than that of
C-C bond and if it occurred, the positive charge will be
carried by carbon atom and not by hetero atom (due to
more electron density)

7) Compounds containing a carbonyl group tend to

undergo fragmentation and positive charge remaining
with carbonyl group portion.
8) Stevenson Rule
If two fragment ions compete with each other, the fragment
with the lowest ionization energy will be formed more frequently and
more intense than other.

In this example propenium ion fragment showing more

intense peak due to lowest ionization energy.
 Types of Fragmentation
Three types of fragmentation:
1) Homolytic cleavage(α cleavage)
2) Heterolytic cleavage
3)Mclafferty rearrangement
Homolytic cleavage
In homolytic cleavage, a covalent bond breaks and bonding electron
pair is shared between each ion produce in the cleavage.
In homolytic cleavage fragmentation, one radical ion and one
fragment ion (with positive charge) will produce.

In general, radical ion do not detected and not showing m/z value as
well as peak, but fragment ion with positive charge show peak.
2) Heterolytic cleavage
It is mainly occurred in carbon attached with hetero atom as C-X and
during the cleavage both bonding electrons will carry by hetero atom
and carbon get positive charge. C-X  C+ + X.
In this cleavage carbon with positive charge will show fragment ion
peak, but heteroatom radical do not detected and not showing peak.
3) Mclafferty rearrangement
It is one type of fragmentation pattern in mass spectrometry and in
which rearrangement Reaction occurs in the compounds containing
n electrons, π electrons and γ hydrogens. It involves cleavage of α and
β bond followed by transfer of γ hydrogen atom to the hetero atom
with lone pair of electrons.
In this rearrangement one neutral molecule (do not show peak) and
one fragment ion(show peak) will produce.
Mechanism
It can be explained with one carbonyl compound with γ hydrogen.

