Bth 201 enzymology and biochemical techniques

PROF. BALASUBRAMANIAN SATHYAMURTHY 2016 EDITION BTH - 201: ENZYMOLOGY AND BIOCHEMICAL TECHNIQUES
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FOR MSC BIOTECHNOLOGY STUDENTS
2014 ONWARDS
Biochemistry scanner
THE IMPRINT
BTBTBTBTHHHH –––– 202020201111:::: ENZYMOLOGY ANDENZYMOLOGY ANDENZYMOLOGY ANDENZYMOLOGY AND BIOCHEMICAL TECHNIQUESBIOCHEMICAL TECHNIQUESBIOCHEMICAL TECHNIQUESBIOCHEMICAL TECHNIQUES
As per Bangalore University (CBCS) Syllabus
2016 Edition
BY: Prof. Balasubramanian Sathyamurthy
Supported By:
Ayesha Siddiqui
Kiran K.S.
THE MATERIALS FROM “THE IMPRINT (BIOCHEMISTRY SCANNER)” ARE
NOT FOR COMMERCIAL OR BRAND BUILDING. HENCE ONLY ACADEMIC
CONTENT WILL BE PRESENT INSIDE. WE THANK ALL THE
CONTRIBUTORS FOR ENCOURAGING THIS.
BE GOOD – DO GOOD & HELP OTHERS

DEDICATIODEDICATIODEDICATIODEDICATIONNNN
I dedicate this material to my spiritual guru Shri Raghavendra swamigal,I dedicate this material to my spiritual guru Shri Raghavendra swamigal,I dedicate this material to my spiritual guru Shri Raghavendra swamigal,I dedicate this material to my spiritual guru Shri Raghavendra swamigal,
parents, teachers, well wishers and students who always increase myparents, teachers, well wishers and students who always increase myparents, teachers, well wishers and students who always increase myparents, teachers, well wishers and students who always increase my
morale and confidence to share my knowledge to reach all beneficiaries.morale and confidence to share my knowledge to reach all beneficiaries.morale and confidence to share my knowledge to reach all beneficiaries.morale and confidence to share my knowledge to reach all beneficiaries.
PREFACEPREFACEPREFACEPREFACE
Biochemistry scanner ‘THE IMPRINT’ consists of last ten years solved
question paper of Bangalore University keeping in mind the syllabus and
examination pattern of the University. The content taken from the
reference books has been presented in a simple language for better
understanding.
The Author Prof. Balasubramanian Sathyamurthy has 15 years of teaching
experience and has taught in 5 Indian Universities including Bangalore
University and more than 20 students has got university ranking under his
guidance.
THE IMPRINT is a genuine effort by the students to help their peers with
their examinations with the strategy that has been successfully utilized by
them. These final year M.Sc students have proven their mettle in university
examinations and are College / University rank holders.
This is truly for the students, by the students. We thank all the contributors
for their valuable suggestion in bringing out this book. We hope this will be
appreciated by the students and teachers alike. Suggestions are welcomed.
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CONTRIBUTORS:
CHETAN ABBUR ANJALI TIWARI
AASHITA SINHA ASHWINI BELLATTI
BHARATH K CHAITHRA
GADIPARTHI VAMSEEKRISHNA KALYAN BANERJEE
KAMALA KISHORE
KIRAN KIRAN H.R
KRUTHI PRABAKAR KRUPA S
LATHA M MAMATA
MADHU PRAKASHHA G D MANJUNATH .B.P
NAYAB RASOOL S NAVYA KUCHARLAPATI
NEHA SHARIFF DIVYA DUBEY
NOOR AYESHA M PAYAL BANERJEE
POONAM PANCHAL PRAVEEN
PRAKASH K J M PRADEEP.R
PURSHOTHAM PUPPALA DEEPTHI
RAGHUNATH REDDY V RAMYA S
RAVI RESHMA
RUBY SHA SALMA H.
SHWETHA B S SHILPI CHOUBEY
SOUMOUNDA DAS SURENDRA N
THUMMALA MANOJ UDAYASHRE. B
DEEPIKA SHARMA
EDITION : 2016
PRINT : Bangalore
CONTACT : theimprintbiochemistry@gmail.com or 9980494461

