Adnan's Pharmaceutical Instrumentation by Adnan Sarwar Chaudhary - 2nd Edition

2nd Edition
Adnan’s
PHARMACEUTICAL
INSTRUMENTATION
S
ADNAN S. CHAUDHARY
BSc, Pharm-D, MBA
ADNAN’S PHARMACEUTICALS INSTRUMENTATION
‫بسم ہللا الرحمن الرحیم‬
DEDICATIONS
“To Our Parents, Brothers, Teachers, Friends, and
Class Fellows”
2 Adnan Sarwar Chaudhary & Saad Muhammad Rustam

Table of Contents
ATOMIC ABSORPTION & EMISSION SPECTROSCOPY 8
DEFINITION 8
HISTORY 8
THEORY 8
ELEMENTS DETECTED BY AAS: ( IN DARK) 8
PRINCIPLE : 9
INSTRUMENTATION: 9
SOURCES: 10
BURNERS: 11
FLAMES: 12
FLAME EMISSION SPECTROMETRY: 12
PROCESS : 12
INTERFERENCES: 13
APPLICATIONS: 14
REFERENCE: 14
FLAME PHOTOMETRY 15
DEFINATION: 15
FLAME PHOTOMETER: 15
PRINCIPLE: 15
INSTRUMENTATION 16
INTERFERENCES: 17
APPLICATIONS: 18
ADVANTAGES: 18
LIMITATIONS: 18
INFRARED SPECTROSCOPY 19
THEORY & PRINCIPLE: 19

IR SPECTRUM: 19
VIBRATIONAL MODES & ABSORPTION FREQUENCIES 19
NUMBER OF FUNDAMENTAL VIBRATIONS 20
SELECTION RULE: 21
FACTORS INFLUENCING ABSORPTION (VIBRATIONAL FREQUENCIES): 22
TYPES OF INFRARED SPECTROSCOPY: 23
DETECTORS: 24
FOURIER TRANSFORM INFRARED SPECTROSCOPY 26
SAMPLING SYSTEM OF INFRARED SPECTROSCOPY 27
Adnan Sarwar Chaudhary & Saad Muhammad Rustam 3
APPLICATIONS OF INFRARED SPECTROSCOPY: 28
MOLECULAR FLUORESCENCE SPECTROSCOPY 30
FLUORESCENCE 30
PHOSPHORESCENCE 30
PRINCIPLE 30
QUANTUM YIELD OR QUANTUM EFFICIENCY 30
INSTRUMENTATION 31
PHARMACEUTICAL APPLICATIONS 31
LIMITATIONS OF FLUORESCENCE SPECTROSCOPY 32
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (NMR) 33
DEFINITION: 33
PROTON MAGNETIC RESONANCE SPECTROSCOPY: 33
PRINCIPLE: 33
THEORY: 33
BEHAVIOR OF SPINNING NUCLEUS UNDER APPLIED MAGNETIC FIELD: 34
INSTURMENTATION: 35
WORKING OF NMR SPECTROPHOTOMETER 36
APPLICATIONS OF NMR SPECTROSCOPY 37
KEYS OF SPECTROSCPY 39
CHROMATOGRAPHY 40
HISTORY: 40
CHROMATOGRAPHY 40
TERMS IN CHROMATOGRAPHY 40
TYPES OF CHROMATOGRAPHY OR “CLASSIFICATION” 42
ON THE BASIS OF PHYSICAL STATE OF THE MOBILE PHASE: 43
CLASSIFICATION ACCORDING TO THE MECHANISM OF SEPARATION. 43
CLASSIFICATION ACCORDING TO THE SHAPE OF “CHROMATOGRAPHIC BED” 45
COLUMN CHROMATOGRAPHY 47
METHODS OF SEPARATION: 47
COLUMNS 47
SOLVENTS USED WITH COLUMNS: 48
COMMONLY USED ADSORBANTS: 48
INTRODUCTION OF THE SAMPLE: 49
DEVELOPMENT TECHNIQUE (ELUTION): 49
DETECTION OF COMPONENTS: 49
FACTORS AFFECTING COLUMN EFFICIENCY: 50
APPLICATIONS: 50

ADVANTAGES OF COLUMN CHROMATOGRAPHY: 51

DISADVANTAGES 51
THIN LAYER CHROMATOGRAPHY (TLC) 52
INTRODUCTION 52
MECHANISM OF SEPARATION 52
STATIONARY PHASE 53
MOBILE PHASE 53
PREPARATION OF PLATES 54
SAMPLE APPLICATION AND DEVELOPMENT 54
DETECTION METHODS 56
ADVANTAGES OF TLC 57
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC) 57
APPLICATIONS OF TLC 57
GAS CHROMATOGRAPHY 59
INTRODUCTION 59
BASIC PRINCIPLE: 59
INSTRUMENTATION: 59
APPLICATIONS: 63
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 65
INTRODUCTION 65
TYPES OF HPLC TECHNIQUES: 65
INSTRUMENTATION 66
TYPES OF ELUTION IN HPLC 71
APPLICATIONS: 72
LIMITATIONS OF HPLC: 72
GAS CHROMATOGRAPHY –MASS SPECTROMETRY (GC-MS) 73
GAS CHROMATOGRAPHY – MASS SPECTROMETRY 73

APPLICATIONS OF GC-MS 73
CAPILLARY ELECTROPHORESIS 74
INTRODUCTION 74
TYPES OF ELECTROPHORESIS 74
INSTRUMENTATION 75
THE BASIS FOR ELECTROPHORETIC SEPARATIONS: 77
APPLICATIONS 77
KEYS 77
DIFFERENTIAL SCANNING CALORIMETRY 79
INTRODUCTION 79
PRINCIPLE 79
INSTRUMENTATION 79
FACTORS AFFECTING DSC CURVE 80
APPLICATIONS OF DSC 80
THERMO-GRAVIMETRIC ANALYSIS 81
INTRODUCTION: 81
TGA INSTRUMENTATION 81
COMBINED THERMAL INSTRUMENTS 83
COMBINED THERMAL INSTRUMENTS 83

ANALYTICAL APPLICATIONS OF THERMOGRAVIMETRY 83
KEY & REFERENCES 84
DIFFERENTIAL THERMAL ANALYSIS 85
DEFINITION 85
INTRODUCTION 85
PRINCIPLE 85
FACTORS AFFECTING DTA CURVE 85
INSTRUMENTATION 86
APPLICATIONS 86
RADIO PHARMACEUTICALS 88
RADIO PHARMACEUTICALS 88
TYPES OF RADIOISOTOPES (RADIONUCLIDES): 88
TYPES OF RADIOACTIVE RADIATIONS: 88
BIOLOGICAL EFFECT OF RADIATION 88
MEASUREMENT OF RADIATIONS 89
APPLICATION OF RADIOPHARMACEUTICALS 90
KEYS 91
POLAROGRAPHY 92
VOLTAMETRY 92
POLAROGRAPHY 92
ELECTRODE SPECIFIC FOR “POLAROGRAPHY” 92
ILKOVIC EQUAITON 94
APPLICATIONS OF POLAROGRAPHY: 95
KEYS 95
POTENTIOMETRY 96
POTENTIOMETRY 96
REFERENCE ELECTRODES 96
INDICATOR ELECTRODES 97
NERNST EQUATION 98
2. MEMBRANE INDICATOR ELECTRODES 98
KEYS 100
CONTACT US: 100
THANKS 100

ATOMIC ABSORPTION & EMISSION

SPECTROSCOPY
DEFINITION
“Atomic absorption spectroscopy (AAS) is a spectro-analytical procedure for the quantitative
determination of chemical elements using the absorption of optical radiation (light) by free atoms in the
gaseous state.”
HISTORY
Atomic absorption spectroscopy was first used as an analytical technique, and the underlying
principles were established in the second half of the 19th century by Robert Wilhelm Bunsen and Gustav
Robert Kirchhoff, both professors at the University of Heidelberg, Germany.
The phenomenon of atomic absorption (AA) was first observed in 1802 with the discovery of the
Fraunhofer lines in the sun's spectrum. It was not until 1953 that Australian physicist Sir Alan
Walsh demonstrated that atomic absorption could be used as a quantitative analtical tool. Atomic absorption
analysis involves measuring the absorption of light by vaporized ground state atoms and relating the
absorption to concentration.
THEORY:
Deals with the spectroscopy of ATOM. ELECTRONIC TRANSITION within the atom can take
place when energy is absorbed and line spectra are observed. The sample is converted to free atoms by
different ways. Mainly a FLAME as ATOMIZER.
Atomic Absorption Spectrometry is describe when atoms in ground state , absorbs energy or
particular radiation , becomes excited and jump to higher energy level within an atom. In analytical
chemistry the technique is used for determining the concentration of a particular element (the analyte) in a
sample to be analyzed. AAS can be used to determine over 70 different elements in solution or directly in
solid samples used in pharmacology, biophysics and toxicology research.
ELEMENTS DETECTED BY AAS: ( IN DARK)

PRINCIPLE :
The sample solution is aspirated (sprayed) into a flame and sample element is converted to Atomic
vapors. The flame then contain the atoms of that element, some are thermally EXCITED but most in ground
state These ground state atom can made from that element The wavelengths of radiation given off by the
source are the same as those absorbed by the atom in the flame . Atomic absorption spectrophotometry is
identical in principle to absorption spectrophotometry.
The absorption follows BEER S LAW i .e. The absorbance is directly proportional to the path-
length in the flame and to the concentration of atomic vapor in the flame. both of these variables are difficult
to determine , but the path-length can be held constant and conc. Of atomic vapor is directly proportional to
the conc. Of the analyte being aspirated. Different flames are required for diff sample.
INSTRUMENTATION:
Requirements for atomic absorption spectrophotometry are;
1. Light Source
2. A Cell (Flame)
3. Monochromator
4. Detector
5. Readout Device
Sometimes a chopper is also placed in between the flame and the source to split source beam.
The process of atomic absorption spectroscopy (AAS) involves two steps:
1. Atomization of the sample

2. The absorption of radiation from a light source by the free atoms
Then comparing the spectral data of sample to standard spectrum of elements to detect the elements.

SOURCES:
A sharp line source is used in atomic absorption because the width of the absorption line is very narrow
(a few thousandths to one hundredths of a nanometer) So due to small size of absorption line , only small
fraction of radiation from a continuum source passed by the slit and reaching the detector would b absorbed.
Types of Sources include:
1) Electrode – less discharge Lamp

2) Vapor Discharge Lamp
3) Hollow cathode Lamp
Electrode-less discharge lamp:

Electrode – less discharge lamps provide high intensity (10-100 times) and narrow emission lines
which lead to higher signal – to -noise ratios over the lines obtained using hollow cathode lamps. The
benefits of electrode- less discharge lamps are realized especially when analyzing volatile elements like As,
Sb, Bi, Cd, Hg, Rb, Sn, Te, etc.
Electrode – less discharge lamps are further subdivided into two categories – microwave and radio
frequency excited lamps.
Microwave Electrode – less discharge lamps

Microwave lamps are excited by microwaves
generated at frequencies around 100 MHz. The
intensity of microwave lamps is better than
radiofrequency lamps. Seeding with (1-2mg) mercury
leads to mercury vapor preventing adsorption of metal
onto the walls and windows of the lamp thereby
increasing its useful life. However, microwave
discharge lamps require temperature regulation to give
a stable line intensity.
Radiofrequency Electrode – less discharge lamps

The intensities of radio frequency electrode- less discharge lamps are generally lower in comparison
to microwave lamps.
Electrode- less discharge lamps with all their benefits are not as popular as hollow cathode lamps and
are used mainly for analysis of about 15 volatile elements. The reasons are mainly higher cost and difficulty
in operation in comparison to hollow cathode lamps.
Hollow Cathode lamp

Most commonly used source is (HCL) or hollow cathode
lamp. Hollow-cathode lamps are a type of discharge lamp that
produces narrow emission from atomic species. They get their
name from the cup-shaped cathode, which is made from the
element of interest. The electric discharge ionizes rare gas
atoms.

Components
It consist of ;
 Cylindrical hollow cathode (made up of element to be determined or an alloy of it)

 A tungsten anode
 Both enclosed in glass tube with QUARTZ window because the line of inerest are mostly in UV
region.
Working of HCL:
The tube is under reduced pressure and filled with an inert gas (neon , argon). A high voltage is
impressed across the electrodes , causing gas atoms to ionize at anode. These positive ions are accelerated
towards the negative cathode . When they bombard the cathode , they cause some of their metal to sputter
and become vaporized.
The vaporized metal is excited to higher electronic level by continued collision with the high energy gas
ions. When the electrons return to the ground state , the characteristic line that metallic element emitted.
Also lines due to gas emission. These line passed to the sample flame and gives the result.
BURNERS:
Mostly used burner is Premix chamber burner or laminar flow burner
Components are :
 Burner head
 Mixing chamber
 Fuel inlet
 Gas ( oxidant ) inlet
 Sample flow through capillary
 Nebulizer
Working :
There are two types of nebulizers: atomizer jet and ultrasonic. The atomizer, or "compressor
nebulizer," is the most common. This type uses an aerosol compressor to vaporize droplets of
sample. Ultrasonic, or "mesh nebulizers," use high-frequency sound waves to make liquid sample fine
dro
plet
s.

The fuel and support gases are mixed in a chamber before entering burner head where they combust.
The sample solution is aspirated through a capillary by the venturi effect using support gas that is air. The air
creates a partial vacuum at end of capillary, drawing the sample through capillary. It is broken into a fine
spray at the tip a process usually known as Nebulization.
The larger droplets of the resulting aerosol condensed and drain out. The remaining fine droplets mix
with combustion gases and enter flame. The atomization efficiency of that portion of that sample that enters
the flame is high because droplets are finer and path length of flame is long. Combustion of premix burner is
quite and a popular version is Boiling burner.
Another type of burner that is Total consumption burner that works he same principle as that of
premix burner but the sample in premix chamber burner is 90% wasted and only 10% is consumed in
burning thus Total consumption burner is preferred over Premix chamber burner.
FLAMES:
Most widely used flames for this purpose are Air-Acetylene flame that has max speed of 160 cm/sec
and temp is 2250 C. Acetylene – nitrous oxide that has max speed of 180 cm/sec and temp is 2955. These
flames are used with premix burner. The latter high temp flame is mostly used for REFRACTORY
ELEMENTS. The air – acetylene and other hydrocarbon flames absorbs a large fraction of radiation at
wavelength below 200nm. While argon – hydrogen is use for this regions detectability. Cool flames used for
chemical interferences. A hot flame for emission spectrometry for analysis of large elements that is nitrous
oxide flame. For high temp flames a stainless steel thick burner head is used to prevent the burning.
FLAME EMISSION SPECTROMETRY:

Formerly known as flame photometry deals with the detection of emission of photons by excited
electron in the form of a spectral bar graph…only the radiation absorbed by the electron is emitted and
recorded. The source of excitation of electron is also flame which is low energy source and thus the
spectrum of emission is simple and have few emission lines.
PROCESS :
The sample solution is introduced in flame in the form of fine spray. The solvent evaporates from
fine spray in the flame , leaving the dehydrated salt. This salt (sample to be analyzed ) is dissociated into
gaseous atoms in ground state. Then atoms in flame absorbs energy and raised to an excited ELECTRONIC
state , excitation is if short time and excited electrons drop back to ground state emitting photons which are
observed in AES.

The light beam is generated by lamp that is specific for a target metal. The lamp must be perfectly
aligned so the beam crosses the hottest part of the flame. The light passed through the flame is received by
the monochromator, which is set to accept and transmit radiation at the specified wavelength and travels into
the detector. The detector measures the intensity of the beam of light. When some of the light is absorbed by
metal, the beam's intensity is reduced. The detector records that reduction as absorption. That absorption is
shown on output device by the data system. And same the emitted radiations are also recorded and shown on
the output device in the form of spectra.
INTERFERENCES:
Following types of interferences are seen in AAS and AES these are:
1- Spectral interference :
When during the process of both emission and absorption spectral formation the monochromator is
unable to resolve unwanted spectral lines from wanted one spectral interferences takes place. Light scatter or
absorption by solid particles , un-vaporized solvent droplets or molecular species in flame cause positive
interferences. This is a problem for wavelength less than 300nm.When solution of high salt content are
aspirated because the salt may not dissolved or its molecules are may be dissociated into atoms. These are
known as BACKGROUND ABSORPTION and can be corrected for by measuring absorbance of line that
is close to absorption of line of test element but that are not absorbed by element itself. The line used for
correction can be a filter gas line from HCL.
2- Ionization interferences :
A fraction of alkali or alkaline earth metals in hot flame are ionized in flame. Since we are only
measuring un- ionized atoms both emission and absorption signal decreases. However the presence of other
easily ionized elements in sample will add free electrons to flame and suppress ionization of test element.
This result enhanced absorption and emission and a positive interference. Ionization can be suppressed by
adding a solution of a more easily ionized element ( potassium , cesium )
3-Physical interferences :
Most parameters that affect the rate of sample uptake in burner and the atomization efficiency can be
considered physical interferences. Variations in gas flow rate , variation in sample viscosity due to temp and

solution type , high solid content and change in temperature of flame. These can b corrected by frequent
calibration and use of internal standards.
4-Refractory compound formation (chemical interferences):

The sample solution may contain a chemical usually an ANION that will form a heat stable ion
(refractory compound). Example, Phosphate can react with calcium in the flame to produce Calcium
phosphate , this cause reduction in absorbance since to absorb its resonance lines calcium it must be in
atomic form. This interferences can be removed chemically by the addition of high concentration of
strontium chloride or lanthanum nitrate ( a releasing agent ) The strontium or lanthanum will combine to
phosphate and prevent and prevent its reaction with calcium. Also a high concentration of EDTA can be
added to solution to form calcium chelate thus preventing its reaction with phosphate The EDTA-Ca
dissociate in flame to give free Calcium vapors for analysis. A higher temperature flame that is Nitrous
oxide acetylene can also used to prevent this reaction.
A serious situation occurs when the analyte metal reacts with gases present in flame. Refractory
elements such as Aluminum , titanium , molybdenum , and vanadium will react with O and OH species in
flame to produce thermally stable metal oxides and hydroxides. These can be eliminated or decomposed
only using high temperature flames that is NITROUS OXIDE FLAME.
APPLICATIONS:
1. Agriculture – analyzing soil and plants for minerals necessary for growth of pharmaceutical plants.
2. Chemical – analyzing raw chemicals as well as fine chemicals used in drug preparation.
3. Environmental Study – determination of heavy metals in water, soil, and air.
4. Food Industry – quality assurance and testing for contamination in food products.
5. Pharmaceutical – many applications from quality control to detecting impurities in drugs.
6. It is used in petroleum industry, to detect metallic impurities in petrol, lubricating oils.
7. The recent applications of atomic absorption spectroscopy (AAS) in determination of trace level
inorganic impurities in drugs and, plants its extracts.
8. AAS is used for speciation of heavy metals in pharmaceutical products and metallic impurities in
pharmaceuticals, herbal medicine and plant extracts.
REFERENCE:
Analytical Chemistry (Gary D. Christian)

FLAME PHOTOMETRY
DEFINATION:
Flame photometry or flame emission spectrometry is the branch of spectroscopy in which the species
examined in the spectrometer are in the form of atoms.
FLAME PHOTOMETER:
“An instrumented used in inorganic chemical analysis to
determine the concentration of certain metal ions among
the sodium, potassium, calcium and lithium.”
Flame photometry is based on measurement of intensity

of the light emitted when a metal is introduced into the
flame. The wave length of color tells us what the elements
is (“qualitative”). Color's intensity tells us how much of
the element present (“quantities”).
PRINCIPLE:
The basic principle upon which Atomic spectroscopy works is
based on the “Matter absorbs light at the same wavelength at
which it emits light.” When a metal salt solution is burned, the
metal provides a colored flame an each metal ion gives a different
colored flame. Flame tests, therefore, can be used to test for the
absence or presence of a metal ion.
Excitation:
The electrostatic force of attraction

between the electrons and nucleus of the
atom helps them to absorb a particular
amount of energy. The atoms then jump to
the exited energy state.
Emission process:
Since the higher energy state is unstable the atoms jump back to the stable low energy state with the
emission of energy in the form of radiation of characteristic wavelength, which is measured by the photo
detector.
1. The solution containing metal to be measure is first aspirated into the burner.
2. The solvent then evaporated leaving fine divided solid particles.
3. This solid particles move towards the flame, where the gaseous atoms and ions are produced.

