UNIT – V

INSTRUMENTAL METHODS AND ITS PPLICATIONS

Electromagnetic spectrum:
The entire range over which electromagnetic radiation exists is known as electromagnetic spectrum.
The electromagnetic spectrum covers larger range of wavelength.
Below figure shows a diagrammatic representation of the electromagnetic spectrum. A
logarithmic scale is used in this representation. The divisions between the different spectral regions
indicate the origin of radiation. The limits indicated in Fig---are arbitrary.

Electromagnetic spectrum
Beer-Lambert’s Law:

Lambert’s law:

Let a beam of monochromatic radiation pass through a homogeneous absorbing medium.

Now the rate of decrease in intensity of the radiation ‘dI’ with the thickness of the absorbing
medium ‘dx’, is proportional to intensity ‘I’ of the incident radiation.

Beer-Lambert’s Law

Let a beam of monochromatic radiation pass through a homogeneous absorbing medium.

Now the rate of decrease in intensity of the radiation ‘dI’ with the thickness of the absorbing
medium ‘dx’ is proportional to the intensity of the incident radiation ‘I’ as well as the
concentration ‘C’ of the solution.
UV-visible spectroscopy:

UV-Visible spectroscopy involves transition of valence electrons of molecules or ions from

the ground electronic state E0 to a higher electronic state E1. The ultra violet region is 100 to
400 nm. The visible region is 400 to 800 nm. The amount of energy required for the transition
is,

E = hυ = E1 - E0

Type of transitions involved in organic molecules

There are three types of electrons in organic molecules -  bonded electrons,  bonded
electrons and non-bonded electrons (n). All the three types of electrons are present in four
types of Transitions.
Fundamental Vibrations

n  *   * n  *  *

The types of transitions and their order of energies are:

n  * <   * < n  * <  *

1. n  * transitions
These are shown by unsaturated molecules with heteroatoms like N, O, S and halogen. They
are due to the excitation of an electron from non-bonding lone pair (n) to an empty anti-
bonding * orbital.
Example: Acetaldehyde and acetone
n  * transition occurs at 270 to 300 nm. An electron from the non-bonding

2.   * transitions
These are shown by molecules with  electrons. (e.g.) olefins, aromatic compounds. They are
due to the excitation of an electron from  bonding orbital to anti-bonding * orbital.
Example: Ethylene CH2 = CH2
  * transition occurs at 200 nm.
3. n  * transitions
These are shown by saturated compounds containing a lone pair of electrons. They are due to
the excitation of an electron from non-bonding orbital (n) to a * anti-bonding orbital.
Example: Trimethyl amine (CH3)3 N;
n  * transition occurs at 227 nm. An electron from the non-bonding orbital of ‘N’ atom is
excited to a * orbital.

4.   * transitions
These are shown by all saturated compounds. They are due to the excitation of an electron from
 bonding orbital to a * anti-bonding orbital.
Example: CH4

  * transition occurs at 122 nm.   * are high energy transitions. They are weak, and
usually not observed.

Instrumentation of UV- spectroscopy Components

i) Radiation source
Hydrogen discharge and deuterium lamps as well as mercury lamps are the common sources. They
must supply a steady radiation of sufficient intensity.

ii) Monochromator
It disperses the radiation according to the desired wavelength. It comprises an entrance slit, a
dispersing prism or grating and an exit slit.
iii) Cells
They hold the sample and reference solutions. The cell must be uniform in construction, inert to
solvents and transparent.

iv) Detector
Barrier layer cell, photomultiplier tube, photocell are used as detectors. The detector converts the
radiation falling on it into electric current.
v) Recorder
The signal from the detector is recorded on a chart paper by the recorder.

Working
The radiation from the source passes through the monochromator, which selects a narrow range of
wavelength to pass through an exit slit. The radiation from the monochromator is split into two
equal beams. One half (reference beam) passes through cell containing the blank solution. The
other half (sample beam) passes through the cell containing test solution. The detector compares
the intensities of the two beams. If sample absorbs, the intensity of the sample beam “I” will be
less than that of reference beam (I0). The recorder plots absorbance versus wavelength.

