UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS

Lecture-01
ELECTROMAGNETIC RADIATION
Electromagnetic radiation can be described in terms of a stream of mass-less particles called
photons each travelling in a wave like pattern at the speed of light.
Electromagnetic radiation consist of varying electric field and magnetic fields in mutually
perpendicular planes. The radiation propagate in the direction perpendicular to the plane of both
the fields.

The EMR consist of discrete packets of energy called as photons.

The EMR are characterized by wavelength, frequency, wave number & Energy.
 Wavelength (λ): The distance between two crests or two parts of wave in same phase is
called wavelength. Unit: meters.
 Frequency (υ): The number of oscillation of waves per unit time is called frequency. Unit:
cycles/second (or) Hertz.
 Wave Number (ύ): It is the number of waves per unit distance. It is reciprocal of
wavelength. Unit: m-1.
 Energy: The energy associated with EMR is E = h.c / λ and E = h υ
c = 2.99792458 m/s is the speed of light in a vacuum
h = 6.62607015×10−34 Js is Planck’s constant.
There are different types of electromagnetic radiations depending on the energy associated
with the photon that in turns depends on frequency or wavelength.

ELECTROMAGNETIC SPECTRUM

The arrangement of electromagnetic radiations, either in ascending or descending order of the

wavelength and frequency, is called the Electromagnetic spectrum.
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS
Lecture-2:
SPECTROSCOPY
Introduction:
Spectroscopy is the study of the interaction electromagnetic radiation with matter.
Spectrometry is the measurement of the responses of matter during interaction with
electromagnetic radiations and the instrument which accounts these measurements is called
Spectrometer or spectrograph.
Interaction of EMR with matter:
When EMR falls on matter then it interacts with matter which having discrete energy levels.
Types of energy levels:
Electronic Energy Levels: At room temperature the molecules are in the lowest energy levels E0.
When the molecules absorb UV-visible light from electromagnetic radiation, one of the outer
most electrons from pi - bond, sigma bond or a lone pair is promoted to higher electronic energy
state.
Vibrational Energy Levels: Vibrational energy levels are of less energy than electronic energy
levels. When infrared radiation is absorbed, molecules are excited from one vibrational level
to another. Thus, during vibrational excitation molecule begins to vibrate.
Rotational Energy Levels: The spacing between rotational energy levels is very small. When
microwave radiation is absorbed, molecules are excited from one rotational level to another.
Relation between energy: ΔE rotational < ΔE vibrational < ΔE electronic
Energy Level diagram

If E is the total energy of a molecule, it can be expressed as the sum of translational, rotational,
vibrational and electronic contributions.
E = Etrans +Erot + Evib + Eelec
Absorption or emission of EMR causes a change in any of these types of energies.
In molecular spectroscopy, we measure the change in these energy states.
Translational energy – It is due to the overall movement of the molecule. Energy levels are not
quantized. Hence no spectroscopy.
Rotational energy – It is due to spinning of molecules about the axis passing through the centre
of gravity - Rotational Levels are quantized – Rotational spectroscopy (Microwave spectroscopy)
Vibrational energy – It is due to vibrations in molecules – Vibrational Levels are Quantized – IR
Spectroscopy (Vibrational spectroscopy)
Electronic energy – Consists of electronic levels which are quantized – UV/visible spectroscopy
(Electronic spectroscopy)
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS
Lecture-3:
BEER-LAMBERT LAW
Lambert Law:
This law states that when monochromatic light passes through a transparent medium, the rate of
decrease in intensity with the thickness of the medium is proportional to the intensity of the light.
This is equivalent to stating that the intensity of the emitted light decreases exponentially as the
thickness of the absorbing medium increases arithmetically, or that any layer of given thickness of
the medium absorbs the same fraction of the light incident upon it.
Mathematically it can be expressed as
dI
− ∝ I
dl
dI
− =aI
dl
Where,
I=Intensity of the incident light
l=Thickness of the medium
a= Proportionality factor
dI
− = a dl
I
Now, integrating above equation,
𝐼 𝑙
dI
∫− = a ∫ 𝑑𝑙
I
𝐼𝑂 0
log 𝑒 𝐼 − log 𝑒 𝐼𝑜 = −𝑎 𝑙
𝐼
log 𝑒 = −𝑎𝑙
𝐼𝑜
𝐼
= 𝑒 −𝑎𝑙
𝐼0
So, 𝐼 = 𝐼𝑜 𝑒 −𝑎𝑙 --------------------- (1)
Beer Law:
Beer’s law states that the intensity of a beam of monochromatic light decreases exponentially as
the concentration of the absorbing substance increases arithmetically.