4. The ions absorb the energy from the flame and excited to high energy levels.
5. When the atoms return to the ground state radiation of the characteristic element is emitted.
6. The intensity of emitted light is related to the concentration of the element.
INSTRUMENTATION
Parts of flame photometer:
1. Nebulizer and Mixing chamber
2. Source of Flame
3. Optical System
4. Photo Detector & Read out Device
Nebulizer and Mixing chamber:

Help to transport the homogeneous solution
of the substance into flame at a steady rate.
Source of Flame:
A burner that provides flame and can be maintained in a constant form and at a constant temperature.
Burner
Flame may be divided into the following regions or zones.
1. Preheating zone
2. primary reaction zone or inner zone
3. interconal zone
4. secondary reaction zone
Preheating zone: In this, combustion mixture is heated to the ignition

temperature by thermal conduction from the primary reaction zone.
Primary reaction zone: This zone is about 0.1 mm thick at atmospheric

pressure. There is no thermodynamic equilibrium in the zone and the
concentration of ions and free radicals is very high. This region is not
used for flame photometry.
Interconal zone: It can extend up to considerable height. The maximum temperature is achieved just above
the tip of the inner zone.
Secondary reaction zone: In this zone, the products of the combustion processes are burnt to stable
molecular species by the surrounding air.
Mechanism:
In the Flame:
Flame photometry employs a variety of flues mainly air, oxygen nitrous oxide (N2o) as oxidant. The
temperature of the flame depends on fuel-oxidant ratio. The intensity of the light emitted could be describe
by the Scheibe-Lomakin Equation.
I=k×
Where,
I = Intensity of emitted light
c = The concentration of the element
k= Constant of proportionality
n-1 ( At the linear part of the calibration curve)\
Then
I=k×c
That is the intensity of emitted light is directly related to the concentration of the sample.
Optical System (Optical Filter ):

The optical system comprises of: convex mirror, lens (monochromators) and filter.
Monochromators: These consist of slits and dispersion elements. The common dispersion element in
modern ﬂame atomic absorption and emission spectrometers is a diffraction grating.
Filters: The alkali metals in a low-temperature ﬂame emit only a few lines and therefore have a simple
emission spectrum.
Photo Detector & Read out Device:

Detect the emitted light and measure the intensity of
radiation emitted by the flame. The produced
electrical signal are directly proportional to the
intensity of light.
INTERFERENCES:
In determining the amount of a particular element present, other elements can also affect the result.
Such interference may be of 3 kinds:
1)Spectral interferences: occurs when the emission lines of two elements cannot be resolved or arises from the
background of flame itself. They are either too close, or overlap, or occur due to high concentration of salts in the
sample..
2)Ionic interferences: High temperature flame may cause ionization of some if the metal atoms, e.g.
Sodium. The ion possesses an emission spectrum of its own with frequencies, which are different from
those of atomic spectrum of the Na atom.

3)Chemical interference: The chemical interferences arise out of the reaction between different
interferents and the analyte. Includes;
Cation-anaion interference:
The presence of certain anions, such as oxalate, phosphate, sulfate, in a solution may affect the intensity of
radiation emitted by an element E.g.; Calcium +phosphate ion forms a stable substance, as Ca3(PO4)2
which does not decompose easily, resulting in the production of lesser atoms.
Cation-cation interference:
These interferences are neither spectral nor ionic in nature. E.g. Aluminum interferes with calcium and
magnesium.
APPLICATIONS:
1- Determine the availability of alkali and alkaline earth metals which are critical for soil cultivation.
2- In agriculture, the fertilizer requirement of the soil is analyzed by flame test analysis of the soil.
3- In clinical field, Na+ and K+ ions in body fluids, muscles and heart can be determined by diluting
the blood serum and aspiration into the flame.
4- Analysis of soft drinks, fruit juices and alcoholic beverages can also be analyzed by using flame
photometry.
ADVANTAGES:
1- 1.Simple quantitative analytical test based on the flame analysis.
2- 2.Inexpensive.
3- 3.The determination of elements such as alkali and alkaline earth metals is performed easily with
most reliable and convenient methods.
4- 4.Quite quick, convenient, and selective and sensitive to even parts per million (ppm) to parts per
billion (ppb) range.
LIMITATIONS:
1- Alteration of light emission because of altered flame temp.
2- It needs perfect control of flame temperature.
3- Interference by other elements is not easy to be eliminated
4- Heavy and transition metals , the number of absorption and emission lines is enormous and the
spectra are complex
5- Inadequate selectivity of WL.
6- Differences in viscosity between standards and sample.

Infrared Spectroscopy
It deals with the n absorption of electromagnetic radiations by organic molecule in the infrared
region between 2.5 to 1mm wavelength (4000- 625 cm).
THEORY & PRINCIPLE:

Organic molecules are in a constant state of vibration at ordinary temp, each bond vibrating with a
characteristic frequency which depends on the bond strength & masses of atoms. The frequency of most
molecular vibrations corresponds to the y frequency of electromagnetic s radiations in IR region.
When n IR radiation passed through a compound, s some of the frequencies are absorbed while
others are transmitted without being absorbed. Only those frequencies are absorbed which match with the l
vibrational frequencies of the bonds.
This absorption is associated with excitation of the molecules from ground state to higher vibrational
energy state, such as
ΔE = hν
IR spectroscopy basically vibrational spectroscopy. During absorption of IR radiation only amplitude

of the vibration is d increased but the frequency of vibration does not alter. Finally absorbed energy is d
released as heat & molecule revert from the excited state to original ground state.
IR SPECTRUM:
A plot of e absorbance (A) or percent transmittance (100T) along y-axis & frequency or wavelength
along x-axis, result in an infrared spectrum. The positions of s absorption bands are either expressed by
wavelength λ in micrometer µm or mostly by wave number ύ in reciprocal centimeter (cm‾). T Maximum T
is at the top of e vertical scale so absorbance is observed as a minimum, called a peak.
VIBRATIONAL MODES & ABSORPTION FREQUENCIES

Fundamental Vibrations:
The vibrations due to individual bonds or functional groups are called fundamental vibrations. While
others must be considered as vibrations of the molecule as a whole.
Two main modes of vibrations, Such as: Stretching and Bending.
Types:
Stretching vibrations:
It is t rhythmical movement of two bonded atoms along bond axis i.e. g changing bond length
(increase or decrease).
Types of stretching vibrations:
i. Symmetric stretch: Movement of atoms with respect to a particular atom in a molecule in the
same direction.
ii. Asymmetric stretching: One atom s approaches the l central atom while other departs from it.
Bending vibrations:
It is the rhythmical movement of atoms that causes a change in bond angles with a common atom
while bond length remains same. As y more energy is required to stretch a spring than to bend it, so
stretching absorption occurs at higher frequencies.
Bending vibrations are also two types:
Types:
In plane bending vibrations
In plane bending vibrations are
Scissoring: In this type, two atoms approach each other.
Rocking: Movement of the atoms takes place in the same direction.
Out of plane bending vibrations:
Wagging: Two atoms move up & below the e plane with respect to the central atom.
Twisting: One of the atom moves up the plane while other moves down the plane with respect to central atom.
Vibrational frequency:
Hook’s law explain the value of stretching vibrational frequency of a bond. The stretching frequency
(ύ in cm‾) is related to the masses of the atoms & the resistance of a bond to stretch.
v/c = ύ = ½ πc [k/m1 m2 /m1 +m2 ]½
= ½ πc √k/µ
µ= m1 m2 /m1 +m2
µ = reduced mass.
m1 & m2 = masses of atoms.
k = force constant 5x105 gm sec-2
C = velocity of radiation 2.99x1010 cm sec-1.
Thus the value of l vibrational y frequency or wave number s depends upon h bond strength & masses. If
bond e strength increase or e masses reduce the value of l vibrational frequency increases.
NUMBER OF FUNDAMENTAL VIBRATIONS

The IR light is absorbed when the oscillating dipole moment of molecule interacts with the
oscillating electric vector of an IR beam, i.e. The dipole moment of beam should be different from the dipole
moment of a molecule.
For IR-absorption, the vibration should not be centro- symmetric, only those vibrations are IR-active
which are not centro symmetric. The total number of vibrations in a molecules depends on the s number of
atoms present in the molecule. Each atom has three degrees of freedom because three coordinates ( x,y , &
z) are required to describe its position. e.g. a molecule consisting of “n n” atoms therefore has a total of s 3n
degrees of freedom. Out of these 3n degrees of freedom, in case of linear molecule, there are only s 2
degrees of rotation. It is due to the n rotation of molecule about its axis of linearity.
For a linear molecule (3n – 5):
Total degree of freedom = 3n
Translational degree of freedom =3
Rotational degree of freedom =2
Vibrational degree of freedom = 3n – 3 – 2
= 3n – 5
Example: In case of CO2. (O=C=O)
By applying formula 3n – 5 = 3x3 – 5 = 9 – 5
=4
Where n is the number of atoms. So there will be 4 modes of fundamental vibrations for CO2 (linear).
Non- - linear molecule (3n – 6):
In case of non-linear molecule, there are three degrees of rotation.
Total degrees of freedom = 3n
Translational degrees of freedom =3
Rotational =3
Vibrational degrees of freedom = 3n – 3 – 3
= 3n – 6
Example:
In case of H2O
By applying formula 3n – 6 = 3x3 – 6 = 3
So there will be 3 modes of fundamental vibrations for H2O (non-linear).
SELECTION RULE:
It is not necessary that every fundamental vibration results in the n absorption of IR- radiation.
According to selection rule, only those vibrations which cause t dipole moment to fluctuate (change electric
field) will interact with the fluctuating electric field of electromagnetic radiation (matching frequency) &
hence will cause absorption, such vibrations are said to be IR-active vibration.
Such as: in C = O etc.

Vibrations that do not cause a change in the dipole moment will not interact with the infrared radiation,
known as IR- inactive vibrations.
e.g. in symmetrical diatomic molecules such as H2 , N2 , Cl2 etc.
Number of absorption bands in an IR spectrum may be reduced if;
a) A fundamental vibration does not cause a change in the dipole moment of the molecule.
b) Some of fundamental vibrations are degenerate & absorb at the same frequency.
c) Some of the absorption bands are so close to each other that they merge.
d) An absorption band is too weak.
e) A fundamental frequency falls outside the IR-region.
FACTORS INFLUENCING ABSORPTION (VIBRATIONAL FREQUENCIES):

The calculated value of absorption is never exactly equal to its experimental value. This difference
arises due to the presence of other structures in the immediate neighborhood of the bond. Thus, IR spectrum
tells us whether certain groups are present or not.
Frequency shifts also occur on working with same substance in different state, in vapors state a substance
usually absorbs at higher frequency.
There are many other factors, which are responsible for l vibrational shifts.
1)- Coupled vibrations & Fermi resonance:

An isolated C – H bond has only one stretching n vibration but stretching vibrations of C – H bonds
in a methylene group (-CH 2 ) couple together to produce 2 vibrations of different frequencies, one higher &
one lower.
Coupling can also e take place between a fundamental vibration & overtone (combination tone) of
some other fundamental vibration i.e., a molecule transfers its energy from fundamental to overtone & back
again. This type of coupling or resonance called“ Fermi Resonance”
The basic requirement

for Fermi R, is that
coupling vibrations
must be the same
symmetry & must
originate from group
close to each other in
molecule. e.g. CO2 .
2)- Electronic Effects:

When substituents in the
neighborhood of a
particular group is
changed, its absorption

frequencies are altered. The frequency shifts are o due to the electronic effects such as; Inductive effect, c
mesomeric effect & field effect etc.
Inductive effect: The n introduction of– CH3 group cause +I which results in g lengthening or weakening of
bond & thus wave number of absorption decreases.
The introduction of electronegative group causes -I effects by increasing force constant & finally wave
number of absorption rises.
Mesomeric effect: The mesomeric (resonance) effect has contribution in g shifting C=O stretching
frequency. Any substituent effect single bond character, decrease bond h strength & hence lower C=O
stretching frequency. A +M group (MeO), in g aromatic ring will lower C=O stretching while –M group
(NO2) will have opposite effect.
Field effect: These effects can be seen when two functional groups influence each
other’s vibrational frequencies through space interaction that may be either steric or
electrostatic in nature. e.g. ortho haloacetophenone. The oxygen & halogen atom
cause electrostatic repulsion due to non-bonding e. This causes C=O p group out of
plane thus conjugation diminished & absorption occurs at higher wave number.
3)- Angle strain

Double bonded carbon atoms in unstrain molecule
have bond angle is 120°. The absorption frequencies of C=C
& C=O are regarded as normal. But, if the bond angle is
reduced below 120° ° e.g. in five-member & smaller rings, the n absorption frequencies rise due to increase
in rings strain, bond length, force constant & g stretching vibrations. If bond angle increases above 120°, the
opposite effect is observed.
4)- Hydrogen bonding:

H bonding brings about remarkable downward frequency shifts. Inter molecular hydrogen bonds
give broad bands whereas intra-molecular hydrogen bonds give sharp & well defined. Alcohols & phenols
usually exist as polymeric association, due to strong H-bonding, & show a broad absorption band.
The H-bonding results in lengthening of the original O – H. So, bond is weakened & force constant
is decreased & hence stretching frequency is lowered. H-bonding in chelates (intra-molecular H-B) e.g. in
methyl salicylate, is particularly strong, & O –H stretching frequency may be very low.
TYPES OF INFRARED SPECTROSCOPY:

Divided into two classes: Continuous wave or dispersive spectroscopy & Fourier transform or time
domain spectroscopy.
Continuous wave or dispersive spectroscopy

In this type; infrared radiation dispersed & only small portion of frequencies or wavelengths available is
viewed by the detector at one time. The resulting record of intensity verses wave number is conveniently
understand.
Fourier transform or time domain spectroscopy

Measuring the intensity or radiant power of many wavelengths simultaneously as a function of time. This
time-domain spectroscopy most commonly called Fourier-transform, because the time domain spectrum is
converted by a
fourier transform to a
conventional
frequency domain
spectrum.
Instrumentation
-Continuous
wave
Source of infra red
radiations;
a) The Nernst
glower:
It is most commonly used source. Which is a cylinder

composed of rare-earth oxide has diameter about 2 mm
& approximately 20 mm long. The operating temp as
high as 1800 K.
b) The Globar:
It is silicon carbide rod about 5mm diameter & 50mm long, operated at 1600 K . It provides greater out put
than Nernst glower in region below 1500 cm‾¹.
c) Nichrome wire:
It is tightly bound coil of nichrome wire, which is electrically heated to

incandescence. Provide lower heat but has longer life.
DETECTORS:
A). Photon detectors:
Based on the photoconductive effect. These have much faster response & greater sensitivity to infrared
radiation but operate only over a very restricted range of λ.
B). Thermal detectors:
Absorption of infrared radiation produces heating effect, which in turn alters physical property of the
detector. These are unstable over a wide wavelength range.
A. Photon detectors
It consists of a thin film of semiconducting material such as lead sulfide doped with copper & put into an
evacuated envelope to protect from reaction with atmosphere. Absorption of photon of sufficient energy
promote some electrons from non-conducting to the conducting state, resulting decrease in resistance of the

material. These detectors are sensitive to radiation b/w 1-3 µm in λ, (10,000-2000cm‾¹) & have a respective
time of about 10µsec.
B. Thermal detectors
Classified into four types based on the properties of the material that are altered upon exposure to
infrared radiation.
I- Thermocouples detector:
II- Thermistors or Bolometers:
III- The Golay or pneumatic detector:
IV- The Pyroelectric detector:
I- Thermocouples:
Based on electrical current flows in two dissimilar metal wires connected together at both ends & a
temperature difference exists between two ends. The end exposed to the infrared radiation is called “hot
junction”. While other end called “cold junction” which is thermally insulated. A small piece of blackened
gold foil welded to the hot junction which absorb IR radiation. The thermocouple is enclosed in an
evacuated steel casing with KBr to avoid loss of energy by convection.
The cold junction is kept at a constant temp while the hot junction is exposed to IR radiation & increases
its temp. The temp difference between two junction generate potential difference which depends on how
much IR radiation falls on the hot jun. The response time is 60 – 100 µsec.
II- Thermistors or Bolometers:

It is made of fused mixture of metal oxides. When the temp of mixture increases, its electrical resistance
decrease. Its resistance changes by 5% per C°. The response time is slow.
III- The Golay or pneumatic detector:

It consists of a small metallic cylinder which is closed by
a black ended metal at one end & by a flexible metalized
diaphragm at the other. The cylinder is filled with inert gas
(xenon) & sealed it. When IR radiation fall on the black end
metal plate, it heat the gas, which expands & cause the
diaphragm to swell outward.
The bend of diaphragm can be measured by the light

beam reflects from it to a phototube. The signal seen by the
phototube is modulated in accordance with the power of
radiant beam incident on the gas cell. This detector is
superior for the region below 200 cm‾¹ & is useful for Far-IR
region.
IV- The Pyroelectric detector:

This is the most recently developed infrared detector. Consists of thin layer of deuterated triglycine
sulfate (DTGS) crystals placed between two electrodes. It acts as a temperature-dependent capacitor. Upon
exposure to IR radiation, the temperature & polarization of the crystal change.
The change in polarization is detected as

a current in the circuit connecting the
electrodes. The signal depends on the rate
of change of polarization with
temperature. This detector operates with
a much faster time & is widely used for
FT-IR spectroscopy.
DISPERSIVE INFRARED
SPECTROMETERS
Dispersive infrared spectrometers:
Most commercial instruments are double beam, which cancels background absorption caused by
atmospheric CO2 & H2O. The radiation from the source is split into 2 beams, one passing through sample
cell & other serve as reference beam. Absorption by the sample is measuring directly from the difference in
intensity of two beams. This intensity difference is usually measured by optical null method. The sample &
reference beam changed to alternate pulse through optical chopper. Which dispersed by the monochromator
& focused on the detector. No signals if two intensities are equal but if they differ, an alternating voltage is
developed, amplified & used to move attenuator (wedge) & finally recorded as absorption spectrum of the
compound.
FOURIER TRANSFORM INFRARED SPECTROSCOPY