In this rearrangement, γ hydrogen interact with carbonyl group and

carbon – γ hydrogen bond is shifted to oxygen. Then carbonyl bond is
shifted to next carbon atom followed by cleavage of next single bond
and shifted to next carbon atom to form double bond.
Note: It will identify the functional group in the molecule by showing
corresponding m/z value. e.g Aldehyde- m/z=44, Acids-m/z=60,
Amides-m/z=59.
Types of ions produced in mass spectrometry
1) Molecular ion or parent ion
Ion formed from organic molecule by the loss of one electron when it
undergo electron bombardment and that ion carrying positive
charge. This ion is called as Molecular ion and also called as parent
ion. M  M+(Molecular ion)
2)Fragment ion/Daughter ion
It is generated by the fragmentation of molecular ion in the ionization
chamber. This fragment ion formed by two reasons as follows:
i) Due to instability of M+ion , it form fragment ions such as m1+ ,m2+
ii) Due to excess energy of ionization potential (out of 70eV, only 10eV
used for formation of M+ion) Molecular ion fragmented to form
fragment ions.
3)Metastable ion
The ions resulting from decomposition of molecular ion in field-
free region (in between the ionization chamber and magnetic
analyzer) are called as metastable ions. These ions appear as broad
peaks called as metastable ion peaks. These peaks are weaker in
intensity but useful to study fragmentation mechanism.
4)Quasi molecular ions
A protonated molecular ion or An ion formed by removal of one
hydrogen from molecule is called as quasi molecular ion.
M+H [MH]+ (M+1) M [M-H] -+H+(M-1)
These ions mimics the characteristics of molecular ion and M+1
peak will be intense peak than M peak to find out exact molecular
weight of compound.
5) Multiple charged Ion
If the ions lost more than one electron during
ionization process in mass spectrometry and then the
ions are called as multiple charged ions.
Effect of multiple charged ions
Normally m/z value is equal to molecular weight
because charge (z) is one, but in the case of multiple
charged ions , molecular weight will be half (z is 2). It
is more common in hetero aromic molecule and it is
useful in confirming molecular weight.
6) Negative ions
In ionization process of sample, along with
positive ions, the negative ions may formed. The
formation of negative ions is very rare and it may
produce in thermal ionization method. The
negative ions can be produced in three ways:
i) AB+e  A+B-
ii)AB+e  AB-
iii)AB+e  A+ +B- (ion pair)
Base peak
The most intense/tallest peak in the mass spectrum. It is
due to the ion with the greatest relative abundance. Some
time molecular ion peak and base peak are same but in
some time both will differ.
Isotope ion peak
Due to presence of heavier isotope elements
very less intense peaks will appear. In general
hydrogen, carbon and nitrogen posses isotopes,
but their relative abundance is very less (less
than 1%). Hence in mass spectrum the isotope
peak appear as small peak near molecular ion
peak and it may be called as M+1 peak or M+2
peak.
 Applications of Mass spectrometry
1) Determination of Molecular weight
Mass spectrometry is useful technique to determine or confirm
the molecular weight of compound that can be easily volatilize.
2) Determination of Molecular formula
The molecular ion peak and molecular weight of compound
must be identified by mass spectrum and then Molecular
formula can be derived from the molecular peak and other
peaks of fragment ions.
3) Determination of structure of compounds
Due to electron bombardment, the molecule undergo
fragmentation to produce many ions and from the knowledge of
molecular ion peaks as well as other ion peaks, possible
structure of compounds can be determined.
4) Determination of isotopic abundance
The isotopic abundance (relative proportions of stable isotopes of
each element) of molecule can be determined by mass
spectrometry. This information is useful for :
i) Tracer studies of isotopes
ii) Determination of atomic weight of compound
iii)Study of origin as well as nature of solar system.
5) Determination of isotopic ratio
Isotope ratio is the ratio of abundance of two isotopes of same
element e.g 18O/16O. This isotopic ratio can be determined by mass
spectrometry and it is used to determine concentration of
individual components present in complex mixture.
6) Differentiation of cis and trans isomer
The intensity of molecular ion peak obtained for cis and trans
isomer is different. Molecular ion peak of trans isomer is more
intense than cis isomer.
7)Detection of impurity
The mass spectrometry is able to detect the presence of
low concentration impurities (few ppm level) in the
sample if the structure of the impurity is quite differ from
main component. E.g detection of trace amount of xylene in
acetyl acetone. In the mass spectrum of acetyl acetone
sample, apart from molecular ion peak(m/z100), extra
peak observed at m/z 106 indicate presence of high
molecular weight impurity xylene.
8)Identification of unknown compounds
The mass spectrometry use to identify unknown
compound by comparing mass spectrum of unknown
compound with the mass spectrum of standard compound.
9) Identification of proteins