𝐼 = 𝐼𝑜 𝑒 −𝑏𝑐 ------------------------- (2)

From combination of equation (1) and (2), we get,

𝐼 = 𝐼𝑜 𝑒 −𝑘𝑐𝑙
𝐼
= 𝑒 −𝑘𝑐𝑙
𝐼0
𝐼
𝑙𝑜𝑔10 𝐼 = −𝜀𝑐𝑙
0
𝐼
A = log10 𝐼𝑜 = ɛ𝑐𝑙 ------------------------ (3)
Here ɛ= molar absorption coefficient Equation (3) often known as beer-lambert law.
Limitation of Beer-Lamber’s law:
 The electromagnetic radiation should be monochromatic.
 The light beam should not be scattered.
 The solution should be diluted.
Applications of Beer-Lambert law:

 to conduct a qualitative and quantitative analysis of biological compounds

 to determine the concentration of various substances in cell structures by measuring their
absorbing spectra in the cell
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS
Lecture-04
UV- VISIBLE SPECTROSCOPY

In electromagnetic spectrum, which is falling in the region 100-400 nm are called UV rays.
In electromagnetic spectrum, which is falling in the region 400-700 nm are called visible region.
UV Visible spectroscopy is a type of absorption spectroscopy in which EMR of region (100-700
nm) is absorbed by the molecule which results in the excitation of the electrons from the ground
state to a higher energy state.

ELECTRONIC TRANSITIONS

σ – σ* transition: In this transition, the electrons present in σ bonding orbital (lower energy)
excited to σ* anti bonding orbital (higher energy). This transition requires higher energy. This
transition is only observed in compound containing σ bond / electrons and not lone pair electrons
on any atoms.
E.g. Transition in saturated compounds like CH4, C2H6 etc.
The absorption band occurs in the far ultraviolet region in the range of 100 to 135 nm Wavelength.

π – π* transition: In this transition, the π electrons in bonding orbital’s (lower energy) are excited
to π* anti bonding orbital (higher energy).
This transition is only observed in double bonded compounds containing π electrons.
The compound containing isolated double bond shows large absorption in the range of 160- 175
nm. This band is called as E band. The compound containing conjugated double bond shows very
large absorption in the range of 210 -280 nm. This band is called as K band.
The aromatic and hetero aromatic compounds absorbed at 220 – 270 nm Wavelength. This band
is called as B band.

n - π* transition: In this transition nonbonding or lone pair of electrons are excited from
nonbonding orbital to π* anti-bonding orbital.
This transition is observed in unsaturated compound containing double bond and lone pair of
electrons on one atom. e.g. C = O, C = N, C = S etc.
The absorption band occurs in region of 270 – 320 nm. This band is called as R band.
e.g. Aldehyde, ketone shows absorption in the range of 270 – 320 nm but unsaturated Aldehyde
and ketone are absorbs at 300 – 350 nm wavelength.
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS

n - σ* transition: In this transition nonbonding or lone pair of electrons are excited from
nonbonding orbital to σ* anti bonding orbital. This transition is only observed in compound
containing σ bond and lone pair electrons on at least one atom. e.g. C – O, C – N, C – S
This band is observed in near UV region (180 – 225) nm.

σ- π* and π - σ*transition: These transitions are forbidden transitions and are only theoretically
possible. The relative energies of excitations are below as,

Selection Rules: The various electronic transitions which are governed by certain restrictions are
called selection rules. These are:

(i) The transitions which involve a change in the spin quantum number of an electron during the
transition do not occur. Thus, singlet-triplet transitions are forbidden.