Dispersive IR spectrophotometer has some limitations. It takes several minutes to record an infrared
spectrum. Wavelength produced by dispersion suffers from inaccuracies, associated with mechanical
movement of the grating. Most of the light coming from the source is lost due to the narrowness of the
focusing slit, resulting poor sensitivity.
Thus, it is necessary to modify the frequency of incoming radiation to a frequency that can be easily tracked
by detectors. The overall selected range of radiations are made to fall on the sample with spf time, and
obtained spectrum is a time domain spectrum which shows a
peak value at a particular time. The time domain signal is then
converted to a frequency domain spectrum by an
interferometer (Michelson interferometer).
FT-IR Spectrometer
a) Source
b) An optical system (Interferometer)
i) Beam splitter
ii) Stationary (fixed) Mirror
iii) Moving Mirror
c) Laser – (He-Ne)

d) Sample cell
e) Detector
Michelson Interferometer:
It consits of 3 basic components:
A beam-splitter, a fixed mirror & a moveable mirror.
Interferometer is the key to an FT-IR spectrometer, & moveable mirror is the key to an interferometer.
Beam-splitter is a semi-reflecting device so that 50% invading light is transmitted while remaining 50% is
reflected. Incoming radiation wavelength λ 1 is split into two identical beams by the beam-splitter.
“A” travels a fixed distance.
“B” whose distance can be varied by changing the position of mirror B.
When beam “A” & “B” are recombined after reflection, interferences can occur. If path distance of beams
A & B are identical the constructive interference takes place & detection has a maximum output. If mirror B
is moved by distance of ¼ then beam B will have different distance that traveled by A & destructive
interference occurs, produce a minimum detector output. If mirror is displacing further by a distance ¼ yield
path difference 0, constructive interference again occurs & detector gives maximum response.
In this way, the interferometer generates a low frequency response that is proportional to the frequency of
radiation that can be followed by the detector.
Advantages of using Fourier transform method:
1) Whole rang of infrared frequencies pass through the sample simultaneously, much time is saved (< 1s).
2) No loss of radiation as using slits, called Fellgett’s advantage, all radiant energy is utilized. This increase
the sensitivity of the instrument & an excellent spectrum is obtained from small sample (nanogram).
3) As interferometer equipped with lasers for referencing the path-length difference, are capable of higher
resolution than dispersive instrument.
4) Many moving parts result in mechanical slippage in dispersive, but only mirror moves in FTIR during
experiment.
5) Any emission of IR radiation by sample will not be detected in FTIR.
SAMPLING SYSTEM OF INFRARED SPECTROSCOPY

1- Gases:
A gas sample is introduced into evacuated gas cell, which is made of

cylindrical glass body about 10cm in length. For trace analysis at ppm
level (air-pollution & breathe analysis) very long path length (upto
30m) can be obtained by using multiple-reflection cells.
2- Liquids & Solutions:
A neat sample may be examined as a thin film (0.1mm thickness)

squeezed between two optically polished circular flat plates
(5mm thickness & 25mm diameter) of rock salt (NaCl, KBr). The

plates held together by capillary action & mounted in the sample beam. Plates must be cleaned immediately
after use by rinsing with suitable solvent (chloroform), kept dry, store in well closed desiccator & should be
handled by their edges.
3- Solids:
Two common techniques for solid sample, mulls & pellets.
a) Mulls: The finely ground solid sample (2-10mg) mixed with Nujol (mineral oil) to make a thick
paste, which is spread b/w 2 plates (alkali halides) . Then mounted in a path of infrared beam & the
spectrum is run.
b) Pellet: Finely ground sample is thoroughly mixed with same wt of powdered KBr. The mixture is
then pass under high pressure (8000-20,000 psi) in a press (13mm diameter) to form a small pellet
(transparent). An evacuable, heated (40°c) metal desiccator is useful for storing die components &
KBr.
APPLICATIONS OF INFRARED SPECTROSCOPY:

1- Structure determination:
Structure of organic compound determined by correlation & interpretation of spectra.
2- Identification of compounds:
By comparison of infrared spectrum with an authentic sample.
3- Detection of impurities:
The presence of absorption bands at position where the compound is not expected, indicate the presence of
impurities. For example; cyclohexanone is readily detected in cycolohexanol by intense carbonyl band.
4- Progress of a reaction:
Can be determined by examining IR spectra of sample drawn from the reaction medium. For example:
oxidation of secondary alcohol to ketone determined by disappearance of O-H band near 3600cm‾¹ &
appearance of C=O band near 1715cm‾¹.
5- Percentage composition of a mixture:
If Beer’s law is obeyed, the spectrum of mixture resulting from the known composition of pure components
is determined (multiple standard). Then measure the spectra of unknown sample & by graphic method (%
age purity find).
6- Study of atmospheric contaminants:
Infrared spectroscopy have been employed for variety of compounds for which limits of exposure have been
set by health administration.
7- Determination of total amine contents:
The total primary & secondary amine content of aliphatic amines can be determined easily & rapidly by
functional group analysis in the near-infrared.

8- Analysis of petroleum, hydrocarbons, oil & grease contents by EPA:
Semi quantitative determination of petroleum, hydrocarbons, oils & grease can be done by comparison of IR
spectra of sample with standards.
Qualitative analysis:
Infrared spectrum is divided into two parts.
1. Functional group region: 4000 – 1600 cm‾¹.
2. Finger print region: 1600 – 625 cm‾¹.
Various functional groups absorb at specific positions in the functional group region of the spectrum. Finger
print region gives unique characters of each compound. The most common use of IR spectroscopies are for
qualitative identification & structure determination of organic compounds.
Quantitative analysis:
IR spectroscopy was considered to provide only qualitative & semi quantitative analysis of common
samples, particularly when data were acquired by use of conventional dispersive instrument.However, the
development of FT-IR instrumentation & computerized data-processing capabilities have highly improved
the performance of quantitative IR work.
The basis for quantitative analysis of absorption spectrometry is the Beer’s law.
A = abc
a = molar absorptivity
b = path length
c = concentration
Instead of transmittance scale, absorbance is used in quantitative analysis, absorbance “A” is defined as
negative logarithm of the transmittance “T”.

Molecular Fluorescence Spectroscopy

FLUORESCENCE
“Fluorescence is a photoluminescence process in which atoms or molecules are excited by
absorption of electromagnetic radiation. The excited species then relax to the ground state, giving up
their excess energy as photons.”
Fluorescence methods are much less widely applicable than absorption methods because of the relatively
limited number of chemical systems that show appreciable fluorescence
 When a beam of light is incident on certain substances, they emit visible light of longer
wavelength than incident light.
 The energy of emitted radiations is lesser than incident radiation because a part of energy is
lost due to vibrational transitions.
 Substances which show the fluorescence are referred to as fluorescent substances.
PHOSPHORESCENCE
The phenomenon where the emission of light is continuous by some compounds even when the
incident light source is cut off is referred to as phosphorescence.
PRINCIPLE
Molecular fluorescence is measured by exciting the sample at the absorption wavelength, also called
excitation wavelength, & measuring the emission at a longer wavelength called the emission or fluorescence
wavelength. Usually, fluorescence emission is measured at right angles to the incident beam so as to avoid
measuring the incident radiation.
The short-lived emission that occurs is called fluorescence, whereas luminescence that is much longer
lasting is called phosphorescence.
QUANTUM YIELD OR QUANTUM EFFICIENCY

The fluorescence intensity is described in terms of quantum yield or quantum efficiency. The quantum
yield “Q” is the ratio of the number of photons emitted to the number of photons absorbed. Fluorophores
with highest quantum yields display brightest emission (Rhodamines) & Chemical species that do not
fluoresce appreciably have zero quantum yield.
Intrinsic Fluors
Some biomolecules are intrinsic fluors or they are fluorescent themselves. The amino acids with
aromatic groups e.g., phenylalanine, tyrosine, tryptophan are fluorescent. Hence proteins containing these
amino acids have intrinsic fluorescence. The purine & pyrimidine bases & some coenzymes “NAD & FAD”
are also intrinsic fluors. Intrinsic fluorescence is used to study protein conformation changes, to probe the
location of active site & coenzymes in enzymes.
Extrinsic Fluors

These are fluorescent molecules that are added in biochemical system under study. These have been
used to study the binding of fatty acids to serum albumin, & to characterize the binding sites for cofactors.
Ethidium, proflavine & acridines are used for nucleic acid characterization. Ethidium bromide has enhanced
fluorescence when bound to double stranded DNA.
INSTRUMENTATION
The basic instrument is a spectrofluorometer consists of:
 Light source
 Two monochromators
 Sample holder & a

detector.
 There are two

monochromators, one
for the selection of
excitation wavelength,
another for analysis of
the emitted light.
 The detector is at 90
degrees to the
excitation beam.
Light source: The lamp source is a xenon arc that emits radiation in the UV, visible & near-infrared
regions.
Excitation Monochromator: The light is directed by an optical system to the excitation

monochromator, which allows scanning of certain wavelength range.
Sample holder: The exciting light then passes into the sample chamber (fluorescence cuvette). A
special fluorescent cuvette with four transparent quartz or glass sides is used. When the excited light
impinges on the sample cell, molecules in the solution are excited & some will emit light.
Emission Monochromator: Light emitted at right angles to the incoming beam & is analyzed by the
emission monochromator.
Detector: The wavelength of emitted light is measured by a photomultiplier tube serves as the
detector to measure the intensity of the light. The output current from the photomultiplier is fed to
some measuring device that indicates the extent of fluorescence.
PHARMACEUTICAL APPLICATIONS
Fluorescence & phosphorescence methods have naturally low limits of detection than absorption based
spectrophotometeric methods. These are among the most sensitive analytical techniques.
 Determination of fluorescent drugs in low dose in the presence of non-fluorescent excipient.

 In carrying out limit tests where the impurity is fluorescent.

 Useful for studying the binding of drugs to components in complex formations.

 Widely used in bio-analysis for measuring small amounts of drugs & for studying drugs protein
binding.
LIMITATIONS OF FLUORESCENCE SPECTROSCOPY

 This method is applicable only to a limited number of molecules i.e. molecules that are fluorescent or
can be rendered fluorescent.
 The accuracy of the method is affected by a number of interferentes such as temperature, pH,
complex formation etc.

Nuclear Magnetic Resonance Spectroscopy

(NMR)
DEFINITION:
The study of absorption of electromagnetic radiation by a nucleus in radiofrequency region of
spectrum (10 7 -10 8 Hz) in the presence of magnetic field is known as NMR spectroscopy.
PROTON MAGNETIC RESONANCE SPECTROSCOPY:

It is a powerful technique used in the investigation of molecular structures. When study is
concentrated on hydrogen (proton), the spectroscopy is called Proton Magnetic Resonance Spectroscopy
(PMRS).
PRINCIPLE:
Absorption of electromagnetic radiations in radiofrequency region of spectrum results in change in
orientation of the spinning nuclei in a magnetic field.
THEORY:
A nucleus is spinning around an axis. This spinning of a nucleus is designated by spin quantum
number, denoted by “l”. The spin Quantum number may have any value 0, 1/2, 1, 3/2 etc. This spin quantum
number in turns depends upon the total number of protons and neutrons in a nucleus. e.g.
Atomic Number Mass Number Spin Quantum No.
Even Even Zero
Odd Even Integer
Even or Odd Odd Half-integer
The nucleus with l = 0 is considered as NMR inactive.
The nucleus with l ≠ 0 is considered as NMR active.
Since a nucleus is spinning continuesly along an axis, it generates a magnetic field along spin axis.
Thus a nucleus behaves like a tiny bar magnet with angular momentum “µ”
Spinning charge generate a magnetic dipole

BEHAVIOR OF SPINNING NUCLEUS UNDER APPLIED MAGNETIC FIELD:

We know that nuclei act as tiny bar magnet oriented randomly in the absence of external magnetic
field. But in the presence of magnetic field they become oriented specifically depending upon their spin
quantum No. e.g., Nuclei with “l” is 1/2, two orientations are possible. One along the axis of applied field
while other is against (opposite) the applied magnetic field. The best example is Hydrogen nucleus with one
proton ( l = ½). The nucleus (proton) will have two spin states. One is called α spin state along the magnetic
axis (with lower energy) and other is β spin state opposite to applied magnetic axis (with high energy).
Energy difference:
The difference in energy (∆E) between the α and β-spin states is directly proportional to the strength
of the applied magnetic field, H0 .
Energy from α-spin (lower energy) = E1 = - 1/2 (γh/2λ)µ 0
Energy from β-spin (high energy) = E2 = + 1/2 (γh/2λ)µ 0
∆E = E2 – E1
= 1/2 (γh/2λ)µ 0 - x - 1/2 (γh/2λ)µ 0
= 1/2 (γh/2λ)µ 0 + 1/2 (γh/2λ)µ 0
∆E = (γh/2λ)µ 0 but ∆E = hv
hv = γh/2λ µ 0
v = γ µ 0 / 2λ
where
v = frequency
µ 0 = strength of magnetic field
γ = constant → Gyromagnetic ratio
h = Planks constant
As
γ and 2λ are constant, So
V = µ0
Spin Flipping:
The transition of proton from

the lower energy α-spin state to the
higher energy β-spin state takes place
by absorption of electromagnetic
radiation of appropriate frequency.

This transition is known as spin flipping. When nucleus is irradiated with a beam of hv , the low energy
nuclei will absorb energy from radiofrequency source only if the processing frequency is same as the
frequency of radiofrequency beam. Energy difference occurs and resonance is produced.
How to get NMR signals:
There are two modes of inducing the spin-flipping:
i. Frequency-sweep mode
ii. Field-sweep mode.
NMR signals (absorption) can occur by keeping one of both v or µ0 constant and change the second one. i.e.
in the mod of frequency-Sweep the strength of applied magnetic field is kept constant and frequency of
electromagnetic radiation is varied or
Frequency-Sweep = ( v is varied and µ0 is constant), and in the mode of field-sweep the frequency of
electromagnetic radiation is kept constant and the strength of the applied magnetic field isvaried. Or
Field-sweep = ( v is constant and µ0 is varied).
As we know, v α µ0 , NMR signals can be achieved by keeping frequency

constant and making a change in the applied magnetic field. Here absorbance
increases with increase in the µ 0 and then decreases. While frequency v
remains constant.
INSTURMENTATION:
There are two types of spectrophotometers which are commonly used for the NMR study; continuous wave
CW-NMR spectrophotometer and Fourier transform FT-NMR spectrophotometer. In the CW-NMR
spectrophotometer, the frequency of the electromagnetic radiation is kept constant and the strength of the
applied magnetic field is gradually varied to sequentially bring the processional frequency of all the nuclei in
resonance. The spectrum is recorded directly as absorption verses frequency, and it takes time to complete.
In the FT-NMR spectrophotometer, the strength of the applied magnetic field is kept constant and the radio-
frequency is varied. The signal detected in this case is recorded, digitized and stored in a computer.
In the CW-NMR spectrophotometer, the absorption of radiant energy is measured, where as in FT-NMR the
energy emitted by relaxing the nuclei is measured. Thus, the CW-NMR provides an absorption spectrum
whereas the FT-NMR provides an
emission spectrum.
The main components of a typical NMR

spectrophotometer include,
1. sample inlet,
2. magnet,
3. sweep coils,
4. radiofrequency transmitter,
5. radiofrequency receiver,

6. read out device.
1) Sample inlet:
It is usually 15cm long with 5mm diameter borosilicate glass tube. The tube should be chemically
inert, durable and transparent to the radio frequency radiations.
2) Magnet:
Magnet used may be permanent magnet, electromagnet, and superconducting magnet. Permanent
magnet is cheapest and is convenient to use, but it lacks flexibility. Electromagnet is flux density magnet in
which field strength can be changed by current. It is insensitive to temperature change, while temperature
can be controlled in other two types. Superconducting magnet is relatively compact and more powerful than
other magnets, therefore provide high resolution. All NMR spectrophotometers above 100 MHz are based
on helium-cooled superconducting magnets. It is very stable and allows measurement under homogenous
magnetic field over a long period of time.
3. Sweep coils
It consists of coils which are wrapped around or placed between the poles of magnet. The applied
field must be both stable and homogenous across them. The strength of applied magnetic field can be varied
by passing the current in sweep coils.
4. Radiofrequency Transmitter
It is often a highly stable crystal controlled oscillator, the output of which is multiplied to the desired
frequency. The coil of oscillator is wounded around the sample container perpendicular to the applied field,
so that applied radiofrequency field should change the effective magnetic field in the process of irradiation.
5. Radiofrequency Receiver
It consists of a receiver coil which are coiled around the sample tube at right angle to the applied
field and transmitter coil. Radiation from transmitter is absorbed by the sample and detected by
radiofrequency receiver.
6. Read out device

The signal from the receiver coil is weak and it is amplified and recorded mechanically or pre-
calibrated charts.
WORKING OF NMR SPECTROPHOTOMETER

1. Sample Handling:
About 1.0 ml of approximately 5% sample solution in a suitable solvent is commonly taken in the test tube
(depth of 3 – 4 cm). A few percent of a reference substance, e.g., TMS (tetramethyl silane) is also added to
the sample solution.
An ideal solvent must be;
1. Non-viscous
2. Inexpensive
3. Capable of dissolving the sample
4. Chemically inert
5. Devoid of proton of its own
6. Magnetic isotropy
7. Volatility to recover the solvent

8. Absence of H-atom.
Examples of Ideal Solvents:
Carbon tetrachloride (CCl4 ), Carbon disulfide (CS2 ), deuterium oxide
(D2O), deuterated choloroform (DCl3 ), and occasionally used solvents are
CD3CN. (CD3) 2SO and CD3COCD3 .
2. Working:
 Sample tube is placed between poles of magnet.

 Radiofrequency radiation is made to fall on sample by the radiofrequency generator.
 By increasing the current in the sweep coils, total field strength is increased. As a result, precessional
frequency of each set of protons increases until resonance with radiofrequency source takes place.
Now detector produces a signal.
 A signal from detector is amplified and then recorded.
APPLICATIONS OF NMR SPECTROSCOPY

1. Structure Elucidation:
Structure of an unknown compound can be explained from its NMR spectrum.
a. Number of main NMR signals should be equal to the number of protons.
b. Chemical shift indicates that what type of hydrogen bonds (atoms) are present.
c. Spin-Spin coupling reveals about the possible arrangement of groups.
2. Determination of Optical Purity:
Since diastero-isomers differ in their NMR characteristics, so it is used for practical applications in the
determination of optical purity.
3. Keto-Enol Tautomerism:
NMR spectroscopy is probably the most powerful physical analytical method for qualitative and quantitative
analysis of keto-enol equilibra., e.g., Keto and Enol forms of Acetone.
4. Drug-Macromolecular Interaction:
We can study micelle formation by drugs in solution and drug macromolecule (enzymes, metals)
interactions. e.g., Study of Tetracycline and Metal interaction by using NMR.
5. Quantitative Analysis:
NMR spectroscopy has been used to determine the molar ratio of the components in a mixture. Hence,
percentage of each component can be calculated as:

AUP of component
Percentage of components = ------------------------- x 100
Total AUP
AUP = area under the peak
6. Study of Isotopes:
Several nuclei in addition to protons which have magnetic moments can be studied by magnetic resonance
technique. e.g., Flourine, phosphorous etc.
7. Iodine Value of Triglycerides:
Triglycerides have in their proton magnetic resonance (PMR) spectra,four characteristic sets of signals from
the resonance of oleifinic protons. Using this, iodine value can be found.
8. Moisture Analysis:
Water absorbed in biological materials such as food products, appear in NMR spectra as a relatively sharp
bands.
9. Determination of surfactant chain length:
NMR provides a rapid, accurate, non destructive method for analysis of chain length of surfactive agents
than chemical methods.
10. Hydrogen Analysis:
Percentage of hydrogen in an unknown sample may be determined easily and rapidly from total integral of
its NMR-spectrum. The percentage of hydrogen is given by
Total Integral Area
Hydrogen percentage = ----------------------------
KCV
K = proportional constant for the sample tube
C = concentration
V = effective volume of sample solution
11. Detection of impurities in durgs:
If in the NMR spectrum of the known drug, we discover extra peaks, clearly the test sample is impure and
need to be purified.
12. Determination of ethanol content in Alcoholic Liquor:
In many countries the alcoholic liquors must contain at least 40% alcohol. In order to verify this the analysis
of liquors being sold in the market is done via NMR analysis.
KEYS OF SPECTROSCPY
 Inductive effect: The withdrawal or donation of electrons through a σ bond.
 Incandescence: emission of light by hot object.
 K = °C + 273
 °C = K − 273
 Doped: impurity added to semiconductor.
 Distortion: change of shape
 Modulated: change in shape
 Interference: process of wave interaction.
 Metal oxide: Al oxide, Iron oxide, Zinc oxide.
 Incandescence: light produced by an object heated to a high temperature.
 EPA: environmental protection agency
 ATR: attenuated reflectance spectroscopy
 Polymeric film: DNA molecule

CHROMATOGRAPHY
HISTORY:
The Russian botanist Mikhail Tswett introduced the term chromatography in 1906 to describe his
experiments in separating different colored constituents of leaves by passing an extract of leaves through a
column.
CHROMATOGRAPHY
Chromatography is a physical method of separation in which the components to be separated are
distributed between two phases, one is stationary phase while the other is mobile phase which moves in a
definite direction.
The International Union of Pure and Applied Chemistry

(IUPAC) has defined chromatography as a method used
primarily for the separation of components of a sample, in
which the components are distributed between two phases,
one of which is stationary while the other moves.
 The stationary phase may be a solid, or a liquid

supported on a solid, or a gel
 The stationary phase may be packed in a column,
spread as a layer, or distributed as a film, etc.
 In these definitions “chromatographic bed” is used as
a general term to denote any of the different forms in which the stationary phase may be use
 The mobile phase may be gaseous or liquid.
The word chromatography is derived from two Greek words “chromatus” & “graphein” meaning “color” &
“to write” respectively. Chromatography may be preparative or analytical.
Preparative is to separate the components of a mixture for further use (purification).
Analytical chromatography is done normally with smaller amount & is measuring the relative proportions
of analytes in a mixture.
TERMS IN CHROMATOGRAPHY
Stationary Phase:
It may be a solid, a gel or a liquid. If a liquid, it may be distributed on a solid & solid may or may not
contribute to the separation process. Liquid may be chemically bonded to the solid (bonded phase) or
immobilized onto it.
Mobile Phase:
A phase which percolate through the stationary bed, in a definite direction. It may be a liquid or a gas.
Effluent: The mobile phase leaving the column.