BANGALORE UNIVERSITY SYLLABUS (REVISED 2014)
M.SC BIOTECHNOLOGY II SEMESTER
BTH: 201 – ENZYMOLOGY AND BIOCHEMICAL TECHNIQUES
52 hrs
Unit – 1: Physical Techniques:
Principles and applications of Rayleigh scattering, viscometry. Absorption,
adsorption, crystallization, x-ray crystallography, spectrophotometry,
fluorimetry, flame photometry, mass spectroscopy.
Distillation, liquid – liquid extraction.
Centrifugation, differential, gradient, ultra centrifugation, salt fractionation,
and dialysis. 10Hours
Unit – 2: Chromatographic Techniques:
Principles and applications of -gel filtration- ion exchange chromatography-
thin layer, chromatography- affinity chromatography- gas liquid
chromatography, high performance liquid chromatography (HPLC).
8Hours
Unit – 3: Electrophoresis:
Principles and applications of moving boundary electrophoresis, zone
electrophoresis, gel electrophoresis-PAGE and SDS PAGE agarose gel
electrophoresis, isoelectric focusing and 2D Gel electrophoresis, pulsed field
electrophoresis. 6Hours
Unit – 4: Enzyme catalysis:
Introduction to enzymes; nomenclature and classification of enzymes; chemical
nature and properties of enzymes, activation energy, factors effecting enzyme
activities, active site, allosteric site, coenzymes and factors. Types of enzyme
specificity, units of enzyme activity. Strategies of purification of enzymes,
criteria of purity, molecular weight determination and characterization of
enzymes. Enzyme single and multi substrate reactions. Ping-pong mechanism,
sequential mechanism (ordered and random), enzyme models – host guest
complexation chemistry. 8Hours
Unit – 5: Enzyme kinetics and Mechanism of Enzyme catalysis:

Chemical kinetics, rate of reaction, order of reaction, zero order and first order.
Derivation of michaelis-menton equation, km value and its significance,
lineweaver-bunk plot. Velocity maximum. Mechanism of enzyme action, lock
and key model, induced fit hypothesis, substrate strain theory (with isozyme as
a typical example). Mechanism of enzyme catalysis – Acid-Base catalysis,
Covalent catalysis, metal ion catalysis and entropy effect. Enzyme inhibition-
reversible and irreversible, competitive, uncompetitive, non competitive.
Regulation of enzyme activity- Covalent modulation, Allosteric regulation,
ligand interactions, scatchard plot, co-operative interactions, feedback
regulation. Isozymes. 12Hours
Unit – 6: Coenzymes and their Mechanism of action:
Structure and mechanism of action of some important co-enzymes NAD+,
FAD, FMN, TPP, pyridoxal phosphate, lipoic acid, CoASH and vitamin B12.
8Hours
References:
1. Nelson, D.L., Cox, M.M. Lehninger. (2004). Principles of Biochemistry, 4th
edition Pub WH Freeman Co.
2. Daniel, L, Purich, Melvin, I. Simon, John, N., Abelson. (2000). Contemporary
enzyme kinetics and mechanism.
3. Plowman. (1972). Enzyme kinetics. McGraw hill.
4. Jack kite. (1995). Mechanisms in protein chemistry, Garland publishers.
5. Gerhartz, W. (1990). Enzymes in industry: Production and applications. VCH
publishers, NY.
6. Chaplin, M.F., Bucke, C. (1990). Enzyme technology. Cambridge university
press, Cambridge.
7. Belter, P.A., Cussier, E. (1985) Wiley Bio separations .
8. Asenjo, J. Dekker, M. (1993) Separation processes in biotechnology.
9. Upadhyay and Nath (2003). Biophysical chemistry, principles and techniques,
Himalaya publishing house.