UV-Visible spectroscopy applications:

i) Determination of Structure of Compound

ii) Study of Coordinate Complexes
iii) Determination of Formation Constants
iv) Determination Kinetic Studies
v) Determination of Concentration of Solutions
vi) Determination of Calcium in Blood.
vii) Determination of Molecular weight
 IR spectroscopy:

IR spectra is produced by the absorption of energy by a molecule in the infrared region and transitions
occur between vibrational levels. So, IR spectroscopy is also known as vibrational spectroscopy.
Range of Infrared Radiation
The range in the electromagnetic spectrum extending from 12,500 to 50 cm (0.8 to 200 µ) is commonly
referred to as the infrared. This region is further divided into three sub regions.

λ=0.8 2.5 15 20µ

Near IR IR Far IR
-1
Υ=12500 4000 667 50 cm
Fig. Range of IR radiation
(i) Near infrared: The region is from 12500 to 4000 cm -1
(ii) Infrared (or) ordinary IR: The region is from 4000 to 667 cm -1
(iii) Far infrared: The region is from 667 to 50 cm -1

Selection rules
A selection rule states the possibility of transition from one level to another level and exhibit of spectra
by the compounds. Whenever the matter absorbs the IR radiation, it can induce vibrations in the molecule.
Vibrational modes can be divided into stretching (symmetric and asymmetric) and bending vibrations (in-plane
and out-of plane).

The First selection rule says that all the bonds in a molecule are not capable of absorbing IR energy but
those bonds which are accompanied by a change in dipole moment will absorb in the IR radiation and such
transitions are called IR active transitions. The transitions which are not accompanied by a change in dipole
moment of the molecule are not directly observed and are considered as IR inactive transitions.

The second selection rule tells us about the transitions that are possible (or allowed) amongst the quantized
energy levels. The selection rule for the IR spectroscopy is

v = ±1, ±2, ±3…etc

This means that the molecule may go over from any vibrational level to any other level. This in turn means that
the spectrum would be quite complex. However, the actual IR spectrum for a diatomic molecule is not so
complex. The pure vibration spectrum of a diatomic molecule consists of mainly three signals: an intense signal
for the fundamental vibration, that arise from
the transitions from ground state (v = 0) to the first (v = 1) excited vibrational level. The second is a weak signal
for its first overtone that is observed at roughly double the frequency of the fundamental band; it is due to the
transitions from ground state (v = 0) to the second (v = 2) excited vibrational level. The third and the weakest
signal is observed at roughly triple the frequency of the fundamental band; it is due to the transitions from
ground state (v = 0) to the third (v = 3) excited vibrational level. It is called the second overtone.

Molecular Vibration and Origin of IR Spectrum

Since atoms in a molecule are continuously vibrating molecules are also vibrating. There are two kinds
of fundamental vibrations in the molecule.
1. Stretching vibrations: During sretching the distance between two atoms decreases or
increases, but bond angle remains unaltered.
2. Bending (or) deformation vibrations: During bending bond angle increases and decrease
but bond distance remains unaltered.

Fingerprint region
The vibrational spectral (IR spectra) region at 1400 – 700cm -1 gives very rich and intense absorption
bands. This region is termed as fingerprint region. The region 4000 – 1430cm -1 is known as Group
frequency region.

There are two types of fundamental vibrations in a molecule.

Fundamental Vibrations

Stretching Vibrations Bending Vibrations

Symmetric stretching Asymmetric stretching In- Plane Bending Out- Plane Bending

Sissoring Rocking Wagging Twisting

1) Stretching vibration
In this vibration, the bonds are elongated and compressed. But the bond angle remains
unchanged. There are two kinds of stretching vibrations:
i) Symmetric stretching

In this type the atoms of the molecule move in the same direction
ii) Asymmetric stretching

In this type the atoms of the molecule move in the oppsiite direction

2) Bending or deformation vibrations

Bending vibrations involves a change in the bond angle whereas the bond length remains
unchanged.
i) In plane bending vibrations: Here all the atoms are in the same plane during vibration.

Scissoring: In this type, the atoms move away and come close to each other in the same plane
just likes the blades of a scissor.
Rocking: In this type the movement of atoms takes place in the same direction.
ii) Out of plane bending vibration: Here the atoms go out of plane during vibration.