Sample: The mixture consisting of a number of components, which is attempted on the chromatographic
bed & carried by the mobile phase.
Sample components: The chemically pure constituents of the sample. Also called elute or analyte.
Solute: A term referring to the sample components in partition chromatography.
Solvent: A term referring to the liquid stationary phase in partition chromatography & in liquid
chromatography often used for the mobile phase.
Zone: A region in the chromatographic bed where one or more components of the sample are located, also
called band.
Chromatogram: A pattern formed by substances that have been separated by chromatography lines.
Elution: Carrying the components of the mixture by the mobile phase through stationary phase is known as
elution.
Eluent: The mobile phase is called eluent.
Eluate: The solution of the components in the mobile phase passed through the stationary phase is called
eluate.
Eluotropic series: A list of solvents ranked according to their eluting power. Such series are useful for
determining necessary solvents needed for chromatography of chemical compounds. Normally such series
progresses from non-polar solvents (n-hexane) to polar solvents (methanol or water).
Partition coefficient “k”: It is the ratio of solute concentration in the stationary phase (liquid) to the mobile
phase (gas or liquid).
Concentration of solute in stationary phase
k=
Concentration of solute in mobile phase
Retention time: It is the time required for the mobile phase to sweep away component from the stationary
phase.
Retention volume: It is the volume of the mobile phase required to sweep away component from the
stationary phase.
Retention time of nonretained solute:(tM or to ):
It is defined as the transit time of a non-retained solute through the column.
Retention volume of nonretained solute (VM ):
It represents the void volume or holdup volume of the column.
Adjusted retention time (t´ R ): It is the retention time adjusted for the hold-up time: t´ R = t R - t M where t
R is the retention time & t M is the hold-up time. The hold-up time is the time of an analyte which
completely penetrates the pores & is not retained at all by the stationary phase.
Volumetric flow rate: The volume of fluid passes per unit time. It is expressed in terms of column
parameters as follows:
Fc = ×
Where d = inner diameter of column
L = column length
ɛ = total porosity of column packing
Velocity of the mobile phase: The average linear velocity (µ) of the mobile phase is measured by the transit
time of a non-retained solute through the column.
µ = L/t M
Chromatographic efficiency: A parameter that is used to quantify the width of a chromatographic peak is
called chromatographic efficiency, N.
Chromatographic efficiency is inversely related to the rate of band broadening per unit time.
N = L/H
Where L is the length of the column & H is referred to as the height equivalent of a theoretical plate
(HETP).
Resolution:
The purpose of chromatography is to separate or resolve compounds. The separation or distance between
two peaks is known as their resolution & it depends upon 3 factors: Retention time (time taken for the
analytes to elute”k”), selectivity (how different the analytes are “α”), & efficiency (how good the column is
“N”).
Rs = t R2 – t R1 / 0.5 (w 2 + w 1 )
TYPES OF CHROMATOGRAPHY OR “CLASSIFICATION”

1. On the basis of physical state of the mobile phase.
2. Classification according to the Mechanism of Separation.
3. Classification according to the shape of “Chromatographic Bed”.
ON THE BASIS OF PHYSICAL STATE OF THE MOBILE PHASE:

I. Gas Chromatography
a. Gas-Solid chromatography: Mobile phase is gas while stationary phase is solid.
b. Gas-Liquid chromatography: Mobile phase is gas while stationary phase is liquid.
Gas chromatography (GC): It is the separation technique in which the mobile phase is always a gas. It is
carried out in a column, which is typically packed. Gas chromatography is based on a partition equilibrium
of analyte between a solid stationary phase & a mobile gas phase (helium) It is widely used in analytical
chemistry & is well suited for use in the petrochemical, environmental monitoring as well as industrial
chemical fields.
II. Liquid Chromatography
a. Liquid-solid chromatography (adsorption): Mobile phase is liquid and stationary phase is solid.
b. Liquid-Liquid chromatography (partition): Mobile phase is liquid and stationary phase is also liquid
(distributed on solid support).
i. Normal-phase chromatography: Stationary phase is more polar than mobile phase
ii. Reversed-phase chromatography: Stationary phase is less polar than mobile phase
Liquid chromatography (LC): The separation technique in which the mobile phase is a liquid. It can be
carried out either in a column or a plane.
In HPLC the sample is forced through a column packed with a porous material (stationary phase) by a liquid
(mobile phase) at high pressure.
HPLC is divided into:
Normal phase liquid chromatography (NPLC): In which the stationary phase is more polar “silica” &
mobile phase is less polar “toluene or n-hexane”.
Reversed phase liquid chromatography (RPLC): In which the stationary phase is less polar
“C18=octadecylsilyl” & mobile phase is more polar “water or methanol”. It is considerably more important.
III. Supercrtical-fluid chromatography:

A separation technique in which a mobile phase is a fluid above & relatively close to its critical temperature
& pressure. Under these conditions the mobile phase is neither a gas nor a liquid. Instead, the mobile phase
is a supercritical fluid whose properties are intermediate between a gas & a liquid. The most common
mobile phase for supercritical fluid chromatography is CO 2 . Its low critical temperature “31°C” & critical
pressure “72.9” atm, are relatively easy to achieve & maintain. Supercritical CO 2 is a good solvent for
nonpolar & for the analysis & purification of low to moderate mol wt., thermally labile & chiral compounds.
Example; Extraction of caffeine from coffee beans. The principles of SCFC are similar to HPLC.
CLASSIFICATION ACCORDING TO THE MECHANISM OF SEPARATION.

I. Adsorption chromatography:

In which the separation is based mainly on difference between the adsorption affinities of the sample & the
surface of an active solid. Examples: TLC & column chromatography.
 Column chromatography is a typical example of adsorption chromatography in which the solid

stationary phase is packed in a tubular column, and the mobile phase is allowed to flow through the
solid.
 Thin layer chromatography (TLC) is another example of adsorption chromatography in which the
stationary phase is a plane, in the form of a solid supported on an inert plate.
II. Partition chromatography:
In which separation is based on differences between the solubility of components in mobile phase (gas) &
stationary phase (liquid) called “gas chromatography”. Or Between the solubility of analyte in the mobile
(liq) & stationary phase (liq) “liquid-liquid chromatography”.
Paper chromatography is a type of partition chromatography in which the stationary phase is a layer of water
adsorbed on a sheet of paper.
III. Ion-exchange chromatography:
A separation technique in which separation is based on differences in the ion exchange affinities of the
sample components.
 This technique uses an ion-exchange resin as the stationary phase.

 Ion-exchange resin is a polymeric matrix with the surface of which ionic
functional groups, such as carboxylic acids or quaternary amines, have been
chemically bonded.
 As the mobile phase passes over this surface, ionic solutes are retained by
forming electrostatic chemical bonds with the functional groups.
 The mobile phases used in this type are always liquid.
 When choosing a chromatographic format for the analysis of an ionic compound, ion-exchange is
generally considered after attempts at developing a reversed phase method has proven unsuccessful.
 However, ion-exchange chromatography is the method of choice for the analysis of inorganic ions,
and it is often preferable to reversed phase methods for the analysis of small organic ions.
IV. Size-exclusion chromatography/ Permeation Chromatography:
In which separation based on exclusion effects, such as differences in mol size. The gel filtration & gel-
permeation chromatography are its subtypes in which stationary phase is a swollen gel.
 In this technique, the stationary phase is a polymeric substance containing numerous pores of
molecular dimensions
 Larger molecules that will not fit into the pores remain in the mobile phase and are not retained
 This method is most suited to the separation of mixtures in which the solutes vary considerably in
molecular sire
 The mobile phase in this type may be either liquid or gaseous
 Size exclusion chromatography is used extensively for the preparative separations of
macromolecules of biological origin as well as for the purification of synthetic organic polymers
V. Affinity chromatography:

In which the unique biological specificity of the analyte & ligand interaction is utilized for the separation.
 The immobilized molecules can he an antibody to a particular protein

 When a crude mixture containing a large number of proteins is passed through the column, only the
protein that reacts with the antibody is bound to the column
 After washing all the other solutes off the column, the desired protein is dislodged from the antibody
by changing the pH or ionic strength
Capillary Electro-Chromatography (CEC)
CEC can be defined as a liquid chromatographic method, in which the mobile phase is electro-osmotically
driven through the chromatographic bed
 The mobile phase in CEC has proven to be superior over other chromatographic methods in terms of
its efficiency in separating ionic compounds and biomolecules.
 The classifications given above for the various types of chromatographic processes can be deceptive
in their simplicity.
 Except in isolated cases, pure adsorption or partition chromatography rarely occurs.
 In practice, separations frequently result from combination of adsorption and partitioning effects.
 The ultimate success of a chromatographic separation depends on the ability of analysts to recognize
the limitations of the methods and adjust their experiments accordingly.
CLASSIFICATION ACCORDING TO THE SHAPE OF “CHROMATOGRAPHIC

BED”
“Column & Planar chromatography”
I). Column chromatography:
A separation technique in which the stationary phase is within a tube.
a. Packed column: The particles of the solid

stationary phase or support coated with a liquid
stationary phase filled inside the tube.
b. Open tubular column: The particles of the

solid stationary phase or the support coated with
a liquid stationary phase concentrated inside the
tube wall leaving an open.
II). Planar chromatography:
A separation in which the stationary phase is present on a plane.
The plane may be paper serving as the stationary phase “Paper

chromatography” (PC). The plane may be the layer of solid
particles spread on a support (glass or aluminum) e.g. thin layer

chromatography (TLC). Sometimes also termed as open bed chromatography.

COLUMN CHROMATOGRAPHY
Column chromatography is an extremely valuable technique for purification of synthetic or natural
products. Compounds are separated by CC through the same mechanism as TLC; through differential
intermolecular forces between the components of the mixture with the mobile, & stationary phase. Because
column is used for stationary phase, so, called column chromatography. On preparative applications it can
be used for the isolation of compounds on scale from µg to kg.
METHODS OF SEPARATION:
Four methods of separation in column chromatography.
1). Adsorption: The components are selectively adsorbed on the surface of the packing (alumina,
magnesium oxide, calcium oxide, charcoal), separation depending upon the interaction of solute with
adsorbent surface. The compound with lesser affinity towards the stationary phase moves faster & hence it is
eluted out fast. While the one with greater affinity towards the stationary phase moves slower & hence it is
eluted out slowly.
2). Partition: The components are selectively partitioned between the elute & stationary phase (thin liquid
film). Commonly used mediums are cellulose, silica gel, celite & kieselgur.
3). Ion Exchange: The ion constituents of the sample are selectively retarded by the exchangeable ions of
the packing. Various ion exchange resins are used for the packing of column.
4). Gels: The column is packed with a permeable gel. Components are separated based on their size using
size-exclusion beads; also called size-exclusion chromatography. In gel chromatography the adsorbent must
be soaked in the mobile phase overnight to absorb the mobile phase & swell.
COLUMNS
Chromatographic columns (tubes) are available in different sizes, shapes & designs. They are made
from glass, stainless steel, teflon & polyethylene. The classical preparative column is a glass tube with a
diameter from 5mm to 50mm & height of 5cm to 1m with a tap & some kind of a filter at the bottom.
In general if the sample contains closely related components, long column are used, While larger
diameter columns are used to accommodate larger amounts of the sample. The length : diameter ratio
ranges from 10:1 to 30:1 & for more efficiency 100:1 ratio is used.
The flow of the mobile phase is controlled by a stop cock or pinch clamp. Before packing the
column, a wool glass plug is placed at the bottom to prevent the falling of column material.
Packing of column
The adsorbent is applied to the Column in two ways:
1). Dry Packing:
In this method the dry adsorbent is poured to the column directly. Vibration is applied to get rid of air
bubbles then the mobile phase passed through the adsorbent. This method cannot be applied to gel
Chromatography.
2). Slurry packing (Wet method):

The adsorbent is suspended in the mobile phase & stirred very well to drive off all air bubbles. The resulted
slurry is then poured into the column. At the tap end of the column a piece of glass wool or cotton must be
added before the slurry application. Sand may be added after the slurry. After slurry application the column
must be allowed to settle down.
Precautions:
a) A filter paper or sand usually placed at the top of the column.

b) There should be no any air bubble in the column during packing & packing should never be dry
because it would cause channeling.
c) After carefully packing the column, the flow rate of eluent is maintained at optimum.
SOLVENTS USED WITH COLUMNS:
1. For Partition: There is a very wide choice of pairs of liquids to act as stationary & mobile phase, which
have a low mutual solubility. A hydrophilic liquid may be used as a stationary phase with a hydrophobic
mobile phase or vice versa. Aqueous buffers & alcohols can be used as mobile phase for polar mixtures,
while hydrocarbons, ethers, esters & chlorinated solvents can be used for less polar substances.
2. For Adsorption: The commonly used stationary phase are silica gel or alumina. Mobile phase used are
according to the eluting power of a solvent is approximately proportionate to its polarity. The commonly
used solvents are organic acids, water, methanol, ethanol acetone etc. A typical series according to the
polarity of solvent is: water > methanol > ethanol > acetone > ethyl acetate > diethyl ether > chloroform >
benzene > cyclohexane > hexane.
3. For Ion Exchange methods: the sample is poured in aqueous solution.
COMMONLY USED ADSORBANTS:

The most commonly used adsorbants are alumina & silica gel. The properties of good adsorbants are:
a) Large surface area

b) Availability of polar sites
c) High porosity
d) Reproducibility in the degree of activation.
Alumina (aluminum oxide - Al2 O3):
It is available in many modifications, depending on the extent of drying & preparation. It is activated at
200°c or 400°c for 3. Anhydrous alumina is a poor adsorbent. The adsorbent activity can be controlled by
varying amount of water. Alumina with 3% water provides the most useful surface area.
Silica Gel (silicon dioxide):
It is activated by heating at 160°c for 3 hours, which is not remove all water. It contains silanol groups (-Si –
OH) as adsorption sites. Silica gel is compatible with water & most organic solvents. It is swells upon
adsorption. Although it has higher surface area but less chemically active than alumina, & is used for
separating organic compounds.

Silica Gel (silicon dioxide):
It is activated by heating at 160°c for 3 hours, which is not remove all water.
It contains silanol groups (-Si – OH) as adsorption sites. Silica gel is
compatible with water & most organic solvents. It is swells upon adsorption.
Although it has higher surface area but less chemically active than alumina, &
is used for separating organic compounds.
INTRODUCTION OF THE SAMPLE:

The sample (mixture of components) is dissolved in minimum quantity of the mobile phase used for
preparing the column or a solvent of minimum polarity. The entire sample is introduced into the column at
once & gets adsorbed on the top portion of the column. From this zone, the individual sample can be
separated by a process of elution.
DEVELOPMENT TECHNIQUE (ELUTION):

After the introduction of the sample, the individual components are separated out from the column by
elution techniques. The two techniques are:
a. Ioscratic elution technique

b. Gradient elution technique
Isocratic elution technique (iso-same or similar): In this technique, the same solvent composition or
solvent of the same polarity is used throughout the process.
e.g: Chloroform only, Petroleum ether : Benzene = 1 : 1 etc.
(ii) Gradient elution technique (gradient-gradually): Solvents with gradually increasing polarity or
increasing elution strength are used during the separation process. Initially low polar solvent is used
followed by gradually increasing to a more polar solvent. e.g: Initially benzene, then chloroform, ethyl
acetate, methanol etc.
DETECTION OF COMPONENTS:
The detection of colored components can be done visually. Different colored bands are seen moving down
the column which can be collected separately. But, for colorless compounds, detection depends upon the
properties of the components.
i). By using UV/Vis light
ii). By fluorescence detector
iii). Using flame ionization detector.
iv). Evaporation of solvent & weighing the residue.
v). Monitoring the fractions by thin layer chromatography (TLC) technique.
Any of the above techniques can be used for the detection of compounds (qualitative analysis).
Recovery of components:

1) Earlier, recovery of the components was done by cutting the column into several distinct zones. Later,
extrusion of zones by using plunger.
2) The best technique is to recover the components by a

process called elution.
3) Recovery is done by collecting different fractions of

mobile phase of equal volume like 10ml, 20ml, etc.
4) They can also be collected time wise i.e. a fraction every

10 or 20minutes etc.
5) The recovered fractions are detected by techniques discussed earlier.
6) Similar fractions are mixed (pooling) so that bulk of the compound is collected.
7) If fraction still contains several components, it can be resolved by using another column.
FACTORS AFFECTING COLUMN EFFICIENCY:

For any separation, efficiency of the column is associated with the following factors:
(i) Dimension of the column: A length:diameter ratio of 20:1 or 30:1 are ideal, but 100:1 is more
satisfactory.
(ii) Particle size of the adsorbent: Adsorbent activity depends on the surface area of the adsorbent. By
reducing particle size, surface area increases & hence increase adsorbent activity.
(iii) Nature of the Solvent: The flow rate of solvent is affected by its viscosity. The less viscous solvents
are better efficient than more viscous one.
(iv) Temperature of the Column: Speed of elution is increased, but adsorbent power is decreased at higher
temp. Normally room temperature is used for all samples.
(v) Pressure: High pressure above the column & low pressure below the column increases the efficiency of
separation. Which is achieved by using pressure device above & vacuum pump below the column.
APPLICATIONS:
(i) Separation of mixture of compounds: CC can be used for the separation of several classes of drugs &
constituents like alkaloids, glycosides, amino acids, plant extracts & drug formulations.
(ii) Removal of impurities or purification process: Impurities present in a compound can be removed by
using appropriate stationary & mobile phase.
(iii) Isolation of active constituents: From plant crude extracts active or required constituents can be
isolated.
(iv) Isolation of metabolites from biological fluids: e.g. Ketosteroids from urine & cortisol from blood,
plasma or serum.
(v) Estimation of drugs in formulation or crude extracts:

a) Determination of % w/v of strychnine in syrup of ferrous phosphate, quinine & strychnine.
b) Determination of primary & secondary glycoside in digitalis leaf.
c) Determination of phytomenadione inj & tab.
d) Separation of tautomers & racemates.
ADVANTAGES OF COLUMN CHROMATOGRAPHY:

 Any type of mixture can be separated.
 Any quantity can be separated (µg to mg).
 Wider choice of mobile phase.
 In preparative type, the sample can be separated & reused (up-to kg).
 Automation is possible.
DISADVANTAGES
 Time consuming method.
 More amount of solvents required which are expensive.
 Automation makes the technique more complicated & expensive.