(ii) The transitions between orbitals of different symmetry do not occur.

Lecture-05
INSTRUMENTATION OF DOUBLE BEAM UV SPECTROPHOTOMETER
Principle of UV Visible Spectroscopy:
On passing electromagnetic radiations in the UV and visible region through a compound, a part of
the radiation is absorbed by the compound. After the absorption of energy, the electrons in the
orbitals of lower energy are excited into the orbitals of higher energy.

Light Source:
Tungsten filament lamps and Hydrogen-Deuterium lamps are the most widely used and suitable
light sources as they cover the whole UV Visible region.
 Hydrogen - Deuterium Discharge Lamp (200-400 nm)
 Tungsten Filament lamp (400 - 750nm)

Monochromator:

It is a device used to resolve wide band of Polychromatic light radiation into narrow band of
Monochromatic radiations. Ex: prisms, Gratings etc
Beam splitter:
Splits the light from the source into two beams. One of the two divided beams is passed through
the sample solution and the second beam is passed through the reference solution.
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS

Sample holders (Cuvettes):

Sample dissolved in suitable solutions is taken in an optically transparent sample cell called
cuvette. Quartz or silica cuvettes are used for UV range of spectrum. Glass can’t be used for the
cells as it also absorbs light in the UV region.
Detector:
Detector converts a light signal into an electrical signal. Generally two photocells serve the purpose
of detector in UV spectroscopy.
Eg: Photomultiplier tubes, silicon photodiodes and thermo couple are sensitive in the ultraviolet
and visible wavelength ranges
Amplifier:
The Pulsating or alternate currents which come from the detector transfer into a device called
amplifier which amplify the signals many times.
Recorder:
The amplifier is connected to a recorder. The recorder records the absorption bands automatically
which are displayed on the read out device. It records UV-VISISIBLE spectrograph with
absorbance against the wavelength.

Applications of UV Spectroscopy

• Detection of Impurities
• Structure elucidation of organic compounds
• Quantitative analysis
• Qualitative analysis
• Molecular weight determination
• Distinction between Cis & Trans isomerism
• Detection of functional group
• Kinetics of reaction
• Effect of Conjugation
• UV spectrophotometer may be used as a detector for HPLC.
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS
Lecture-06
IR SPECTROSCOPY
In electromagnetic spectrum, which is falling in the region 12500 cm-1 to 20 cm-1 are called
Infrared region.

The range of infrared region is 12500 ~ 20 cm-1 and can be divided into
 Near-infrared region (12500 ~ 4000 cm-1),
 Mid-infrared region (4000 ~ 400 cm-1)
 Far-infrared region (400 ~ 20 cm-1).

Types of fundamental vibrations:

Vibration is periodic displacement of atoms or nuclei from their equilibrium position.

There are two types of fundamental molecular vibrations viz., stretching (change in bond
length) and bending (change in bond angle).

Stretching vibrations: In stretching vibrations the atoms move along the bond axis. As a
result, the bond length increases or decreases but bond angle remains unchanged.

Stretching vibrations are of two types’ viz., symmetrical Stretching & asymmetrical stretching.
Symmetric stretching: In this type the atoms of the molecule move in the same direction.
Asymmetrical stretching: In this type the atoms of the molecule move in the opposite
direction.

Bending vibrations: Bending vibrations involves a change in the bond angle whereas the bond
length remains unchanged
There are two types of bending vibrations:
a) In - plane bending vibrations:
 Scissoring: In this type, the atoms move away and come close to each other in the same
plane just like the blades of a scissor.
 Rocking: In this type the movement of atoms takes place in the same direction.
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS

b) Out-of-plane bending vibrations:

 Twisting: In this type one atom moves up and the other moves down the plane with
respect to the central atom.
 Wagging: In this type two atoms together move up and below the plane with respect
to the central atom.

Selection rules for Infrared transitions:

For a particular vibration to be infrared active there must be a change in the dipole moment of
the molecule during the vibration. In other words transition dipole moment must not be zero.