Unit – 1 : Physical Techniques:
Principles and applications of Rayleigh scattering, viscometry.
Absorption, adsorption, crystallization, x-ray crystallography,
spectrophotometry, fluorimetry, flame photometry, mass spectroscopy.
Distillation, liquid – liquid extraction
Centrifugation, differential, gradient, ultra centrifugation, salt
fractionation, and dialysis.
PRINCIPLES AND APPLICATIONS OF RAYLEIGH SCATTERING
Rayleigh scattering is a physical phenomenon where light is scattered in
different directions by very small particles. These particles are much smaller
than the wavelength of the light involved and may even be as small as a single
atom. Rayleigh scattering is most commonly seen in gases although it can
occur in both liquids and solids. The amount of scattering present depends on
the polarizing properties of a particular type of particle and can vary depending
on the elements involved
PRINCIPLES OF RAYLEIGH SCATTERING:
Rayleigh scattering, named after the British physicist Lord Rayleigh, is the
elastic scattering of light or other electromagnetic radiation by particles much
smaller than the wavelength of the light. The particles may be individual atoms
or molecules.
It can occur when light travels through transparent solids and liquids, but is
most prominently seen in gases.
Rayleigh scattering is a function of the electric polarizability of the particles.
Rayleigh scattering of sunlight in the atmosphere causes diffuse sky radiation,
which is the reason for the blue color of the sky and the yellow tone of the sun
itself.
Scattering by particles similar to or larger than the wavelength of light is
typically treated by the Mie theory, the discrete dipole approximation and other
computational techniques.

Rayleigh scattering applies to particles that are small with respect to
wavelengths of light, and that are optically "soft" (i.e. with a refractive index
close to 1).
The size of a scattering particle is parameterized by the ratio x of its
characteristic dimension r and wavelength λ:
The amount of Rayleigh scattering that occurs for a beam of light depends
upon the size of the particles and the wavelength of the light. Specifically, the
intensity of the scattered light varies as the sixth power of the particle size, and
varies inversely with the fourth power of the wavelength. .
The intensity I of light scattered by a single small particle from a beam of
unpolarized light of wavelength λ and intensity I0 is given by:
where R is the distance to the particle, θ is the scattering angle, n is the
refractive index of the particle, and d is the diameter of the particle.
The Rayleigh scattering coefficient for a group of scattering particles is the
number of particles per unit volume N times the cross-section. As with all wave
effects, for incoherent scattering the scattered powers add arithmetically, while
for coherent scattering, such as if the particles are very near each other, the
fields add arithmetically and the sum must be squared to obtain the total
scattered power.
Applications:
Rayleigh scattering is the mechanism that causes the sky to be blue. When
sunlight travels through the atmosphere, it is scattered by particles that are
present. Some wavelengths of light, however, are scattered more than others.
In this case, blue light is scattered more efficiently and the sky appears blue

most of the time. The only exception is during sunset or sunrise where the
sun’s rays are passing directly through the atmosphere. In this case, the sky
appears redder as red light isn’t scattered as much as blue and can pass
through the atmosphere unaffected.
There are also several practical applications of Rayleigh scattering that are
used in modern technology. For example, the fact that light is scattered is used
in some optical fibers. For the optical fibers to function correctly there needs to
be some scattering of the optical signals and this is achieved using small
particles.
VISCOMETRY
Definition:
Viscosity is the resistance to flow which is a property of fluids, both liquids and
gases.
When a liquid flows through a tube, layers of liquid slide over each other and
intermolecular forces cause resistance to flow.
Ostwald viscometer is commonly used to determine the viscosity between the
fluids.
Principle:
When a liquid flows by gravity, the time required for the liquid to pass between
two marks, upper mark and lower mark, through a vertical capillary tube is
determined.
The time of flow of the liquid under test is compared with the time required for
a liquid of known viscosity (usually water).
The viscosity of unknown liquid η1 can be determined using the equation,
Where,
ρ1=Density of unknown liquid , ρ2= Density of known liquid,
t 1= Time of the unknown liquid, t 2= Time of the known liquid
η 2= Viscosity of known liquid

Instrumentation:
Procedure:
The Ostwald viscometer rinsed thoroughly many times with distilled water
dried with acetone initially as well as after each run. A constant temperature
water bath 25.C was done.
A clamp was set up so the viscometer is placed inside the water bath, so the
fiducial marks were visible below the water level.
10ml of distilled water was pipette in the viscometer.
Then at least 10 minutes was allowed for the water and viscometer to come to
thermal equilibrium.
With a rubber bulb, the water was drawn up well above the top fiducial mark.
The rubber bulb was removed and the liquid was running down the capillary of
the viscometer.
When the water level reached the top of the fiducial mark a timer was started.
When the water level reached the lower fiducial mark, the timer counter was
stoped.
The run was prepared two more times.
10ml of any one of the given solution was pipette into the viscometer.
The solutions then came to equilibrium and the time flow was measured as
before.
All time measurements were repeated at least 2 more runs for each solution
concentration.