THIN LAYER CHROMATOGRAPHY (TLC)

INTRODUCTION
is an altogether new, versatile, open layer & specialized laboratory technique that was advanced in early
fifties, & since then has become a critical means of separation for analysts & researchers. Kirchner in 1950
was the first, who used adsorption chromatography on glass-plate coated with silicic acid or alumina. Stahl
in 1958, introduced a standard equipment for preparing uniform thin layers of known thickness, which
finally accepted an additional modern tool for analytical chemistry.
Other names of TLC in various literature as: “open-column chromatography” “drop chromatography”
“strip-chromatography” “spread-layer chromatography” “surface chromatography”
“Thin layer chromatography (TLC) is a method of analysis in which the stationary phase, a finely
divided solid, is spread as a thin layer on a rigid supporting plate, and the mobile phase, a liquid, is
allowed to migrate across the surface of the plate.”
 The mobile phase does not flow under the influence of gravity or high pressure but is drawn across
the plate by capillary action
 Although separation efficiencies equivalent to those obtained with gas or HPLC cannot be obtained
by this method, it has the advantages of speed, versatility, and simplicity.
 TLC is the simplest of all the widely used chromatographic methods to perform.
 A suitable closed vessel containing solvent and a coated plate are all that required to carry out
separations and qualitative and semi-quantitative analysis.
 With optimization of techniques and materials and the use of commercially available instruments,
highly efficient separations, accurate and precise quantification can be achieved.
MECHANISM OF SEPARATION
 Mechanism of separation can be Adsorption, Partition (Normal phase and Reverse phase), Ion-
exchange, Size-exclusion
 It depends on the nature of the stationary and mobile phases
Technique Stationary Phase Mobile Phase

Silica gel
Nonpolar or polar organic
Alumina
Adsorption solvents
Charcoal
Polyamide Polar organics
Cellulose Mixed aqueous, organic
Partition
Silica gel solvents
ODS silica gel
Reversed phase partition Coated silica Mixed aqueous, polar solvents
Acetylated cellulose
Ion exchange resins
Ion exchange Buffered aqueous solutions
DEAE- and CM-cellulose
Ion exchange Dextran gels Buffered aqueous solutions

STATIONARY PHASE
A wide variety of stationary phases are available in size ranges suitable for use in TLC.
Silica gel (SiO 2 )
 It is the most frequently used stationary phase

 It is employed as such for adsorption TLC
 It is modified for reversed phase separations by;
o Coating with a thin layer of a nonpolar substance, such as silicone oil, or
o Binding a nonpolar functional group to it, such as octadecylsilyl (ODS)
 The surface of silica is acidic due to the presence of many silanol hydroxyl groups
 Therefore, it is best suited for the analysis of acidic compounds
 It is also preferable for polar compounds such as amino acids and sugars
 The mean particle size of silica gel is 15 µm, with the particle size range 05 to 40 µm now
commonly used
Alumina (Aluminum oxide, Al 2O3)
 It has a basic surface

 It is chosen over silica gel for the separation of basic and weakly polar compounds Polyamide
(nylon)
 It is a long chain polymer
 Because it has many free amide and carboxyl groups on its surface, is an adsorbent with strong
hydrogen- bonding abilities
 It will readily bond phenols, carboxylic acids, quinones, and nitro-compounds, all of which require
polar solvents such as methanol and dimethylformamide to displace them
Cellulose
 It is a polysaccharide, has numerous neutral hydroxyl groups on its surface and can adsorb water or
polar solvents by hydrogen bonding, making it useful for partition TLC
 Less active and less frequently used sorbents are;
Calcium phosphate, Calcium carbonate, and Diatomaceous earth (kieselguhr)
 To ensure that the stationary phase adheres firmly to the backing plate and does not flake off during
the development, binders such as calcium sulfate (gypsum), starch carboxymethylcellulose (1-2%),
or polyvinyl alcohol (1-5%) are added to the adsorbent
MOBILE PHASE
 If possible, it is preferable to use a single solvent to develop the chromatogram rather than a
multicomponent mixture
o Because solvents are adsorbed preferentially by the stationary phase, and as the mixture
moves up the plate, the composition of the mobile phase is always changing
o Compounds that travel a greater distance up the plate, therefore, will be exposed to a different
mobile phase than those that are retained strongly
 The solvents also should be volatile so they can be evaporated from the plate after the development
is completed

 The selection of the optimum solvent or mixture for use as the mobile phase depends also on the
nature of the solutes and stationary phase and largely is empirical
 A useful procedure for initial trials is to run two separate plates, one using a very polar solvent (e.g.
ethanol), and the other employing a nonpolar liquid (e.g. hexane)
 After observing which type of mobile phase moves the solutes from the origin and determining their
R f values, the solvent may be modified to increase selectivity and resolution in a number of ways
 The polarity may be altered by adding other solvents chosen by their polarity
 Substances with functional groups similar to those of the solutes, such as ethers, alcohols, or
carboxyls, may be added to increase the R f value by promoting solubility in the mobile phase
 Acids or bases (acetic acid or ammonia) may be added to affect the charges on the solutes to prevent
tailing
 Solvents used in chromatography, in increasing order of polarity;
o Heptane, Hexane, Isoctane, Cyclohexane, Carbon tetrachloride, Toluene, Benzene, Ethyl
ether, Chloroform, Methylene chloride, Tetrahydrofuran, Acetone, Dioxane, Ethyl acetate,
Acetonitrile, Pyridine, I-Propanol, Ethanol, Methanol, Acetic acid, Water
PREPARATION OF PLATES
 Plates with dimensions of 20×20 cm are necessary to attain the greater efficiency required for more
difficult separations
 These are usually made of glass, but plastic, stainless steel, or aluminum backings also are used
 The material must be cleaned scrupulously to prevent interaction of the solutes with contaminants on
the backing
 To reduce band-broadening, the stationary phase should consist of small particles of uniform size so
as to provide a large area for interaction and a small void volume
 The particles are mixed with water or an organic solvent to form a slurry, a suitable binding agent is
added, and fluorescent indicators such as zinc silicate may be included to aid in detection of the
solutes after the development
 The slurry is coated on the plates using a spreader, which will apply a uniform layer of adsorbent of
desired thickness over the surface of the entire plate, and the plates are dried
 Instead of coating a plate with one sorbent, two different substances may be applied simultaneously
so that the layer is made of a gradient mixture of both
o For example, silica gel and alumina can be used to prepare a pH gradient across the width of
the plates
o This may yield separations that otherwise would be impossible
 The thickness of the layer of stationary phase is important to the success of the chromatography, as
excessively thick layers allow the solutes to diffuse laterally
 Layers from 0.1 to 2.0 mm in depth are used most often, with thinner ones (250 µm) being most
suitable for precise separations and thicker coatings for preparative work, due to their greater solute
capacity
SAMPLE APPLICATION AND DEVELOPMENT

 Samples are spotted usually with a capillary tube or a microliter syringe
 Samples may be applied as spots or as thin streaks
 It is essential that all of the solvent be evaporated between repeated applications

 The area of sample application should be kept as small as possible because the bands will broaden as
they travel up the plate
Ascending Development
 For ascending development, the plate is placed in a rectangular jar that contains developing solvent
to a depth of about 0.5 cm
 The atmosphere of the jar should be saturated completely with the mobile phase before development,
a process usually performed by lining the jar with a piece of filter paper that has been wet with
mobile phase
 The plate then is removed from the tank, the mobile phase front is marked by scratching the surface,
and the solvent is evaporated in an oven or, if the sample is heat labile, in the air
Descending Development
 Spots are located at the top of the plate
 Mobile phase is fed to the plate, from a rough, via the wick
 Components of the sample are separated as the mobile phase moves from top to bottom
Multiple Development
 After the plate is dried, it is returned to the chamber and redeveloped in the same direction, using the
same mobile phase
 The process may be repeated as many times as is necessary to ensure effective separation
Two-dimensional Development
 The sample is applied as a small spot in the lower left corner of the plate, about 2.5 cm from each 1
edge
 After the plate has been developed in the usual manner, it is dried, rotated 90° counterclockwise, and
placed in another chamber with a different developing solvent
 The separated spots produced by the first elution are now located at the origin of the second
 This method is useful especially for complicated mixtures containing many components or groups of
substances with different functionalities because the selectivity effects of the mobile phases can be
exploited more efficiently using two solvents
Radial Development
 Samples are applied in a circle close to the center of the plate
 Mobile phase moves by capillary action through the wick at the center of the plate and moves the
sample radially to form the sample spots of different compounds as concentric rings
DETECTION METHODS
Once the chromatogram has been developed, the solute spots must be made visible in order to determine
their R f values
Directly by visible light
 If the substances are highly colored (e.g. dye pigments), there is no difficulty in visual detection
Fluorescence quenching
 An alternative detection method using a TLC plate that has been treated with a fluorescing reagent
and, thus, the whole plate fluoresces when exposed to excitation radiation
 The plate is used to separate the solutes of interest in the usual way and then is exposed to UV light
 The plate is seen as a bright fluorescent sheet and the solutes as dark non-fluorescent where the
solutes have quenched the plate fluorescence
By use of Chemicals
Nonspecific methods
1. Use of iodine vapor

2. Charring of organic compounds
Use of iodine vapor
 Iodine associates with practically all organic compounds, especially with unsaturated or aromatic
compounds forming charge transfer complexes
 In any case the solutes will become visible as brown spots
Charring of organic compounds
 Charring is a very widely employed technique for the detection of carbon containing compounds,
because it is effective for almost all organic compounds
 The process involves spraying the plate with sulfuric acid, usually as a 50% (v/v) mixture with
methanol, and then heating it in an oven at 110 °C for 10 to 30 minutes
 The organic compounds are destroyed by the acid, and a dark deposit of carbon (charcoal)
remains at the spot
 Though this method is effective for most organic solutes, it is destructive and, hence, cannot be
used if the compounds are to be removed from the plates
Specific methods
The more specific methods of detection involve spraying the plates with reagents designed to react with
specific functional groups to produce visible derivatives

These reactions may produce products of three types;
1. Those that are detected directly in visible light (2,4-dinitrophenyl-hydrazones of carbonyls)

2. Those that absorb UV and quench fluorescence (benzoate esters of alcohols)
3. Those that fluoresce directly (phthalaldehyde derivatives of amino acids)
Manual methods
 Compare the spot sizes and intensities between unknown and standard
 Trace the spot outline on paper and weigh it
 They are tedious and give high levels of variability
Spectrodensitometry
 An automated method, much more convenient and is capable of yielding quantitation in the
submicrogram range
 In this method, the plate is placed on a movable stage that is driven by a motor so that the lane of
spectrodensitometric measurements may be made on substances that are colored or absorb UV, those
that have been charred, those that quench fluorescence, and even on photographs or x-ray films
ADVANTAGES OF TLC
1. Simple & equipment cost is low.
2. Rapid than column C.
3. Separation of minute substances (µg).
4. Any type of compound can be analyzed.
5. Detection is very easy.
6. Corrosive spray reagents can be used without damaging plates.
7. Need solvents & stationary phase in less amount than column chromatography.
8. To find solvent system that will separate mixture before proceeding to column chromatography.
9. No spots spreading & making resolution better.
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC)

 HPTLC is a form of TLC, which represents a considerable advance in the practice of TLC
 It is a more rapid, efficient, and sensitive technique than conventional TLC
 It offers an increase in performance that is an order of magnitude greater than TLC
 Thus it is possible to carry out separations on HPTLC that were not possible before on TLC plates
 HPTLC provides superior separation power using optimized coating material, novel mobile-phase
feeding procedures, layer conditioning, and improved sample application
 It promotes for higher separation efficiencies, shorter analysis time, lower amounts of mobile phase,
and efficient data acquisition and processing
 HPTLC is now frequently used in the identification of hydrocarbons, alcohols, phenols,
carbohydrates, organic acids, lipids, steroids, saponins, alkaloids, amino acids, peptides, proteins,
enzymes, nucleic acids, vitamins, antibiotics, pesticides, etc.
APPLICATIONS OF TLC
Purity of any sample

 Purity of sample can be carried out with TLC

 Direct comparison is done between the sample and the standard or authentic sample
 If any impurity is detected, then it shows extra spots and this can be detected easily
Identification of compounds
 TLC can be employed in purification, isolation and identification of natural products like volatile oil
or essential oil, fixed oil, waxes, terpenes, alkaloids, glycosides, steroids etc.
Examination of reactions
 Reaction mixture can be examined by TLC to access whether the reaction is complete or not
 This method is also used in checking other separation processes and purification processes like
distillation, molecular distillation etc.
Biochemical analysis
 TLC is extremely useful in isolation or separation of biochemical metabolites or constituent from its
body fluids, blood plasma, serum, urine etc.
In chemistry
 TLC methodology is increasingly used in chemistry for the separation and identification of
compounds which are closely related to each other
 It is also used for identification of cations and anions in inorganic chemistry
In pharmaceutical industry
 Various pharmacopoeias have adopted TLC technique for detection of impurity in a pharmacopoeia
chemical
 Various medicines like hypnotics, sedatives, anticonvulsant tranquillizers, anti-histamines,
analgesics, local anesthetics, steroids have been tested qualitatively by TLC method
 One of the most important application of TLC is in separation of multicomponent pharmaceutical
formulations
In food and cosmetic industry
 TLC method is used for separation and identification of colors, preservatives, sweetening agent, and
various cosmetic products.
 This are some of the applications of Thin layer Chromatography (TLC)

GAS CHROMATOGRAPHY
INTRODUCTION
Gas chromatography is a technique used for separation of volatile substances, or substances that
can be made volatile, in a gaseous mixture at high temperatures.
Martin & Synge in 1941 first introduced, the gas used as a mobile phase. In 1962, James & Martin
performed first chromatographic separation (mixture of amino acids & fatty acids) by using gas as mobile
phase.
The development of LC & especially the introduction of capillary or open tubular column by
“Galary” modernize the analysis of complex mixture of volatile compounds. The use of GC-MS & GC-IR
system has made the application of GC more important in recent years. Micro column (5-10µm) with
chemically bound liquid & micro cell detector are used now a days.
BASIC PRINCIPLE:
A sample needs to be separated is injected into the gas chromatograph. A mobile phase moves
through a column that contains a wall coated or granular solid coated stationary phase. Mobile phases are
generally inert gases such as helium, argon, or nitrogen. The injection port consists of a rubber septum
through which a syringe needle is inserted to inject the sample. The injection port is maintained at a higher
temp than the BP of the component in the sample mixture. Since the partitioning behavior is dependent on
temp, the separation column is usually contained in a thermostat-controlled oven.
Separating components with a wide range of BPs is accomplished by starting at a low temp & increasing the
temp over time to elute the high BP components.
Gas - Solid Chromatography (GSC)

The stationary phase, in this case, is a solid like silica or alumina. It is the affinity of solutes towards
adsorption onto the stationary phase which determines, the retention time. The mobile phase is, of course, a
suitable carrier gas. The use of GSC in practice is marginal when compared to gas liquid chromatography.
Gas - Liquid Chromatography (GLC)

The stationary phase is a liquid with very low volatility while the mobile phase is a suitable carrier gas.
GLC is the most widely used technique for
separation of volatile species. The presence of a
wide variety of stationary phases with
contrasting selectivities & easy preparation of
column, add to the assets of GLC or GC.
INSTRUMENTATION:
Gas chromatograph consists of the following:
1) A Carrier gas supply,

2) Sample injection port,
3) Column,

4) Oven,
5) Detector,
6) Recorder/integrator system.
1. A Carrier gas:
The mobile-phase (gas) in GC is called carrier gas which must be chemically inert. Helium is the most
commonly used gas, although argon, nitrogen, & hydrogen are also used. These gases are available in
pressurized tanks. Pressure regulators, gauges, & flow meters are required to control the flow rate of the gas.
In addition, the carrier gas system often contains a molecular sieve to remove impurities & water.
2. Sample injection port

The most common method of sample injection involves the use of
micro syringe to inject a liquid or gaseous sample through a self-
sealing, silicone-rubber diaphragm or septum into a flash vaporizer
port located at the head of the column. The sample port is ordinarily
about 50°C above the BP of the least volatile sample.
For quantitative work, more reproducible sample sizes for both liquids
& gases are obtained by means of a rotary sample valve. Rotation of
the valve by 45 degree then introduces the reproducible volume into
the mobile phase.
1) Columns:
Two basic types of columns are currently being used: Packed column & Capillary column.
i) Packed Column: A packed column is constructed from glass, stainless steel, copper or aluminum & is
typically 2–6 m in length, with an internal diameter of 2–4 mm. The column is filled with a particulate solid
support, with particle diameters ranging from 37–44 µm to 250–354 µm. The most widely used solid
support is diatomaceous earth, which is composed of the silica skeletons of diatoms.
ii) Capillary column/open tubular columns:
These are constructed from fused silica coated with a protective polymer. Columns may be up to 100 m in
length with an internal diameter of approximately 150–300 µm. Capillary columns are of two principal
types. Wall-coated open tubular columns (WCOT) contain a thin layer of stationary phase, typically 0.25
µm thick, coated on the capillary’s inner wall.
In support-coated open tubular columns (SCOT), a thin layer of a solid support, such as a diatomaceous
earth, coated with a liquid stationary phase is attached to the capillary’s inner wall.

Support Coated Open Tubular Column (SCOT) Packed Column Wall Coated Open Tubular Column (WCOT)
2) Column OVEN:
Column temperature is an important variable that must be controlled for precise work. Thus, the column is
ordinarily hosed in a thermostat oven. The optimum column temperature depends upon the BP of the sample
& the degree of separation required. Roughly, a temperature equal to or slightly above the average BP of a
sample results in a reasonable elution time (2 to 30 min).
For samples with a broad boiling range, it is often desirable to employ temperature programming, whereby
the column temperature is increased either continuously or in steps as the separation proceeds.
3) Detectors used in GC:

Each chromatograph has a detection device at the exit of column. The purpose of the detector is to monitor
the carrier gas as it leaves the column & response to change its components as solutes are eluted. An ideal
detector should have the following characteristics.
i). Rapid response (less than second).
ii). High sensitivity (ppm & ppb).
iii) Operational temperatures up to 400° C.
iv) Should be stable.
v) Should have small internal volume.
vi) Low noise.
vii) Simple, inexpensive & safe to operate.
viii) Low detection unit.
Unfortunately, no detector exhibits all of these characteristics.
Some of the more common detectors are:
i) Flame Ionization Detector. Flame Ionization Detector
ii) Thermal Conductivity Detector.

iii) Mass Spectrometer (MS)
iv) Electron Capture Detector (ECD)
v) Thermionic Detector
vi) Electrolytic Conductivity (Hall)
vii) Photoionization Detector
viii) Fourier Transform IR (FTIR).
Flame Ionization Detector (FID):
This is one of the most sensitive & reliable destructive detectors. Separate two gas cylinders, one for fuel &
the other for O 2 or air are used in the ignition of the flame of the FID. The fuel is usually hydrogen gas. The
flow rate of air & hydrogen should be carefully adjusted in order to get successful ignition. This detector is
mass sensitive where solutes are ionized in the flame & electrons emitted are attracted by a +ve electrode,
where a current is obtained.
The FID detector is not responsive to air, water, carbon disulfide. This is an extremely important advantage
where volatile solutes present in water matrix can be easily analyzed without any pretreatment.
Advantages:
1. Rugged
2. Sensitive (10-13 g/s)
3. Wide dynamic range (107)
4. Signal depends on number of carbon atoms in organic analyte which is referred to as mass sensitive
rather than concentration sensitive.
Disadvantages:
1. Weakly sensitive to carbonyl, amine, alcohol groups.