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

Instrumentation of IR Spectrometer

Components:
i) Radiation Source
Nichrome wire and Nernst glower are the main sources of IR radiation. Nernst glower is filament
made of oxides of Zr, Th and Ce held together using a binder.

ii) Monochromator
It permits only radiation of desired wavelength to pass through and absorbs other wavelengths.

iii) Cell
The cell holding the sample solution should be transparent to IR radiation. NaCl cells are used.

iv) Detector
The IR radiation falling on the detector is converted into an electric signal.
Some detectors are:
i) Thermocouple

ii) Pyroelectric detectors

v) Recorder
The electrical signal from the detector is recorded as percentage transmittance against
wavelength.

Working
The radiation from the source is split into two beams of equal intensity. One beam passes
through the sample and the other through the reference.
When the sample absorbs, the two emerging beams will have different intensities. This produces
an oscillating signal to be measured by the detector. The signal from detector gets amplified and
goes to the recorder which gives a print out or display.

IR spectroscopy applications:

1.Identification of Compounds
Each molecule gives its own unique IR spectrum. If two samples give the same IR spectra,
they contain identical molecules. Thus the identity of a compound can be established by
comparing the IR spectrum of a compound with the standard IR spectra available in the
literature.

1. Detection of functional groups

Different functional groups absorb at different IR regions. So from the absorption frequency, the
nature of functional group present in the molecule can be identified.

2. The Progress of the reaction can be studied by observing the increasing or decreasing in the
Intensity of particular peak at different time intervals.
3. Structure determination: We can easily find out whether a molecule is linear or not.
4. Cis-trans isomers can be identified.
5. Purity of samples can be identified
6. Molecular weight of a compound can be determined by measuring endgroup concentrations in
the IR spectrum.
7. The crystallinity of a solid can be ascertained by changes in the IR spectrum.
Chromatography

The term “chromatography” is derived from Greek, chroma meaning, “colour,” and graphein
meaning “to write”. The Russian botanist Mikhail Tswett coined the term chromatography in 1906.

Chromatography is an analytical separation technique used to separation of individual components from the
mixture of components as well as their qualitative and quantitative determination. In chemical laboratories, it is
the most used separation technique where it is used for analysis, isolation, and purification.

In this method of separating a mixture of components into individual component through equilibrium distribution
between two phases, stationary phase and mobile phase. Chromatography involves a sample (or sample extract)
being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The substance that has
to be separated during the process of chromatography is known as an analyte.

The stationary phase is a phase that is fixed in a place in a column or on a solid surface. The mobile phase is
passing through the stationary phase carrying with it the sample solution

Principle:
The chromatography is dependent on the principle of partitioning the constituent components (analyte)
between two phases, i.e., a Mobile Phase and a Stationary Phase.

The stationary phase in chromatography is composed of either a solid or liquid substance attached to a glass or a
metal surface on which the components of the mixture to be separated.

The mobile phase in chromatography is either a liquid or gas, which is passed through a chromatographic column
where the components of the mixture are separated at different rates by adsorbing to the stationary phase.
Classification:

Chromatography can be classified into two basic types based on the stationary phase bed.

1. Column chromatography
2. Planar chromatography

The stationary phase is supported on a flat surface or in the pores of a paper and the mobile phase passes
through the stationary phase by capillary action or under the influence of gravity

A more fundamental classification of chromatographic separations is based on the types of mobile and stationary
phases and the kinds of equilibria involved in the transfer of solutes between the phases. Based on the type of
mobile phase used, the chromatography divided into three types, Gas chromatography, Liquid chromatography
and supercritical fluid chromatography. The mobile phases are gas, liquid and supercritical fluids respectively.

Based on separation mechanism, Chromatography is divided into affinity chromatography, Adsorption

chromatography, Partition chromatography and Ion-exchange chromatography.

In adsorption chromatography, a solid stationary phase and liquid or gaseous mobile phase are used. The solute
is adsorbed on the surface of the solid particles. The more strongly a solute is adsorbed, the slower it travels
through the column.

In partition chromatography, A liquid stationary phase is bonded to a solid surface, which is typically inside the
pores of the silica (SiO2) column. The different components in a mixture equilibrate between the stationary phase
and liquid phase.