It was made sure that after each run, the viscometer was cleaned with water
and then with acetone.
Applications:
Viscosity is one of the important physical properties of liquids. It helps to
determine the viscosity of any liquids.
ABSORPTION
Absorption is a physical or chemical phenomenon or a process in which atoms,
molecules, or ions enter some bulk phase - gas, liquid, or solid material.
Absorption is a condition in which something takes in another substance.
If absorption is a physical process not accompanied by any other physical or
chemical process, it usually follows the Nernst partition law:
"The ratio of concentrations of some solute species in two bulk phases in
contact is constant for a given solute and bulk phases"
Laboratory absorber. 1a): CO2 inlet; 1b): H2O inlet; 2): outlet; 3): absorption
column; 4): packing

ADSORPTION
Adsorption is the adhesion of atoms, ions, or molecules from a gas, liquid, or
dissolved solid to a surface. This process creates a film of the adsorbate on the
surface of the adsorbent
Application:
Adsorption, ion exchange, and chromatography are sorption processes in
which certain adsorbates are selectively transferred from the fluid phase to the
surface of insoluble, rigid particles suspended in a vessel or packed in a
column.
CRYSTALLIZATION
Crystallisation concept:
Protein solubility affected by adding "precipation agents" Eg. salt,
polyetheleneglycol etc.
In a controlled way take protein to supersaturation.
Adding percipitant.
Drying out the drop.
Exchanging the buffer (dialysis).
Wait & regulatly observe the experiment under a microscope.
Crystallization methods:
There are three principle crystallization methods currently in use.
Microdialysis: Solution containing desired conditions(external solution) diffuses
across the semi permeable membrane into the protein solution. Properties of
external solutions can be gradually varied leading to crystallization.
Hanging drop: Vapour diffusion method, sample is suspended as a hanging
drop (3-10µl) over a much larger volume of (eg:500µl) of a desired
crystallization condition.
Sitting drop vapour diffusion method: Sample (10-20µl) is placed in depression
formed in a 9 well plate. This is supported over the crystallization chamber.
Sample equilibrates with the crystallization solution through the vapour phase.

Applications: X – RAY CRYSTALLOGRAPHY
X – RAY CRYSTALLOGRAPHY
Introduction:
X-Rays are the form high energy electro-magnetic radiation with wave length in
range of 0.1-100 x 10ֿ¹º m.
Also called as Roentgen rays in honor of their discoverer, Wilhelm Roentgen.
For Crystallographic studies radiation of wave-length 1.53Å (also called Kα
rays) are selected.
X-ray having single wave length are desirable in crystallography because they
give single pattern of strong reflections.
BRAGG’S LAW:
In 1913 Bragg proposed that a crystal may be regarded as series of planes
which behave as a mirror reflecting x-ray.
In real crystal, these lattice planes cut through the crystal lattice in 3-
dimensions.
This lattice planes link corresponding atoms through the crystal lattice.
Miller indices allow identification of individual planes.
They are defined as 3 intercepts (h, k, l) that the plane makes with the cell
axes, in units of cell edge.
These planes must intersect the cell edges rationally, otherwise the diffraction
from the different unit cells would interfere destructively.
We can index them by the number of times h, k and l that they cut each edge.
BRAGG’S LAW OF DIFFRACTION:
X-ray incident on successive lattice planes at an angle θ are reflected when nλ
= 2d sinθ. When d is the distance between two planes this is because
successive lattice planes which obey Bragg’s law give rise to constructive
interference.
Bragg’s law make clear that there is a fixed relationship between the pattern of
Bragg reflections (X-ray diffraction pattern) obtained during x-ray diffraction
and the spacing atom within the crystal lattice.

Sources of X-ray:
Sealed tube generators
Rotating anode generators
Synchrotron
Crystallisation concept:
Protein solubility affected by adding "precipation agents" Eg. salt,
polyetheleneglycol etc.
In a controlled way take protein to supersaturation.
Adding percipitant.
Drying out the drop.
Exchanging the buffer (dialysis).
Wait & regulatly observe the experiment under a microscope.
Crystallization methods:
There are three principle crystallization methods currently in use.
Microdialysis: Solution containing desired conditions(external solution)
diffuses across the semi permeable membrane into the protein solution.
Properties of external solutions can be gradually varied leading to
crystallization.
Hanging drop: Vapour diffusion method, sample is suspended as a hanging
drop (3-10µl) over a much larger volume of (eg: 500µl) of a desired
crystallization condition.
Sitting drop vapour diffusion method: Sample (10-20µl) is placed in
depression formed in a 9 well plate. This is supported over the crystallization
chamber. Sample equilibrates with the crystallization solution through the
vapour phase.