2. Not sensitive to non-combustibles – H2O, CO2 , SO2 ,
NO.
3. Destructive
Thermal Conductivity Detector (TCD)
The TCD which was one of the earliest detectors for GC, is still
widely used. This device contains an electrically heated source
whose temp at constant electrical power depends on the thermal
conductivity of the surrounding gas. The heated element may be
a fine platinum, gold, or tungsten wire. Because of high thermal
conductivity, helium is the choice of mobile phase when using a
TCD.
The advantage of the TCD is its simplicity, its large linear

dynamic range (~10 5 ), its general response to both organic &
inorganic species, & its nondestructive character, which permits collection of solutes after detection.
Its chief limitation is its relatively low sensitivity. Also, keep away any oxygen since oxygen oxidize the
filament & results in its destruction of detector.
Mass spectrometer detector (GC-MS)
Mass spectrometer is used as detector. Effluent from the GC introduced into the Mass spectrometer & after
fragmentation, the mass path of this separated component can be determined.
Electron capture detector (ECD)
The electron-capture detector has become one of the most widely used detectors for environmental samples,
halogen containing compounds, such as pesticides & poly- chlorinated biphenyls. The effluent from the
column is passed over a β emitter, usually nickel-63. An electron from the emitter causes ionization of the
carrier gas & production of a burst of electrons. In the absence of organic species, a constant standing
current between a pair of electrodes results. The current decreases markedly, however, in the presence of
those organic molecules that tend to capture electrons.
The ECD is selective in its response to molecules containing electronegative functional groups such as
halogens, peroxides, quinones, & nitro groups.
APPLICATIONS:
A). Quantitative Applications:
Gas chromatography is widely used for the analysis of wide range of samples in environmental,
biochemical, food science, forensic & petrochemical laboratories.
i) Environmental Analysis: One of the most important environmental applications of GC is for the analysis
of numerous organic pollutants in air, water, & wastewater.
ii) Biochemical Analysis: GC can be used to analyze the human body fluids. Scientists believe that in future
sample of breath, blood & urine could be analyze in few minutes. This would give a complete diagnosis of
disease.
iii) Food Science: Many flavors, spices, & fragrances are readily analyzed by GC, using headspace analysis
or thermal desorption. All food & beverage products must be carefully assayed for contamination with
pesticides, herbicides & many other materials that are considered health risk. GC is the ideal technique for in
food & beverage assays & tests.
iv) Forensic Analysis: Forensic analysis frequently use GC to characterize drugs of abuse.
v) Petroleum Industry: Gas chromatography is ideally suited for the analysis of petroleum products,
including gasoline, diesel fuel, & oil.
Quantitative Calculations: In a quantitative analysis, the height or area of an analyte’s chromatographic peak
is used to determine its concentration. Although peak height is easy to measure, its utility is limited by the
inverse relationship between the height and width of a chromatographic peak.
B) Qualitative Applications
GC can also be used for qualitative purposes.
When using an FT–IR or a mass spectrometer as the detector, the available spectral information can be used
to identify individual solutes.
Retention times can be compared with values measured for standards, provided that the operating
conditions are identical.

HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)
INTRODUCTION
High-performance liquid chromatography is in some respects more versatile than GC, since
(a) It is not limited to volatile & thermally stable samples.
(b) The choice of mobile & stationary phases is wider.
In HPLC, a liquid sample, or a solid sample dissolved in a suitable solvent, is carried through a
chromatographic column by a liquid mobile phase.
Separation is based on solute/stationary-phase interactions, including liquid–solid adsorption,

liquid–liquid partitioning, ion exchange & size exclusion, as well as by solute/mobile-phase interactions.
In each case, however, the basic instrumentation is essentially the same. It is called high
performance liquid chromatography as performance is improved when compared to classical CC. It is also
called high pressure liquid chromatography since high pressure is used when compared to classical CC.
High performance liquid chromatography is basically a highly improved form of column

chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced
through under high pressures of up to 400 atmospheres. That makes it much faster.
It also allows you to use a very much smaller particle size for the column packing material which
gives a much greater surface area for interactions between the stationary phase and the molecules flowing
past it. This allows a much better separation of the components of the mixture
TYPES OF HPLC TECHNIQUES:

A. Based on modes of chromatography
1. Normal phase mode
2. Reversed phase mode
B. Based on principle of separation
1. Adsorption chromatography
2. Affinity chromatography
3. Ion exchange chromatography
4. Size exclusion chromatography
5. Chiral phase chromatography
C. Based on elution technique
1. Isocratic separation
2. Gradient separation
D. Based on the scale of operation
1. Analytical HPLC
2. Preparative HPLC
E. Based on the type of analysis:
Qualitative & Quantitative

analysis
INSTRUMENTATION
1. Solvent delivery system
2. Pump
3. Sample Injector
4. Columns
5. Detector
6. Data System
Solvent delivery system

It consists of solvent reservoirs, a degasser & a pump. The “solvent reservoir” are
vessels of glass or stainless steel that hold the mobile phases of different
polarities. “Degasser” removes the air bubbles from the solvent. Air bubbles may
result in the malfunctioning of pressure (check valve). Solvent used must be
pure, HPLC grade & filtered through 0.2 µm nylon filter.
HPLC Pump:
The pump can deliver solvent at high pressure from the reservoir to the detector
with a flow rate over 50 ml/min. An ideal pump should have following properties.
i) Provide high pressure up to 6000 psi.
ii) Should be pulse free.
iii) Should be corrosion resistant.
iv) Should provide constant flow.
Types of pumps:
1. Reciprocating piston pumps
2. Displacement pumps

3. Penumatic pumps
1. Reciprocating piston pump:

It consists of a chamber in which solvent is pumped by front & back motion of a motor driven piston.
Two ball check valves open & close alternatively & control the flow rate of solvent. It has low internal
diameter & high pressure. It can be used for gradient elution & provide constant flow rate. The disadvantage
is that it has pulsatile flow.
2. Displacement Pump:
This pump consists of syringe like chamber equipped with a plunger that is activated with a screw
driver mechanical motor. The advantage is pulse free & constant flow. Its disadvantages are limited solvent
capacity & error with gradient elution.
Penumatic pumps
The solvent is placed in a collapsible vessel hosed in a vessel which can be pressurized by an inert
gas. These pumps are inexpensive & pulse free. Their disadvantage is that they provide limited pressure upto
2000 psi. The flow rate is not constant & cannot be used for gradient elution.
SAMPLE INJECTOR:
A typical injector consists of a stainless steel loop with six different ports. A moveable teflon cone in
the ring has three open segments each of which is connected with an external sample loop of fixed volume.
In one configuration, the cone permits direct flow of solvent into the column & the loop can be filled with
the sample & remaining sample is flushed to the waste. This is the load position. In the next step the position
of control knob is changed to inject position to make sample loop part of the moving stream. It sweeps the
sample in to the column. Sample can be injected manually by syringe or with the help of auto sampler.
Auto injectors provide continuous injection of samples & more accuracy to manual system. Much more
expensive initially but more convenient upto 100 samples & standards can be taken at once with
microprocessor control.
HPLC COLUMNS:
Guard column/precolumn:

These are small 3-10 cm long & placed between the injector & analytical column or between the pump &
injector. It contains same packing as analytical column.
It has following functions.
i) Retain particulate matters in the mobile phase.
ii) Retain highly sorbed compounds.
iii) Increase the life of analytical column.
Analytical column:
Straight lengths of stainless steel tube in different size & diameter makes excellent column. Usual internal
diameter is 3.9-4.6mm & a length ranging from 10 to 30 cm. A well packed column with internal diameter
4.6 mm & particle size 5µm should give a plate count of 60000-90000 plate/meter & flow rate of 1ml/min.
Fast operation can be performed by using larger bores but narrow columns give better results.
Most commonly used particles are microporous or diffusive particles permeable to solvent. The use of
smaller particle size reduce the eddy diffusion & minimizing the band broadening.
STATIONARY PHASES:
Silica gel: Silica tend to dissolve above pH 8 so it cannot be used for the
separation of basis. Polystyrene & polymethacrylate particles can be used for
this purpose. Silica has surface silanol groups that are bound with the
stationary phase support by silination reaction. The middle free OH group is
encapped with the trimethylsilyl group making it inert.
ODS: The octadecylsilane (ODS) & octasilane (C18 & C8) are the most
commonly used packing materials for reverse phase chormatography.
DETECTORS:
Detectors visualize separated compounds & translate the concentration changes into signals.
Characteristics of an ideal detector:
1) Adequate sensitivity.
2) Good stability & reproducibility.

3) Gives linear response to analytes.
4) Short response time.
5) High reliability & ease of use.
6) Non-destructive.
Most common HPLC detectors
1. UV-Visible absorbance detector.
2. Fluorescence detector.
3. Refractive index detector (RI).
4. Electrochemical detector.
5. Conductivity detector.
6. Mass Spectrophotometer detector (MS).
7. FT-IR detector.
UV-Visible absorbance detector
It works on the principle of the UV or visible radiations absorb by the sample components. Organic
compound having chromophores absorbs at a particular wavelength & the intensity of absorbance is directly
proportional to the concentration of the analyte. It has better sensitivity (108 g/ml). It is temp resistant,
simple, reliable & inexpensive. It used for gradient & isocratic elution. This is non-destructive detector. It
can be used only for the compounds having chromophores.
Types:
i) Fixed Wavelength Detector:
It consists of a lamp which emits at a certain wavelength. Mercury vapor lamps are probably the most
common & emit intense light at 254 nm. Compounds containing carbonyl groups, multiple double bonds, or
aromatic rings can be detected.
ii) Variable Wavelength Detector:
The variable type uses a deuterium lamp that produces a broad spectrum
of wavelengths that are separated by diffraction grating. The instrument is
adjustable & only the wavelength of interest passes through the slit &
sample & finally absorbance is measured.
iii) Diode Array Detector:

The photodiode array detector passes a wide spectrum of light through the sample & then light is separated
into individual λ. The spectrum of light is directed to an array of photosensitive
diodes. Each diode can measure different wavelengths which allows for the
monitoring of many wavelengths at once. Monitoring two peaks instead of one
can provide information on the purity of the peak, or it can be used to quantify
a peak when an interfering peak is present.
Fluorescence detector
The fluorescence detector is based on the principle that some compounds
fluoresce when bombarded with UV light. The analyte is excited by light
commonly at 253.7nm from a low pressure mercury lamp. The light is absorbed, molecule is excited & gives
off light of a different wavelength. This wavelength is monitored by a detector. Fluorescence detectors are
among the most sensitive of HPLC detector.
Refractive index detector

It is also called universal detector which detects change in
refractive index of elute as it appears from the column.
Their use is limited to isocratic elution. These are temp
sensitive & detection limit is upto 10 g/ml. Organic acids,
sugars & fungal metabolites can be analyzed by this
detector.
The disadvantage is that they have less sensitivity & are

not suitable for gradient elution.
Electrochemical detector
ECD used for compounds undergo oxidation or reduction reactions.
Usually accomplished by measuring gain or loss of electrons from
migrating samples at given pot difference between electrodes. The
advantages are; high sensitivity, simplicity, convenience, &
widespread applicability.
Conductivity detector
It is based on the principle that the electrical conductivity of a
liquid is proportional to the ionic concentration in the mobile
phase. The design is simple & the detector can be made small
enough for an effective sensing volume of a few nano liters. It is
used for detecting electrically charged ions.
Mass spectropotometer detector

In this detector, the analytes are first ionized since the detector only measures charged particles. The charged
particles then enter the separator that separates ions on the basis of mass using a magnetic field. The masses
of different fragments hit the detector where they give up their charge creating an electric signal which is
recorded. The magnitude of electric current is directly proportional to the concentration of analyte.
Infrared Absorbance Detectors

Two types of infrared detectors are offered commercially. The range of the first instrument is from 2.5 to
14.5 µm or 4000 to 690 cm-1. The second type of infrared detector is based upon Fourier transform
instruments.
INTEGRATOR/READ OUT SYSTEM

Integrator collects the data from the detector & converts it into a readable & recordable form. Usually it
shows the output in the form of a peak.
Types of recording devices for HPLC:
1. Strip chart recorder.

2. Integrator.
3. Computerized data collection method is currently used in which output signal of the detector can be
seen on the computer monitor.
TYPES OF ELUTION IN HPLC

Isocratic elution:
The mobile phase composition remains fixed during the whole process of elution. This type of elution is
used to separate simple mixtures, which have sufficiently different retention time.
Gradient elution:
The composition of mobile phase is changed during the elution process. It is used to separate the complex
mixtures which do not separate with isocratic elution. The change in mobile phase, change the retention
behavior of the components & they separate from each other.
Quantitative analysis of formulation:
The majority of operations of HPLC in the pharmaceutical analysis are to determine quantitatively the active
ingredients. The analysis becomes time consuming if more than one active ingredient is present whose
retention time is very different from each other.
Assay of drug formulation is carried out using a reference standard of known purity. The standard is
dissolved in precise volume of solvent to prepare a known stock solution of standard. The stock solution is
then diluted appropriately to get the calibration series of standard solution. These calibration solutions are
run in the increasing order of conc. on the HPLC with a suitable mobile phase. The peak area obtained for
each calibration standard is used to draw a standard curve. The sample solution is also prepared in the same
solvent & run on the HPLC using the same mobile phase & elution parameter. It is emphasized that the
sample solution should be completely recovered from the column.
From the area of the sample, the quantity of the active drug is calculated using linear regression equation.
a = bx + c
or x = a – c /b
a = area of the curve
b = slope
c = intercept
x = concentration/quantity of analyte.
APPLICATIONS:
1. Widely used in the research, industry &biomedical sciences.
2. Good for separation of non-volatile substances, amino acids, carbohydrates, nucleic acid &
hydrocarbons.
3. Pesticides, pigments, antibiotics & other pharmaceuticals can be separated.
4. Generally, any component that is soluble in common solvents such as alcohol, methanol, acetone can
be analyzed using HPLC.
5. HPLC is highly popular due to its sensitivity & accuracy.
6. It is the method of choice to separate thermolabile substances.
LIMITATIONS OF HPLC:
1. HPLC cannot be used for very volatile substances.
2. It is not a suitable method for the analysis of high mw proteins & nucleic acids, as well as large
polymeric compounds. These molecules may be entrapped in the column packing & damage the
column.

Gas Chromatography –Mass Spectrometry

(GC-MS)
GAS CHROMATOGRAPHY – MASS SPECTROMETRY
The use of MS as detector in GC was developed during the 1950s by Roland Gohlke & Fred
McLafferty. GC has an exceptional potential of separating the natural & synthetic organic compounds while
MS provide a definite structural information. Due to the high sensitivity & fast scan speed of MS, its
combination with GC can provide a very powerful analytical technique for the identification of components.
Mass spectra can be obtained for every eluted component even in nanogram quantity & eluted for a period
of only a few seconds.
GC & MS are quite compatible & are well matched in almost every aspects, except the pressure
difference at column outlet in gas chromatograph & at ionization chamber in mass spectrometer. Hence, the
volume of carrier gas must be reduced before it enters the ionization chamber, that can be solved by using
interface. Interface removes most of the carrier gas & introduces the remaining gas enriched with the sample
components into the ionization chamber.
The commonly used interface is “all glass single stage jet separator” in which elute from the GC
column is pushed through a nozzle. The low mol wt carrier gas e.g., helium, diffuses outward more rapidly
than higher mol wt organic compounds. A bulk of carrier gas is pumped out, while sample-enriched carrier
gas is accepted by another nozzle opposite to first one. The temperature of the interface is maintained at
about 250˚c to prevent condensation of the sample components.
APPLICATIONS OF GC-MS
1. Environmental Monitoring & Cleanup: GC-MS is becoming the tool of choice for tracking organic
pollutants in the environment.
2. Criminal Forensics: GC-MS analyze the particles from a human body in order to help the link of
criminal to crime.
3. Law enforcement: GC-MS is increasingly used for the detection of illegal narcotics & it is
commonly used in forensic toxicology to find drugs & poisons in biological specimens of suspect &
victims or deceased.
4. GC-MS has been used for testing of athletes for steroids, stimulants & other performance- enhancing
drugs.
5. Food, Beverage & Perfume Industry: GC-MS is extensively used for the analysis of these
compounds which contain esters, fatty acids, alcohols & aldehydes etc.
6. It is also used to detect & measure the harmful contaminants and adulterants in beverage & various
pharmaceutical formulations.

Capillary Electrophoresis
INTRODUCTION
Electrophoresis is a separation technique based on the differential rate of migration of charged
species in an applied dc electric field. This separation technique was first developed by the Swedish chemist
Arne Tiselius in the 1930s for the study of serum proteins. For many years, electrophoresis has been the
powerhouse method for separating proteins (enzymes, hormones, antibodies) & nucleic acids (DNA, RNA).
An electrophoretic separation is performed by injecting a small band of the sample into an aqueous
buffer solution contained in a narrow tube or on a flat porous support medium such as paper or a semisolid
gel. A high voltage is applied across the length of the buffer by means of a pair of electrodes located at each
end of the buffer. This field causes ions of the sample to migrate toward one or the other of the electrodes.
The rate of migration of a given species depends on its charge & its size. Separation based on differences in
charge-to-size ratios for the various analytes in a sample. The larger this ratio, the faster an ion migrates in
the electric field. The larger this ratio, the faster an ion migrates in the electric field.
TYPES OF ELECTROPHORESIS
1. Slab electrophoresis
2. Capillary electrophoresis
1. Slab electrophoresis:
It is used to separate complex, high-mol-

mass species of biological &
biochemical interest. Separation is carried out on a thin flat layer or slab of a porous semisolid gel
containing an aqueous buffer solution within its pores. Samples are introduced as spots on the slab, & a dc
electric field is applied across the slab for a fixed period. When the separations are completed, the field is
discontinued & species are visualized by staining method as in TLC.
2. Capillary Electrophoresis
Slab electrophoresis does not yield very precise

quantitative information. During the mid-to-late 1980s,
there was explosive growth in electrophoresis which
performed in capillary tubes.
Capillary electrophoresis (CE) yields high-speed, high-

resolution separations on exceptionally small sample
volumes (0.1 to 10nL in contrast to slab electrophoresis
µL range). Additionally, the eluted compounds are
quantitatively detected by suitable detector as in HPLC
instead, staining technique in slab E.

INSTRUMENTATION
A buffer-filled fused-silica capillary, typically 10 – 100 µm in internal diameter & 30 – 100 cm
long, extends between two buffer reservoirs that also hold platinum electrodes. The outside walls of the
fused-silica capillary are typically coated with polyimide for durability, flexibility, & stability.
The sample is introduced at one end & detection occurs at the other. A voltage of 5 to 30 kV dc is
applied across the two electrodes. Electrophoresis compartments are usually sealed off to protect the user.
Sample Introduction
 Electrokinetic injection
 Pressure injection
Electrokinetic injection:
One end of the capillary & its electrode are removed from their buffer compartment & placed in a small cup
containing sample. A voltage is applied, causing the sample to enter the capillary by ionic migration &
electro-osmotic flow. The capillary end & electrode are returned to the regular buffer solution for the
separation.
Pressure injection:
In pressure injection, the sample-introduction end of the capillary is placed in a small cup containing sample,
but here a pressure difference drives the sample solution into the capillary. The pressure difference can be
produced by applying a vacuum at the detector end.
For both electrokinetic & pressure injection techniques, the volume injected is controlled by the duration of
the injection. Injections of 5 to 50 nL are common, & volumes below 100 pL have been reported.
Detection (Detectors)
Separated analytes move to a common point in most types of CE. Detectors are similar in design & function
to the HPLC detectors.
Absorption
Methods:
Both fluorescence
& absorption
detectors are
widely used,
although
absorption
methods are more
common. Several
cell designs have
been used to
improve the

sensitivity of absorption methods. Three types of absorption improvement shown:
Fig.(a) The end of the capillary is bent to a Z shape, which produces a path length as long as ten times the
capillary diameter. Increases in path length can lead to better resolution.
Fig.(b) The absorption path length increases, when a bubble (150 µm diameter) is formed near the end of the
capillary, which gives a threefold increase in path length.
Fig.(c) In this technique, a reflective coating of silver is deposited on the end of the capillary.
The source radiation then undergoes multiple reflections during its transit through the capillary, which
significantly increase the path length.
Indirect Detection
Indirect absorbance detection has been used for species of low molar absorptivity that are difficult to detect
without derivatization. An ionic chromophore is placed in the electrophoresis buffer. The detector receives a
constant signal due to the presence of chromophore. The analyte displaces some of these ions, just as in ion-
exchange chromatography, So, the detector signal decreases during the passage of an analyte. The analyte
is then determined from the decreased absorbance.
Electrochemical Detection.
Two types of electrochemical detections are:
Conductivity & amperometry detectors;
One of the problems with electrochemical detection is that isolation of detector electrodes from the high
voltage (required for the separation). The isolation was done by inserting a porous glass between the end of
capillary & detector.
Mass Spectrometric Detection (CE-MS)

The very small volumetric flow rates of less than I µL/min from electrophoresis capillaries make it feasible
to couple the
effluent directly
to the ionization
source of a mass
spectrometer. A
typical
electrospray
interface coupled
to a quadrupole
mass
spectrometer.