In ion-exchange chromatography, anions such as -SO 3 or cations such as -N(CH3)3+ are covalently attached to
the stationary solid phase, usually a resin. Solute ions of the opposite charge are attracted to the stationary phase.
the mobile phase is a liquid.

In gel filtration or size exclusion or gel permeation chromatography, this technique separates the molecules by
size, with no attractive interaction between the stationary phase and solute.

The affinity chromatography is most selective kind of chromatography employs specific interaction between one
kind of solute molecule and a second molecule that is covalently attached to the stationary phase.

Applications:
1. To identify and analyse samples for the presence of trace elements or chemicals.
2. Separation of compounds based on their molecular weight and element composition.
3. Detects the unknown compounds and purity of mixture.
4. In drug development.
High performance liquid chromatography

1. High performance liquid chromatography, also more commonly referred to in the industry as
HPLC. This Technique is used to separate, identify or quantify the elements within a
mixture.
2. The mixture is then separated by the fundamental method of column-chromatography,
and afterwards, it is identified and quantified using spectroscopy.
3. HPLC is, in essence, an improved version that uses column liquid chromatography. Instead
of allowing a solvent to flow through a column by gravity, it is forced through it under
intense pressures that can reach 400 atmospheres.

Types of HPLC

HPLC

Normal Phase HPLC Reverse Phase HPLC Size - Exclusion Ion- Exchange

PRINCIPLE:
The purification is performed in a separation column that is two phases: a stationary and mobile phase.
A stationary stage is an amorphous substance that has tiny particles of porous material in the separation
column. The mobile phase however is a solvent or solvent mix that is forced by extreme pressure into
the column. The sample is introduced into the flow of the mobile phase from the pump into the
separation column with the Syringe.

Then, the various components of the sample travel across the column in various speeds due to being
retained to different degrees through interactions that occur with the stationary phase. Once the column
has been removed in the end, the different components are identified by a suitable detector and
transmitted as a signal into the HPLC software running on the computer.

INSTRUMENTATION

1. Solvent Reservoir
 Mobile phase contents are contained in a glass reservoir.
 The mobile phase, or solvent, in HPLC, is usually a mixture of polar and non-polar liquid
components whose respective concentrations are varied depending on the composition of the
sample.
2. Pump
 A pump aspirates the mobile phase from the solvent reservoir and forces it through the system’s
column and detecter.
 Depending on several factors, including column dimensions, the particle size of the stationary
phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000
kPa (about 6000 psi) can be generated.
3. Sample Injector
 The injector can be a single injection or an automated injection system.
 An injector for an HPLC system should provide an injection of the liquid sample within the
range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000
psi).
4. Columns
  Columns are usually made of polished stainless steel, are between 50 and 300 mm long, and
have an internal diameter between 2 and 5 mm.
 They are commonly filled with a stationary phase with a particle size of 3–10 µm.

5. Detector
The HPLC detector, located at the end of the column, detects the analytes as they elute from the
chromatographic column.
6. Data Collection Devices
 Signals from the detector may be collected on chart recorders or electronic integrators that vary
in complexity and their ability to process, store and reprocess chromatographic data.

HPLC applications:
The information that HPLC can obtain includes resolution, identification, and quantification of a
compound.
Pharmaceutical Applications
1. To control drug stability.
2. Tablet dissolution study of pharmaceutical dosages form.
3. Pharmaceutical quality control.
Environmental Applications
1. Detection of phenolic compounds in drinking water.
2. Bio-monitoring of pollutants.
Applications in Forensics
1. Quantification of drugs in biological samples.
2. Identification of steroids in blood, urine, etc.
3. Forensic analysis of textile dyes.
4. Determination of cocaine and other drugs of abuse in blood, urine, etc.
Food and Flavour
1. Measurement of Quality of soft drinks and water.
2. Sugar analysis in fruit juices.
3. Analysis of polycyclic compounds in vegetables.
4. Preservative analysis.
Applications in Clinical Tests
1. Urine analysis, antibiotics analysis in blood.
2. Analysis of bilirubin, biliverdin in hepatic disorders.
3. Detection of endogenous Neuropeptides in the extracellular fluid of the brain etc.