Homonuclear diatomic molecules are inactive in the infrared spectrum. They do not have a
dipole moment and during the vibration also the dipole moment is zero. eg: H2, O2, N2 etc.

Heteronuclear diatomic molecules such as CO, NO are active in IR.

Symmetrical polyatomic molecules such as CO2, the symmetric stretching vibration is infrared
inactive whereas the asymmetric stretching vibration is IR active ∆ν = ± 1, transition can take
place between Adjacent vibrational levels, 0 to 1, 1 to 2 etc.
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS
Lecture-07
INSTRUMENTATION OF INFRARED SPECTROPHOTOMETER
Working Principle Irradiation of sample with IR radiation brings about vibrational changes
in molecules. The transition of molecule is from lower vibrational energy level to higher
vibrational energy level. The transition is induced by absorption of photon of the IR radiation
of appropriate frequency, which matches with energy gap between the two levels.
IR absorption by molecules happens only when there is a change in the dipole moment of the
molecule.

 Radiation source: The Nernst glower and globar are the most common source of
radiation. Globar is a silicon carbide rod when heated electrically at 1200-2000°C, it
glows and produces IR radiations.
 Monochromator: The radiation source emits radiations of various frequencies. As the
sample absorbs only at certain frequencies, it is therefore necessary to select desired
frequencies from the radiation source. This has been achieved by monochromators.
Prisms and gratings are commonly used for this purpose.
 Sample holder (Cuvette): The sample holder made up of sodium chloride or
potassium bromide. It is used to contain sample solutions as well as reference solution
because they are transparent to IR radiation.
 Detector: The detectors generally convert photo signals into photo electric signals.
Thermocouples, Bolometers, thermisters, Golay cell, and pyro-electric detectors are
used for this purpose.
 Amplifier: Amplifier will amplify the photoelectric signals received from the detector.
The amplified photo signals are then sent to recorder.
 Recorders: This records the amplified photo electric signals received from the
amplifier and the results are given in the form of a spectra. (Functional group region &
Finger print region)
Applications of Infrared (IR) Spectroscopy

 Identification of functional groups

 Structural elucidation of Organic compounds.
 Detection of impurities in a compound
 studying the progress of reactions
 It is used for the quantitative analysis of a number of organic compounds.
 It is used to determine the ratio of cis-trans isomers in a mixture of compounds.
 It is used to study the presence of water in a sample.
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS
Regions of the Infrared spectrum:

Most of the bands that indicate what functional group is present are found in the region from
4000 cm-1 to 1300 cm-1. Their bands can be identified and used to determine the functional
group of an unknown compound.
Bands that are unique to each molecule, similar to a fingerprint, are found in the fingerprint
region, from 1300 cm-1 to 400 cm-1. These bands are only used to compare the spectra of one
compound to another.
Lecture-08
CHROMATOGRAPHY
The Russian botanist Mikhail Tswett coined the term chromatography in 1906.
Definition: Chromatography is a technique for separating mixtures into their components in
order to analyze, identify, purify, and/or quantify the mixture or components is known as
chromatography.
Principle: chromatography is based on a principle of selective distribution of the different
components of a mixture between two phases, namely stationary phase and mobile phase. The
stationary phase can be a solid or liquid; while the mobile phase is a liquid or gas. When the
stationary phase is solid, the selective distribution is based on adsorption; while it is a liquid
the basis of selective distribution is partition.

Types of chromatographic techniques:

 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS
Lecture-09:
Different terms used in the chromatography technique.
Stationary phase: Stationary Phase in Chromatography is the one that doesn’t move with the
sample. It is generally a porous solid that absorbs components from the mobile phase.

Mobile phase: Mobile Phase in Chromatography is the component that moves with the
sample. It is either a gas or a liquid and is passed through the column where the components
of the mixture are absorbed.

Separated molecules The type of interaction between the stationary phase, mobile phase, and
substances contained in the mixture is the basic component effective on the separation of
molecules from each other.