Mass spectrometry is one of the important tool for study of
structure and function of proteins (Proteomics). Electrospray
ionization and MALDI ionization methods used for this purpose.
10 Phytochemical analysis
Mass spectrometry is used in phytochemical analysis. It is used
to identify and measure the low molecular weight metabolites
present in low concentration.
11) The mass spectrometry combined with some of the
separation techniques such as capillary electrophoresis, Gas
chromatography & HPLC and known as CE-MS, GC-MS &LC-MS.
These techniques are very much useful in phytochemical and
biomolecule analysis.
12) Thermolabile and volatile phytoconstituents are
analysed by ESI and MALDI ionization techniques.
13) Clinical studies
MS is sensitive analytical method and so it is used to
analysis very low quantity of analyte in clinical studies.
14) Forensic applications
GC-MS and LC-MS are mainly used in Forensic analysis to
investigate the use of illegal drugs such as
opiates,cocaine and amphetamine by the analysis of hair,
urine, blood samples.
15) Metabolite analysis
Mass spectrometry use to analysis metabolites of drug
and also different metabolic pathways.
Basic concepts in Mass spectrometry
Definition
Mass spectrometry is one of the spectroscopic
technique used to determine molecular weight
of the compound by measuring mass-to-charge
ratio (m/z) value of one or more molecule
present in a sample.
Molecular ion or Parent ion
Ion formed from organic molecule by the loss of
one electron when it undergo electron
bombardment and that ion carrying positive
charge. This ion is called as Molecular ion and
also called as Parent ion. M  M+(Molecular ion)
Fragment ion/Daughter ion
It is generated by the fragmentation of molecular
ion in the ionization chamber. This fragment ion
formed due to instability of M+ion or due to
excess energy of ionization potential ,Molecular
ion fragmented to form fragment ions.
Metastable ion
The ions resulting from decomposition of
molecular ion in field-free region are called as
metastable ions. These ions appear as broad
peaks called as metastable ion peaks. These peaks
are weaker in intensity but useful to study
fragmentation mechanism.
Quasi molecular ions
A protonated molecular ion or An ion formed by
removal of one hydrogen from molecule is called
as quasi molecular ion.
M+H [MH]+ (M+1)
M [M-H] -+H+(M-1)
Multiple charged Ion
If the ions lost more than one electron during
ionization process in mass spectrometry and then
the ions are called as multiple charged ions.
Effect of multiple charged ions
Normally m/z value is equal to molecular weight
because charge (z) is one, but in the case of
multiple charged ions , molecular weight will be
half (z is 2). It is more common in hetero atomic
molecule and it is useful in confirming molecular
weight.
Negative ions
In ionization process of sample, along with
positive ions , the negative ions may formed. The
formation of negative ions is very rare and it may
produce in thermal ionization method.
Base peak
The most intense/tallest peak in the mass
spectrum. It is due to the ion with the greatest
relative abundance. Some time molecular ion
peak and base peak are same but in some time
both will differ.
Molecular ion peak (M+peak)
The molecular ion peak is the peak in a mass
spectrum that represents the molecular ion (M+
peak) and it is the most intensed peak with
highest m/z value.
Isotope ion peak (M+1 peak )
In mass spectrum the isotope ion peak appear
as small peak near molecular ion peak and it may
be called as M+1 peak
Importance of M+1 peak
1) M+1 peak is a small line (of 1m/z unit) and it
will appear on right side of molecular ion peak.
2) It is used to notify any isotope of carbon
present in the molecule.
3) Using relative peak height of M+1 peak (it is
percentage of peak height of M+ peak) we can
predict the number of carbon atoms and confirm
the molecular weight of compound.
Classification of ionization technique(Based
on energy)
Hard ionization technique
It requires high energy and it produce increased
no of fragmented molecular ion. E.g Electron
Ionization method.
2) Soft ionization technique
It requires comparatively less energy and
comparatively less no of fragmentation of
molecular ion.E.g Chemical ionization method,
Fast Atom bombardment(FAB)method & Matrix
Assisted Laser Desorption
Ionization(MALDI)method.
Classification of ionization techniques based
on methods
1) Gas phase ionization methods
Sample is vaporized before ionization.
Eg: Electron ionization and chemical ionization
methods
2)Desorption methods
Liquid /solid samples are directly converted into
gaseous ions. Eg: Fast Atom
bombardment(FAB)method & Matrix Assisted
Laser Desorption Ionization(MALDI)method.
Electron ionization method
It is also known as electron bombardment
ionization technique. In this method a high
energy(70eV)of beam of electrons passes through
the gaseous sample. The electrons are collided
with neutral molecule and remove one electron
from the molecule to produce positive charged
ion (M+). The excess electrode potential use to
attack the Molecular ion to produce fragment
ions.
Chemical ionization method
It is a ionization technique involving formation of