THE BASIS FOR ELECTROPHORETIC SEPARATIONS:

The migration rate v of an ion (cm/s) in an electric field is equal to the field strength E (V cm‫־‬¹) & the
electrophoretic mobility .
Such as: Equation #1----
The electrophoretic mobility is in turn proportional to the ionic charge on an analyte & inversely
proportional to frictional retarding factors.
The electric field acts on only ions. If two species differ either in charge or in the frictional forces they will
be separated from each other while moving through the buffer. Neutral species are not separated. The
frictional retarding force on an analyte ion is determined by the size & shape of the ion & the viscosity of
the migration medium.
Migration rate in CE
As Equation 1 shows, the migration rate of an ion “v” depends on the electric field strength. The electric
field in turn is proportional to the magnitude of the applied voltage V & inversely proportional to the length
L. Thus: This relationship indicates that high applied voltages are desirable to achieve rapid ionic migration
& a fast separation.
Electro-osmotic flow
When a high voltage is applied across a fused-silica capillary tube containing a buffer solution,
electroosmotic flow usually occurs, in which the bulk liquid migrates (cations) toward the cathode. As a
result of electroosmosis, order of elution in a typical electrophoretic separation is, first, the fastest cation
followed by slower cations, then all the neutrals in a single zone, and finally the slowest anion followed by
successively faster anions.
APPLICATIONS
1. On a macro scale it has been applied for the analytical separation of amines, drugs, vitamins,
carbohydrates, peptides, proteins, nucleic acids, nucleotides, polynucleotides, and numerous other
species.
2. Its unique ability is to separate charged macromolecules of interest in biochemical, biological,
biomedical research & the biotechnology industry.
KEYS
Dead volume: Dead volume is the total volume of the liquid phase in the chromatographic column.
Elution: Passing of mobile phase through a stationary phase for separation.
Eluent: It is the solvent used to separate mixture of components (mobile phase).
Elute: Desired solute taken out from the chromatographic column.
Activation: just removing of moisture from the surface of adsorbent by heating, so the adsorbent activity is
retained.
Edge effect: solvent front in the middle of TLC plate move faster than edge.

Psi: pounds pressure per square inch.
Corrosion: destruction by the chemical action.
Plunger: a tool for clearing clogged drains, consisting of a rubber suction cup attached to a long handle.
Derivatization is a technique used in chemistry which transforms a chemical compound into a product of
similar chemical structure, called a derivative.
REFERENCES:
1. Remington's Pharmaceutical Sciences; The Philadelphia College of Pharmacy and Science, 1985,
Philadelphia, Pennsylvania.
2. Principles of Instrumental analysis By Douglas A Skoog
3. Undergraduate instrumental analysis By Robinson

DIFFERENTIAL SCANNING
CALORIMETRY
INTRODUCTION
Differential scanning calorimetry or DSC is a thermolytical technique in which the difference in the
amount of heat required to increase the temperature of the sample and reference is measured as a
function of temperature. Temperature is same for both sample and reference throughout the experiment.
This technique was developed by E.S Watson and M.J.O’ NEILL in 1962. It is the study of thermal
transition of polymer or sample (change that takes place on heating) and differential calorimeter is
instrument which measures the heat of sample relative to reference. Both the sample and reference are
maintained at same temperature.
ENDOTHERMIC REACTION
If sample absorbs some amount of heat during phase transition it is called endothermic reaction. In
endothermic more energy is required to maintain zero temperature difference between sample and reference.
E.g.: meting, boiling, vaporization.
EXOTHERMIC REACTION
If sample release some amount of heat during phase transition it is called exothermic reaction. E.g:
crystallization, degradation, polymerization.
PRINCIPLE
Energy necessary to maintain zero temperature difference between the sample and reference material as a
function of temperature. By heating 2 types of reactions take place i.e.; exothermic and endothermic
reactions.
INSTRUMENTATION
There are 2 types of instruments.
1. Power Compensated DSC

2. Heat Flux DSC
POWER COMPENSATED DSC

In this the temperature of the sample
and the reference are kept equal to each
other while both are increased or decreased. The power needed to maintain the temperature of sample equal
to the reference temperature is measured.
HEAT FLUX DSC

In this DSC the difference in heat flow into
the sample and reference is measured while
the sample temperature is changed at a
constant rate. Both the sample and
reference are heated by single heater .

Difference between DTA and DSC

In DSC, the difference in heat energy is measured
between the sample and reference. While in DTA, the
difference in the temperature is recorded.
FACTORS AFFECTING DSC CURVE

2 types of factors
INSTRUMENTAL
1. Furnace heating rate

2. Furnace atmosphere
3. Geometry of sample holder and composition of sample container
SAMPLE CHARACTERISTICS
1. Amount of sample
2. Nature of sample
3. Heat of reaction
4. Sample packing
APPLICATIONS OF DSC
1. Purity determination of sample
2. Detection of polymorphism
3. Detection of isomerism
4. Stability studies
5. Lyophilization studies

THERMO-GRAVIMETRIC ANALYSIS
INTRODUCTION:
In a Thermogravimetry or thermogravimetric analysis (TGA)
the mass (weight) of a sample in a controlled atmosphere is
recorded continuously as a function of temperature or time, as
the temperature of the sample is increased (linearly with time).
A
plot of mass or mass percentage as a function
of temp (time) is called a thermogram or a
thermal curve. An example of a TGA
(thermal) curve for the decomposition of
calcium carbonate is; Weight or weight% is
plotted along the y-axis & temperature (or
time for a linear temp. ramp) along the x-
axis.
The change in weight of a sample with temperature tells us several things;
1st Determines the temp. at which the material loses or gains weight. Loss of weight indicates
decomposition or evaporation of the sample. A gain in weight indicates adsorption of a component in the
atmosphere or a chemical reaction with the atmosphere.
2nd the temperatures at which no weight change takes place, indicates the temp stability of the
material. These weight changes at certain temperatures are physical properties of chemical compounds &
TGA is used to determine thermal stability, purity, quality, quantity & kinetic reaction of compounds at
certain temperature ranges.
TGA INSTRUMENTATION
Commercial instruments consists of:
(1) A sensitive microbalance (thermobalance).

(2) A furnace.
(3) A purge-gas system for providing an inert, or
sometimes reactive, atmosphere.
(4) A computer system for instrument control, data
acquisition & processing.
 The balance beam is shown as A.
 The sample cup & holder are B.
 C is a counterweight.
 D is a lamp & photodiodes.
 E is a magnetic coil.

 F is a permanent magnet.
 The computer data-acquisition, data processing, & control systems are G, H, & 1.
 Component J is the printer & display unit.
Thermobalance
A number of designs available commercially, providing quantitative information about samples ranging in
mass from less than I mg to 100 g. Many balances can detect changes in mass as small as 0.1 µg. The
sample holder must be held in the furnace, the rest of the balance must be thermally isolated from the
furnace.
A change in sample mass causes a deflection of the beam, which throw in a light shutter between a lamp &
one of two photodiodes. The resulting imbalance in the photodiode current is amplified & fed into coil E,
which is situated between the poles of a permanent magnet F. The amplified photodiode current is
monitored & transformed into mass (gain or loss) information by the data-processing system.
Furnace
The furnace surrounds the sample & sample holder is programmed for a linear heating rate. Modern
instruments can be heated & cooled rapidly. Commercial instrument can heat at rates up to 200˚C/min from
room temperature to about 1200˚C; cooling by forced air can be done at 50˚C/min. Furnaces with upper
temperatures limits of 1500˚C, 1700˚C, or 2400˚C are also available.
The furnace purged with a desired gas, to provide correct atmosphere for the experiment & to remove gases
from the compartment. Argon or nitrogen is used when an inert atmosphere is required, air is often used for
oxidation & combustion studies & hydrogen gas may be used to provide a reducing atmosphere. Modern
instruments permit the purge gas to be switched automatically, so the sample can be heated in an inert
atmosphere & be switched to air or other reactive gas at high temperatures.
Sample Holder
Samples are typically contained in sample pans made of platinum or aluminum. Platinum is most often used
because of its inertness & ease of cleaning. The volume of sample pan ranges from 40 µL to more than 500
µL and majority of samplers are automated under software control.
Temperature Control & Data Processing

The temperature recorded in a thermogram is ideally the actual temperature of the sample. This temperature
can be attained by immersing a small thermocouple directly in the sample or located close to the sample
container. Modern TGA systems use a computerized temperature control thermocouple with a voltage
versus-temperature table stored in computer memory. In some systems the same thermocouple behaves as
the heating element and the temperature sensor.

COMBINED THERMAL INSTRUMENTS

COMBINED THERMAL INSTRUMENTS
Several manufacturers offer systems that provide simultaneous measurement of heat flow & mass change.
Such instruments can not only track the loss of material or vaporization phenomenon with temperature but
also reveal transitions associated with these processes.
TGA/MS & TGA/FTlR

TGA is used to determine the loss in mass at particular temperatures, but TGA cannot identify the species
present. To obtain this type of information, the output of a thermogravimetric analyzer is often connected to
a Fourier transform infrared (FTIR) or a mass spectrometer (MS).
Several instruments offer devices to interface the TGA unit to a spectrometer. Some even claim true
combination of the software & hardware of the TGA/MS or TGA/FTIR systems.
ANALYTICAL APPLICATIONS OF THERMOGRAVIMETRY

(1) One of the first important applications of thermogravimetry was the determination of correct drying
temperatures for precipitates used in gravimetric analysis.
(2) A second important application was the identification of the gases given off while a sample’s
temperature is increased.
(3) In addition, the composition of the residue can be determined using techniques such as XRD, XRF,
and other techniques.
(4) TGA is very important in determining the upper use temperatures of materials such as polymers by
identifying the temperature at which oxidative degradation occurs on heating in air.
Examples
TGA thermal curve for pure

anhydrous calcium carbonate,
CaCO3 . The loss in mass is due
to the loss of CO2 (g) & the
compound remaining is CaO.
TGA thermal curve of hydrated

calcium oxalate, Ca(COO)2.xH 2
O of both adsorbed &
crystallized water. There is a loss of adsorbed water starting at about 90˚C, & loss of bound water at about
150˚C. The stable compound above 225˚C is anhydrous calcium
oxalate, Ca(COO)2. This loses CO at about 450˚C to form CaCO3 .
The calcium carbonate is stable until approx. 600˚C, when it loses
CO2 to form CaO.

KEY & REFERENCES

(1) Principles of Instrumental analysis by Douglas A. Skoog 6 th Edition.
(2) Undergraduate instrumental analysis by James W. Robinson 6 th Edition.
 XRD: X-ray diffraction
 XRF: X-ray fluorescence

DIFFERENTIAL THERMAL ANALYSIS

DEFINITION
Differential thermal analysis is a technique in which difference in temperature between a substance
and a reference material is measured as a function of temperature while the substance and reference
material are subjected to a controlled temperature program.
INTRODUCTION
Heating the sample and reference material in such a way that the temperature of the sample (Ts) increase
linearly with time. The temperature of reference material is denoted by ( Tr ) The difference in temperature
∆T=Ts – Tr is monitored and plot against sample temperature to give a differential thermogram
PRINCIPLE
The basic principle involve in DTA is the temperature
difference ∆T between the test sample and an inert reference
sample under controlled and identical condition of heating or
cooling is recorded continuously as a function of time.
Glass Transition temperature

Initial decrease in ∆T is due to glass transition. The glass
transition temp Tg is the characteristic temperature at which
glass amorphous polymers become flexible or rubber like. Transition involves no absorption or evolution of
heat so that no change in enthalpy that is ∆H=0. Heat capacity of rubber is different from glass which result
in lowering of the baseline No peak appear during this transition due to zero 0 enthalpy change The
difference in temperature is called differential temperature ∆T is plotted against temperature.
Physical changes usually results in a endothermic peak whereas chemical reactions those of an oxidative
nature are exothermic
Exothermic Reaction
Exothermic reaction (liberation of energy) includes oxidation, polymerization, crystallization and gives
upward peak.
Endothermic Reaction
Endothermic reaction (absorption of energy) includes vapourization , sublimation, absorption, & gives
downword peak.
The third peak is exothermic and encountered only if the heating is performed in the presence of air or
oxygen. This peak is the result of exothermic oxidation of the polymer.
Endothermic Decomposition
Final negative change in ∆T is due to endothermic decomposition of the polymer.
FACTORS AFFECTING DTA Curve

Two types
1- Instrumental factors
2- Sample characteristics
INSTRUMENTAL FACTORS
1. Size & shape of the sample and furnace holder
2. Material from which sample holder is made
3. Heating rate (Furnace heating rate)
SAMPLE CHARACTERISTICS
1. Amount of the sample ( Sample weight)

2. Particle size of the Sample
INSTRUMENTATION
1. Sample & reference holder
2. Sample & reference chamber
3. Furnace
4. Temperature programmer
5. Amplifier and recorder
SAMPLE & REFERENCE HOLDER

Sample and reference substances are contained in small aluminum dishes located above sample and
reference thermocouples in an electrically heated furnace. The reference material is an inert sub stance such
as alumina and silicon carbide.
SAMPLE & REFERENCE CHAMBER

It is designed to permit the circulation of an inert gas such as nitrogen or a reactive gas such as oxygen.
FURNACE
Furnace electrically provides heat through furnace heater leads. It should provide a stable and sufficient
large hot zone.
TEMPERATURE PROGRAMMER
 It is essential in order to obtain constant heating rate.
AMPLIFIER & RECORDER
APPLICATIONS
1. Identification of substances
2. Identification of products
3. Melting points

4. Quantitative analysis
5. Quality Control
6. Study of stability of pharmaceutical product.
Identification of substances
We know that the DTA curve for 2 substances is not identical. Therefore, these serve as finger prints for
various substances. Particularly, DTA has become an established technique for the identification of clays.
Identification of products
When a substance reacts with another substance, the products are identified by their specific DTA curves.
Therefore, this technique has been termed Reaction DTA.
Melting Points
Melting points can easily be determined so DTA can be used as a direct check of purity of compound
Quantitative analysis
The area of DTA peak is proportional to the total heat of reaction and hence to the weight of sample.
Therefore quantitative analysis is possible with help of standard curves of peak area vs weight.
Quality control
DTA technique has been widely used for the quality control of a large number of substances like Cement,
Glass, Soil, Catalysts, Textiles, Explosives, Resins etc.

RADIO PHARMACEUTICALS
(Radio Chemical Techniques)

RADIO PHARMACEUTICALS
Terminology:
Isotopes: The atoms of same element have same atomic number but different mass number. e.g., 12C6 , 13C6
, 14C6 .
Isobar: The atoms of different elements have different atomic number but same mass number. e.g., 40K19,
40
Ca20.
Isotones: The atoms of different elements have different atomic number but same number of protons. e.g.,
14
C6, 15N7 .
Half-life: It is time required to disintegrate to half of its active isotope.
TYPES OF RADIOISOTOPES (RADIONUCLIDES):

i- Natural radionuclides: Substances having high atomic wt (>80) & produce radiation without stimulation.
ii- Artificial radionuclides: Originally inert but become active when bombarded with controlled radiations.
TYPES OF RADIOACTIVE RADIATIONS:

α-particles: These are identical to Helium with least penetrating power & more ionization capacity.
β-particles: These are +ly charged (positron), can penetrate to the tissue up to 1cm.
γ- radiation: These are electromagnetic radiation (visible light), have shorter λ but high energy with great
penetrating power (neutral).
Units of Radio-activity
Curie: This is equal to 1 gm of radium which undergoes 3.7x10¹º disintegration per sec.
Milli-Curie (mc): It is 10‾³ of Curie.
Micro-Curie (µC): This is 10-6 of Curie.
Bacquerel ( Bq): One disintegration per sec.
Electron Volt (eV): It is the KE acquired by e which is accelerated through a PD of 1 volt.
BIOLOGICAL EFFECT OF RADIATION

 Radiations have very dangerous effect on biological tissue depending upon their ability to penetrate,
energy of radiation, surface area & type of tissue.
 These changes initiate free radical chain reactions that form different toxic compounds, that leads to
necrosis as well as alteration of DNA molecules in body.
 The most common side effects of radiations are nausea & vomiting.
MEASUREMENT OF RADIATIONS
Radiation detectors are divided into 2 major groups depending on:
A. Collection of ions
B. Collection of photons.
A. Detectors utilizing ion-collection:
1. Gas filled-detectors
i. Electroscopes
ii.Ion-chambers
iii. Proportional-counters
iv. Geiger-muller counters
2. Solid state detectors
i. Barrier-layer detector
ii. Lithium-drifted detector
B. Photon-collection detector(scintillation detector).
i. Inorganic scintillators
ii. Organic scintillators
iii. Photographic-emulsions
iv. Cerenkov detector
v. Thermo-luminescence dosimeters
vi. Track-etch detector
Ion chamber detector

 Based upon the creation of current by direct ionisation within the gas, which may be He, Ar, air etc.
 It consists of gas filled chamber fitted with 2 electrodes & an electric current measurindevice.
 These are used as radiation monitors/dosimeters etc.
Geiger-Muller Counter (G.M.counter)

 It consists of a stainless steel cylinder body which also act as cathode, while anode is a fine mounted
wire.
 A special gas mixture is filled between electrodes & due to ionization process +ly charged ions are
produced.
 Each particle of radiations cause a brief pulse current which is recorded by a device (scaler) that
indicate total number of particle.
 It is very efficient for detection of β-particles.
Scintillation-Detectors
Organic Scintillators: The most widely used are anthracene (high efficiency) & stilbene (low efficiency).
Plastic scintillators with 100% efficiency & fast response are very useful in nuclear spectroscopy.
Inorganic Scintillators: NaI(Ti) is the widely used scintillator for γ-counting due to its availability in
desired shape & size. Other scintillators are CsI(Ti), LiI(Eu), BaF 2 , CsI(Na) are used.
Handling & Storage of Radioactive materials

Radioactive materials may have harmful effects & lead to serious complications in the body. Since,
following precautions are taken while working with radio detector, radio assays, manufacturing or handling
of radioactive compounds.
i. Should be handled with suitable instrument & direct contact should be avoided.
ii. Food, drinks & smoking should be avoided in laboratory.
iii. Sufficient shielding must be provided over clothing.
iv. Should be stored in lead bricks container in a remote corner.
v. Radioactivity of the surrounding environment should be monitored constantly.
vi. Final disposal of radioactives should be done with great care to animals & environment.
APPLICATION OF RADIOPHARMACEUTICALS
Therapeutic Radiopharmaceuticals:
1. Chromic phosphate P32 for lung, ovarian, uterine, & prostate cancers.
2. Sodium iodide I 131 for thyroid cancer.
3. Samarium Sm 153 for cancerous bone tissue.
4. Sodium phosphate P 32 for polycythemia.
5. Stronium chloride Sr 89 for cancerous bone tissue.
6. Chromic phosphate P 32 is a suspension that is delivered through a catheter, or tube inserted into the
sac surrounding the lungs, or into the abdominal or pelvic cavities.
7. The usual dose is 15-20 mc for abdominal administration & 10 mc for lung sac.
8. It may be injected into the ovaries or prostate.
9. Sodium Iodide I 131 is taken by mouth as a capsule or a solution. The usual dose for treating thyroid
cancer is 30-200 mc, depending on age and body size.
10. Treatment usually requires two to three days of hospitalization. TSH should be injected prior to
treatment.
Diagnostic Radiopharmaceuticals:
1. Chromated albumin Cr51: for detection of protein loss & placenta localizaion.
2. Ferric salt is used in lung imaging but ferrous citrate Fe59 is used for determination of metabolism of
iron.
3. Cyanocobalamin Co60: for absorption & deposition of vit.B12 in megaloblastic anemia.
4. Sterlization: Cobalt 60 is used for the sterlization of syringes, needles, gloves & clothes etc.
References
Pharmaceutical Chemistry By Anil Bhandari
KEYS
Radio frequency: frequencies of electromagnetic radiation in the range between 10 kHz and 300 MHz
Dosimeter: radiation measuring instrument.
Scintillation: emissions
Eu: europium
Ti: titanium
Anthracene: Crystalline solid made from coal tar. Crystalline solid used in manufacturing dyes.