Analyte:- Analyte is the substance that is to be separated from the mixture during
chromatography.
Elution :- is a process of removing adsorbed material from stationary phase by the movement
of mobile phase.
Eluent :- It is a solvent that used for separation of absorbed material from stationary phase

Elute: Elute is the fluid containing the sample that exits the chromatographic column

Column: A Chromatography column is a device used in chromatography for these paration of

chemical compounds

Chromatogram: A chromatogram is the trace generated by the detector signal and requires a
carefully controlled flow rate of the carrier gas (mobile phase) and a carefully controlled
temperature of the column (stationary phase) to yield repeatable results.

Solvent : Any substance capable of solubilizing another substance.

Retention time : Time takes for a particular analyte to pass through the system (from the column
inlet to the detector) under set conditions.

HPLC (HIGH PERFORMANCE LIQUID CHROMATOGRAPHY)

Principle of HPLC:

High Performance Liquid Chromatography [HPLC] is based on principle is Adsorption as well

as partition which are depending on the nature of stationary phase, if stationary phase is in solid
phase is based on adsorption chromatography and if stationary phase is liquid phase is based
on partition chromatography.
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS

Lecture-10
INSTRUMENTATION OF HPLC

Solvent reservoir
The solvent reservoir store the solvent or mobile phase to supply to the column as necessary.
Degasser
The eluent used for LC analysis may contain gases such as oxygen that are non-visible to our
eyes. When gas is present in the eluent, this is detected as noise and causes an unstable baseline.
Degasser uses special polymer membrane tubing to remove gases. The numerous very small
pores on the surface of the polymer tube allow the air to go through while preventing any liquid
to go through the pore.
Pump:
The pump is used to deliver the mobile phase at a constant flow rate through the stationary
phase in the column.
Injector:
The injector is used to introduce the sample into the mobile phase. The sample can be injected
manually or automatically. The simplest method is to use a syringe, and the sample is introduced
to the flow of eluent.
Column:
The column contains the stationary phase, which is typically a solid material that interacts with
the sample components based on their physicochemical properties. The separation is performed
inside the column.
Column Heater:
The LC separation is often largely influenced by the column temperature. In order to obtain
repeatable results, it is important to keep consistent temperature conditions. Also for some
analysis, such as sugar and organic acid, better resolutions can be obtained at elevated
temperatures (50 to 80°C). Thus columns are generally kept inside the column oven (column
heater).
Detector:
The detector monitors the eluent from the column and produces a signal that is proportional to
the concentration of the eluted components. Separation of analytes is performed inside the
column, whereas a detector is used to observe the obtained separation.
The composition of the eluent is consistent when no analyte is present. While the presence of
analyte changes the composition of the eluent. What detector does is to measure these
differences. This difference is monitored as a form of an electronic signal. There are different
types of detectors available.
Record: The data processing unit records and analyzes the output of the detector, and produces
a chromatogram that represents the separation of the sample components.
 UNIT-V: INSTRUMENTAL METHODS AND APPLICATIONS
Lecture-11
Applications of HPLC:

 The HPLC has developed into a universally applicable method so that it finds its use in almost
all areas of chemistry, biochemistry, and pharmacy.
 Analysis of drugs
 Analysis of synthetic polymers
 Analysis of pollutants in environmental analytics
 Determination of drugs in biological matrices
 Isolation of valuable products
 Product purity and quality control of industrial products and fine chemicals
 Separation and purification of biopolymers such as enzymes or nucleic acids

Advantages of HPLC

 It is Speed ,Efficiency &Accuracy

 Versatile and extremely precise when it comes to identifying and quantifying chemical
components. It is simple, rapid, and reproducible. & High sensitivity.
 High performance. & rapid process and hence time saving.
 It is important for qualitative and quantitative analysis.

Limitations of HPLC

 Cost: Despite its advantages, HPLC can be costly, requiring large quantities of expensive
organics.
 HPLC does have low sensitivity for certain compounds, and some cannot be detected as they
are irreversibly adsorbed.
 Volatile substances are better separated by gas chromatography.