molecular ions by reaction between analyte and
reactant ions of reagent gas (Methane,isobutane
&ammonia)
Matrix Assisted Laser Desorption Ionization
(MALDI) method
It is one of soft ionization method classified as
desorption ionization method. In this method
laser beam is used to hit the surface of sample
matrix to produce molecular ions.
It is mainly used to determine molecular weight
of peptides, proteins and antibiotics (molecular
weight up to 3,00,000 unit). This method produce
less fragmentation and intense molecular ion
peak.
Application of MALDI Technique
1)MALDI-TOF mass spectrometry (MS) has
become a widely used technique for the rapid and
accurate identification of bacteria, mycobacteria
and certain fungal pathogens in the clinical
microbiology laboratory
2) The MALDI-TOF can be used in profiling and
imaging proteins directly from thin tissue
sections and known as MALDI imaging mass
spectrometry (MALDI-IMS). It provides specific
information about the local molecular
composition, relative abundance and spatial
distribution of peptides and proteins.
Fast Atom bombardment(FAB)method
It is one of the soft ionization technique classified
under desorption technique . In this method
sample is mixed with matrix and mixture of
sample matrix is bombarded by accelerated
neutral atom like xenon.
It is use to determine molecular weight of
polypetides,olegonucleotide (M.Wt of 300 to
6000 daltons).
Application of FAB method
1) It is suitable method for the analysis of highly
polar, thermally unstable compounds, especially
peptides and proteins.
2) It provides detailed sample molecular
structure information and widely used in
biomedical field.
Mass Analyzer
A mass analyzer is the component of the mass
spectrometer that takes ionized masses and
separates them based on charge to mass
ratios(m/z)and outputs them to the detector
where they are detected.
Types of mass analyzer
1. Magnetic field
2. Time of flight mass analyzer (TOF)
3. Quadrupole mass analyzer.
Magnetic field Mass analyzer
It is one of the mass analyzer used to separate the
ions based on their m/z ratio. The magnetic field
deflect the molecular ions based on their mass
/charge, lighter ion deflect more than heavier ion
(High molecular wt). Hence ion with high
molecular weight reaches the dector first.
Time of flight mass analyzer
It is one of the mass analyzer use to separate
different ions of sample based in their mass to
charge ratio without using electric/magnetic
field. This is achieved by accelerating all particles
at the same electric potential and then
measuring the time taken by each ion to cross
same distance to reach detector.
Advantages of TOF analyzer
1) Increased mass accuracy and mass resolution
2) Greater sensitivity
3) Rapid analysis
4) It can analysis sample of unlimited mass
range. 5) Moderate cost.
Quadrupole mass analyzer
It is one type of the mass analyzer use to separate
the ions using electric field. It consists of four
cylindrical metal rods arranged in a square
parallel to the direction of ion beam (adjacent
rods are of opposite charge.) The rods are
connected to Radio frequency and direct
current generators and it is use to measure m/z
value of ions in a mixture.
Advantages of Quadrupole mass analyzer
1)Classical mass spectra.
2) Good reproducibility
3) Relatively small and low cost systems.
4. Rapid analysis
Detector of Mass spectrometer
Detector of MS is a device, which will convert
beam of ions into current signal and amount of
current generated based on ion abundance.
Types of detector
1)Faraday cup collector, 2)Electron multiplier
tube 3)Photographic plate.
Faraday cup collector
They are based on measurement of direct charge
current produced when an ion hits surface. It
consists of insulated conductor directly
connected to an electrometer amplifier.
Electron multiplier
It consists of the primary cathode optimized for
the detection of ions. The sensitivity of this
detector is 1000 times that of Faraday cup.
Photographic plate detector
This detector system is most sensitive than any
other detector, because the photographic plate
integrates the ion signal over period of time. It is
processed with photographic techniques and
read with the aid of densitometer. It is used more
effectively in double focusing spectrometer.
Recorder of MS
It records the relative abundance of each ion
having specific mass/charge ratio to give a mass
spectrum.
Mass spectrum
A mass spectrum is constructed by plotting m/z
ratio of the ions present in the sample against
their relative abundanace (intensity). Each peak
in a mass spectrum shows a component of unique
m/z value (Mol.wt) present in the sample. In a
mass spctrum, molecular ion peaks and fragment
ion peaks are present.
Fragmentation
In mass spectrometry, fragmentation is the
dissociation of energetically unstable
molecular ions (M+ion formed by electron
bombardment in the ion chamber) into
smaller ions called as fragment ions or
daughter ions. This fragment ion gives
information regarding the structure of the
compound.
Types of Fragmentation
Three types of fragmentation:
1)Homolytic cleavage(α cleavage)
2)Heterolytic cleavage
3)Mclafferty rearrangement