POLAROGRAPHY
VOLTAMETRY
An electrochemical method in which we measure current as a function of the applied potential. It
is an analytical technique which is formerly used only to the metals but now it is used for many non-metals,
anions, & organic substances. The requirement of the species to be analyzed is that it is reducible &
oxidizable within operating range of dropping mercury electrode (such as + 0.2 V to -2 V) vs saturated
calomel electrode (SCE).
Techniques which come under the general heading of voltammetry are:
(a) Polarography (d.c. and a.c.);

(b) Anodic stripping voltammetry;
(c) Chronopotentiometry
POLAROGRAPHY
Polarography is the study of the relationship between the current flowing through a conducting
solution & the voltage applied to a dropping mercury electrode (DME).
This method of analysis was invented by
Heyrovsky in 1922. It is used to study ions in a solution that compares the strength of electric current
passing through the solution during electrolysis. The sample in suitable solution is placed in a special
electrolytic cell which contains a polarisable &
a non-polarisable electrode. A gradually
increasing voltage is applied to the electrodes
of the cell, the corresponding current is
measured & the results plotted graphically.
The current-voltage (cv) curve, which has a

characteristic S-shape , may be used for the
qualitative & quantitative analysis of electro-
reducible & electro-oxidisable substances or
ions. The curve is known as a polarogram &
the electrical apparatus called polarograph. In
typical cv curve, i r is the residual current, I
the current at any point on the wave measured with respect to the line AB produced, & i d the diffusion
current. The diffusion current is proportional to the concentration of electro-reducible ions present & thus
forms the basis of quantitative polarography. E , the half-wave potential which produces a current at C along
the steeply rising portion of the curve, is characteristic of reduced system & enables qualitative analyses.
ELECTRODE SPECIFIC FOR “POLAROGRAPHY”

Dropping Mercury Electrode (DME)

The apparatus used in polarography is

basically very simple. It consists of a
potentiometer connected to two
electrodes via a microammeter. Many
types of electrode systems are available
but the simplest & most convenient is a
dropping mercury electrode, which
consists of a mercury reservoir
connected to a short length of fine
capillary tubing (20-80 µm diameter) in
conjunction with a mercury pool anode.
The basic features of the apparatus, R 1

is a shunt for the galvanometer & R 2 is
often a calibrated potentiometer.
Alternatively an uncalibrated potentiometer may be used, in which case the applied voltage, V, may be measured by
means of a high resistance voltmeter.
Care of Dropping Mercury Electrode:

1) Clean, dry & dust free tubing should be used.
2) Pure, double or triple distilled mercury should be employed.
3) The conventional DME should be mounted within ± 5° at vertical.
4) The capillary assembly should be mounted on a heavy stand & on a bench free from vibrations, to prevent
premature displacement of the mercury drops.
5) The capillary should be protected from dust by keeping the tip of capillary immersed in water when not in
use.
6) The mercury reservoir must not be lowered unless the capillary tip has been washed & immersed in
water.
7) The tip should be clean by immersing periodically in 50% v/v nitric acid with the mercury flowing & then
washing thoroughly with a jet of water.
Advantages of the DME:

i) It has a smooth & continually renewable surface exposed to the solution being analyzed.
ii) Each drop formed is unaffected by the reactions which occurred at the surface.
iii) Mercury amalgamates readily with most metals.
iv) The high voltage of Hg enables analyses in acid solution.
v) The diffusion equilibrium at mercury-solution interface is rapidly attained.
Disadvantages of the DME:

i) Mercury has a limited application in more positive potential range.

ii) The surface area of the drop is never constant.
iii) Changes in the applied voltage produce changes in the surface tension of mercury & therefore, changes
in drop size.
iv) The addition of surface active agents produces changes in drop size.
v) Mercury may be toxic in certain biological studies.
ILKOVIC EQUAITON
Ilkovic assumed the following equation known as Ilkovic equation.
Imax = 607 n D1/2 C m2/3 t1/6
Where
i max = Maximum current during the life of a mercury drop measured in microamperes (1µA=10-6 amperes)
n = Number of electrons involved in reduction of one molecule of reducible substance.
D = Diffusion coefficient (cm2/sec)
C = Concentration (mill moles/liter)
m = Weight of mercury flowing through the capillary (mg/sec)
t = Time (sec) necessary for formation of one drop of mercury, normally between 2 & 7 sec.
The value 607 is a combination of numerical constant which is density of mercury. In practice, the average
current is usually measured with ammeter which cannot follow the fluctuation which occurs. Under such
conditions the average diffusion current i d given by the expression as:
I d = 6/7 i max
According to Ilkovic equation with all other factors constant, it will be noted that diffusion current is
proportional to concentration:
I d = Kc
Where K is a constant.
This linear relationship may fail when the drop time is too short, owing to the stirring effect disturbing the
diffusion layer & producing an abnormally large current. However, the addition of small quantity of gelatin,
which increase the viscosity, often counteracts this effect.
The term m t in Ilkovic equation describes the capillary characteristic provided the drop time of capillary is
within the usual limits. It will be appreciated that capillaries used in various laboratories will be different
with respect to:

Bore, Length, Reservoir height.
Thus a knowledge of m & t will enable comparison to be made between different capillaries.
APPLICATIONS OF POLAROGRAPHY:
1) The polarographic method of analysis is applicable to most metals & many organic compounds.
2) Environmental studies are now becoming increasingly important & metals such as lead & cadmium in
samples are readily determined by polarographic analysis.
3) Oxygen can also be determined polarographically to monitor the extent of river pollution.
4) Furazolidone in veterinary feedstuffs can be determined because of nitro group present.
5) Drug metabolites may be determined readily in plasma, urine, bile & saliva by polarographic methods.
6) Some compounds which are polarographically inactive may be studied after preparing a suitable
derivatives. e.g., Chlropromazine treated with bromine, aromatic hydrocarbons require nitration &
certain alkaloids &hormones require nitrosation before polarographic analysis.
References
1) Practical Pharmaceutical chemistry by “A.H. Beckett”.
2) Principles of instrumental analysis by “Skoog”.
KEYS
Amalgamate: to alloy a metal with mercury, or be alloyed with mercury.
Voltmetry: Measuring the electromotive force or potential difference between two points in a circuit.
Potentiomtery: Measuring an unknown potential difference or electromotive force by comparing it to a

known standard.

POTENTIOMETRY
POTENTIOMETRY
Potentiometry involves measurement of the potential, or voltage, of an electrochemical cell. Accurate
determination of the potential developed by a cell requires a negligible current flow during measurement. A
flow of current would mean that a faradaic reaction is taking place, which would change the potential.
Measurement of the potential is accomplished by

connecting the indicator electrode to a reference half-cell
to form a complete cell as shown in Fig.
The total voltage of the cell is measured;
the difference between the voltage of the
reference electrode to the voltage of the
indicator electrode.
E(total) = E(indicator) – E(reference)
The common reference electrodes used in potentiometry are the saturated calamel elcd (SCE) & the
silver/silver chloride electrode. Their potentials are fixed & known over a
wide temperature range. The choice of reference electrode depends on the application. For example, the
Ag/AgCl electrode cannot be used in solutions containing halides or sulfides that will precipitate or react
with silver.
REFERENCE ELECTRODES
The ideal reference electrode has a known potential, constant & completely insensitive to the composition of
the solution under study. In addition this electrode should be rugged, easy to assemble & should maintain a
constant potential even when there is a net current in the cell.
Types: Standard Hydrogen Electrode, Saturated Calomel Electrode, Silver-Silver Chloride Electrode.
Standard Hydrogen Electrode

It is universally accepted reference electrode which consists of a small piece of platinum foil coated with a
finely divided platinum called platinum black. The coated foil is immersed in an acidic medium having
hydrogen ion activity of 0.1 & through which H2 gas is bubbled at a partial pressure of 1 atm.
The pt-black-foil possess a large surface area enabling absorption of appreciable amount of H2 gas,
ultimately bringing it into direct contact with the surrounding H ions at the electrode surface. It is used for
redox titration.
2. Saturated Calomel Electrode (SCE)

It is most widely used reference electrode because of its potential constancy & ease of preparation. SCEs
consist of mercury in contact with a solution that is saturated with mercury chloride (calomel) & also
contains a known concentration of KCl. Calomel half-cells can be represented as follows:
Hg[Hg2Cl2 (sat’d), KCl(xM)]
where x represents the molar concentration of potassium chloride in the solution. The electrode potential for
this half-cell is determined by the reaction
Hg2Cl2 (s) + 2e¯ REVERSIBLE REACTION 2Hg + 2Cl¯
& depends on the chloride concentration x. Thus, the KCl concentration must be specified in describing the
electrode. The potential for the calomel electrode (vs hydrogen E) in saturated N KCl is 250 mV at 20°.
Saturated Calomel
3. Silver-Silver Chloride Electrode

The various components of a silver-silver chloride electrode are, namely:
A = Porous ceramic fiber,
B = Ag wire coated with AgCl,
C = 1 M KCl sat’d with AgCl,
D = Fill-hole,
E = Electrical lead.
SSCE is difficult to prepare but convenient to use as calomel electrode. It consists

of a silver wire coated with silver chloride (B) and is duly placed in a 1M KCl
solution saturated with AgCl (C). The potential of the silver-silver chloride
electrode (vs normal hydrogen electrode) in saturated N KCl is +200 mV at 25°.
INDICATOR ELECTRODES
An ideal indicator electrode responds rapidly & reproducibly to changes in activity of the analyte ion.
Although no indicator electrode is absolutely specific in its response, a few are now available that are
remarkably selective. There are two types of indicator electrodes: metal & membrane indicator electrodes.
1. Metallic Indicator Electrodes

Metal indicator electrodes develop a potential which is usually determined by the equilibrium position of a
redox reaction at the electrode surface. These are further classified into the following three types,
i. First order electrodes
ii. Second order electrodes
iii. Inert electrodes.
i. First order electrodes:
It is just a metal wire, mesh, or solid strip that responds to its own cation in solution. Such as:
Cu/Cu²+, Ag/Ag+, Hg/Hg+, and Pb/Pb²+.

Problems encountered with these electrodes. They have poor selectivity & responding not only to their own
cation but also to any other more easily reduced cation. Some metal surfaces are easily oxidized, giving
inaccurate response.
Some metals can only be used in limited Ph ranges because they will dissolve in acids or bases. Silver &
mercury are the most commonly used electrodes of the this kind.
ii. Second order electrodes:
It consists of a metal coated with one of its sparingly soluble salts. This electrode responds to the anion of
the salt. For example, a silver wire coated with AgCl will respond to changes in chloride activity because the
silver ion activity is controlled by the solubility of AgCl. The electrode reaction is then written as:
AgCl (s) Ag + Cl or
K sp = [Ag ] [Cl ]
iii. Inert electrodes.
Inert electrodes comprise of chemically inert conductors, for instance: Au, Pt & C which do not necessarily
take part either directly or indirectly in the various redox processes. However, the potential developed at an
inert electrode solely depends upon both the nature and prevailing concentration of different redox-reagents
present in the solution.
For example: Pt-electrode placed in a solution consisting of both Fe & Fe ions develops a potential which is
duly represented by the Nernst equation for ions as given below.
NERNST EQUATION
E = E° Fe3+ /Fe2+ - log
2. MEMBRANE INDICATOR ELECTRODES

A wide variety of membrane electrodes are available from commercial sources that permit the rapid &
selective determination of numerous cations & anions by direct potentiometric measurements. Often,
membrane electrodes are called ion selective electrodes because of the high selectivity of most of these
devices.
Types of membrane indicator electrodes

Glass Membrane Electrodes, Polymer Membrane Electrodes, Crystalline Membrane Electrodes, Gas-
Sensing Electrode.
i) Glass Membrane Electrodes:
As shown in the figure, the internal element essentially comprises of a Ag-AgCl electrode
(B) dipped in a pH 7 buffer saturated with AgCl (A). The thin, ion-selective glass membrane (I) is carefully
fused to the bottom of a high resistance non-responsive glass tube (H) so that the entire membrane may be
immersed while taking measurements.

Various components are as follows:
A = pH 7 buffer solution sat’d with AgCl
B = Ag-AgCl internal reference electrode
C = Mercury connection
D = Connecting wire
E = Shield
F = Cap
G = Shielded & insulated connecting wire
H = High resistance non-responsive glass
I = Selective glass membrane
Glass Membrane Electrode
The half-cell of glass-membrane electrode may be expressed as:
Ag (s) | AgCl (saturated), Cl (inside), H+ (inside) | glass membrane | H+ (outside)
According to the Nernst equation, the potential of the electrode is represented by:
[ ]
E = E° AgCl/Ag - log [Cl-] + log [ ]
ii) Crystalline Membrane Electrodes

The crystalline membrane electrodes have a very close similarity to those of GME except that glass has
been replaced with crystalline membrane.
Example: Fluoride-ion Electrode: the membrane comprises of a single crystal of lanthanum fluoride (LaF3 )
usually doped with a slight trace of europium (II) (Eu) 2+
The potential developed at each surface of the membrane is finally determined by the exact status of the
equilibrium.
LaF3 La + 3F¯
Membrane Membrane Aqueous
iii) Gas-Sensing Electrode
The schematic diagram of a gas-sensing electrode is illustrated in fig., that comprises of reference electrode
(E), a specific-ion electrode (B). An internal electrolyte solution (F) contained in a cylindrical plastic tube
(G). One end of the plastic tubing is provided with a thin, replaceable, gas-permeable membrane that
separates the internal electrolyte solution from the external solution containing gaseous analyte.
Various components of GSE are as:
A = Glass permeable membrane
B = Specific ion electrode (glass)
C = Ring to hold the membrane
D = External solution containing dissolved gaseous analyte
E = Reference electrode (Ag/AgCl)
F = Internal electrolyte solution
G = Plastic tube.
Gass-sensing electrode
References
3) Principles of instrumental analysis by “Skoog”.
4) Practical Pharmaceutical chemistry by “A.H. Beckett”.
5) Pharmaceutical drug analysis by “Ashutoshkar”.
KEYS
Nernst equation:The Nernst equation defines the relationship between cell potential to standard potential and to
the activities of the electrically active species.
Au: angstrum.
Faradaic reaction: with irregular alternating current.
Contact us:
THANKS
Your Feedback Highly Appreciated. We will add your name as Editor if you highlight mistakes or missing
topic in these notes.
ADNAN SARWAR CHAUDHARY
Email: deenasaan@gmail.comm 03041038728
SAAD MUHAMMAD RUSTAM
Email: saad.m.rustam@gmail.com 03034565606

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2nd Edition

‫بسم ہللا الرحمن الرحیم‬

2 Adnan Sarwar Chaudhary & Saad Muhammad Rustam

FLAME EMISSION SPECTROMETRY: 12

THEORY & PRINCIPLE: 19

APPLICATIONS OF INFRARED SPECTROSCOPY: 28

MOLECULAR FLUORESCENCE SPECTROSCOPY 30

NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (NMR) 33

4 Adnan Sarwar Chaudhary & Saad Muhammad Rustam

ADVANTAGES OF COLUMN CHROMATOGRAPHY: 51

THIN LAYER CHROMATOGRAPHY (TLC) 52

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 65

GAS CHROMATOGRAPHY –MASS SPECTROMETRY (GC-MS) 73

GAS CHROMATOGRAPHY – MASS SPECTROMETRY 73

DIFFERENTIAL SCANNING CALORIMETRY 79

COMBINED THERMAL INSTRUMENTS 83

COMBINED THERMAL INSTRUMENTS 83

DIFFERENTIAL THERMAL ANALYSIS 85

CONTACT US: 100

Adnan Sarwar Chaudhary & Saad Muhammad Rustam 7

ATOMIC ABSORPTION & EMISSION

ELEMENTS DETECTED BY AAS: ( IN DARK)

8 Adnan Sarwar Chaudhary & Saad Muhammad Rustam

The process of atomic absorption spectroscopy (AAS) involves two steps:

1. Atomization of the sample

Adnan Sarwar Chaudhary & Saad Muhammad Rustam 9

Types of Sources include:

1) Electrode – less discharge Lamp

Electrode-less discharge lamp:

Microwave Electrode – less discharge lamps

Radiofrequency Electrode – less discharge lamps

Hollow Cathode lamp

10 Adnan Sarwar Chaudhary & Saad Muhammad Rustam

 Cylindrical hollow cathode (made up of element to be determined or an alloy of it)

Adnan Sarwar Chaudhary & Saad Muhammad Rustam 11

FLAME EMISSION SPECTROMETRY:

12 Adnan Sarwar Chaudhary & Saad Muhammad Rustam

Adnan Sarwar Chaudhary & Saad Muhammad Rustam 13

4-Refractory compound formation (chemical interferences):

3. Environmental Study – determination of heavy metals in water, soil, and air.

5. Pharmaceutical – many applications from quality control to detecting impurities in drugs.

6. It is used in petroleum industry, to detect metallic impurities in petrol, lubricating oils.

14 Adnan Sarwar Chaudhary & Saad Muhammad Rustam

Flame photometry is based on measurement of intensity

The electrostatic force of attraction

Adnan Sarwar Chaudhary & Saad Muhammad Rustam 15

4. Photo Detector & Read out Device

Nebulizer and Mixing chamber:

Flame may be divided into the following regions or zones.

2. primary reaction zone or inner zone

4. secondary reaction zone

Preheating zone: In this, combustion mixture is heated to the ignition

Primary reaction zone: This zone is about 0.1 mm thick at atmospheric

I = Intensity of emitted light

c = The concentration of the element

n-1 ( At the linear part of the calibration curve)\

Optical System (Optical Filter ):

Photo Detector & Read out Device:

Such interference may be of 3 kinds:

Adnan Sarwar Chaudhary & Saad Muhammad Rustam 17

18 Adnan Sarwar Chaudhary & Saad Muhammad Rustam

THEORY & PRINCIPLE:

IR spectroscopy basically vibrational spectroscopy. During absorption of IR radiation only amplitude

VIBRATIONAL MODES & ABSORPTION FREQUENCIES

Adnan's Pharmaceutical Instrumentation by Adnan Sarwar Chaudhary - 2nd Edition

Adnan's Pharmaceutical Instrumentation by Adnan Sarwar Chaudhary - 2nd Edition