Homolytic cleavage
In homolytic cleavage, a covalent bond breaks
and bonding electron pair is shared between each
ion produce in the cleavage.

Note: Radical ion do not show any peak and

Fragment ion show positive ion peak.
Heterolytic cleavage
It is mainly occurred in carbon attached with
hetero atom as C-X and during the cleavage both
bonding electrons will carry by hetero atom and
carbon get positive charge.
C-X  C+ + X.
Note: Carbon with positive charge show
positive ion peak and radical ion do not show
peak.
Mclafferty rearrangement
It is one type of fragmentation pattern in mass
spectrometry. It involves cleavage of α and β
bond followed by transfer of γ hydrogen atom to
the hetero atom with lone pair of electrons.
Note: This type of rearrangement occurs in
compounds containing n electrons, π electrons
and γ hydrogen (Carbonyl compounds, acids
and amine)
Nitrogen Rule (Definition for 2 mark)
It is one of the important rule in Mass
spectrometry to identify the molecular ion.
According to this rule, a molecule of even
numbered molecular mass (Mol.Wt) must contain
no nitrogen atom or even no of nitrogen
atoms. An odd numbered molecular mass must
contain odd no of nitrogen atom.
Importance of Nitrogen rule (Additional
points for 5 mark question)
From the Nitrogen rule , the following important
conclusion can be derived,
1. Fragmentation at a single bond gives an odd
numbered fragment ion from the even
numbered molecular ion. e.g
2,4 dinitophenol fragmented to give one
molecular ion with even no m/z value(184)
and one fragment ion with odd no m/z
value(183).(It also satisfy nitrogen rule
that molecular ion with even no molecular
weight contain 2 nitrogen atoms)
2. Odd numbered molecular ion produce even
no fragment ion. e.g
Ethyl amine fragmented to give one
molecular ion with odd no m/z value (45)
 and one fragment ion with even no m/z
value(30). (It also satisfy nitrogen rule that
molecular ion with odd no molecular
weight contain one nitrogen atom)
Ring rule
This rule used to calculate no of unsaturated
sites(R) is equal to the no of rings in the molecule
plus no of double bonds plus twice the no of
triple bonds. Ring rule for molecule CwHxNyOz
R= w+1+y-x/2.
e.g Ring rule for Benzene Mol. Formula
C6H6(w=6,x=6,y=0(No nitrogen),z=0(No oxygen)
R= 6+1+0-6/2 = 7-3=4. Hence R value for
Benzene is 4 and it indicated that Benzene
contains 1 ring and 3 double bonds.
Stevenson rule
It is one of rule for fragmentation pattern in Mass
spectrometry. If two fragment ions compete with
each other, the fragment with the lowest
ionization energy will be formed more frequently
and more intense than other.
 e.g CH3-CH=CH-CH3 CH3-CH = CH++CH3-
(m/z 41) (m/z9.8)
In this example propenium ion fragment
showing more intense peak due to lowest
ionization energy.
Important Applications of Mass spectrometry
1)Determination of Molecular weight and
Molecular formula of unknown compounds.
2)Determination of structure of compounds.
3)Differentiation of cis and trans isomer of
organic compounds.
4)It is used to detect impurity present in the
sample based on different m/z value of impurity
than molecular ion.
5) It is used to identify the unknown compounds
by comparing with mass spectrum of standard
compound.