Mounting of crystal:
Mounting crystals for diffraction in a format suitable for exposure to x-ray
beam.
Crystals of protein or DNA are far less stable than in-organic crystals.
Protein or DNA crystals are usually mounted in thin glass capillaries suitable
for assembling and rotation in the x-ray beam.
Mounted crystal can now assembled on a goniometer head in the apparatus
used for diffraction.
The basic physical principles:
Electrons scatter x-rays. The amplitude of the wave scattered by an atom is
proportional to its number of electrons. Thus, a carbon atom scatters six times
as strongly as hydrogen atom.
The scattered waves recombine. Each atom contributes to each scattered beam.
The scattered waves can reinforce or cancel one another, depending on whether
they are in phase or out of phase.
The way in which the scattered waves recombine depends, only on the atomic
arrangement.
Instrumentation:
Three components of x-ray crystallographic experiment are
a) Source of x-ray
b) A protein crystal
c) Detector
A narrow beam of x-rays of wave length. 1.54 Å is produced by accelerating
electrons against a copper and allowed to strike the protein crystal.

Part of the beam goes through the crystal without any change in direction. The
rest is scattered in different directions. The scattered beam is detected by
photographic films. The blackening of the emulsion is proportional to the
intensity of scattered x-ray beam.
Working:
When x-ray beam is passed through a molecular crystal, produce a diffraction
pattern in the form of spots the spacing of spots allows us to determine the
repeating distances in the periodic structure. Spot intensities are measured
and used which helps in structural studies.
Application:
Quick and accurate structure determination
X-ray diffraction method id used to check the purity of the water, this
technique has given a new subject Mineralogy.
SPECTROPHOTOMETRY
Definition:
A method used to measure the light absorption of a solution and quantitatively
determine the solution's concentration.

Principle:
Beer -Lambert’s Law:
BEER-LAMBERT’S LAW:
A = absorbance directly proportional to the path length, b,
and the concentration of the sample, c.
The extinction coefficient is characteristic of the substance
under study and of course, is a function of the wavelength.
Molecules strongly absorb only in some regions of the
electromagnetic spectrum.
The photon carries a specific amount of energy defined by
its wavelength.
The molecule will only absorb a photon if the energy it
carries matches a certain amount the molecule can use.
In the ultraviolet-visible region, this energy corresponds to
electronic excitations (promotion of electrons from occupied
orbitals to unoccupied orbitals).
The longest wavelength (the least energy) therefore
corresponds to the energy difference between the ground
and the first excited state (or promotion of an electron from
the highest filled orbital (HFO) to the lowest unfilled orbital
(LUO)).
Types:

The most common spectrophotometers are used in the UV and visible regions
of the spectrum and some of these instruments also operate into the near-
infrared region as well.
Visible region 400–700 nm spectrophotometry is used extensively in
colorimetry.
Instrumentation:
Procedure:
Spectrophotometry is the quantitative study of electromagnetic spectra that is
used to measure the light absorption as well as the diffusion or specular
reflectance.
A spectrophotometer is a photometer that can measure intensity as a function
of the color, or the wavelength of the light.
The most common spectrophotometers are used in the UV and visible regions
of the spectrum, as well as into the near-infrared region.
There are two major classes of spectrophotometers; single beam and double
beam.
A double beam spectrophotometer measures the ratio of the light intensity on
two different light paths, and a single beam spectrophotometer measures the
absolute light intensity..
Although ratio measurements are easier, and generally more stable, single
beam instruments have advantages in a larger dynamic range with more
compact results.
The spectrophotometer measures the fraction of light passing through a given
solution.

In a spectrophotometer, light is guided through a monochromator, which picks
light of one particular wavelength out of the continuous spectrum.
This light passes through the sample that is being measured.
After the sample, the intensity of the remaining light is measured with a
photodiode or other light sensor, and the transmittance for this wavelength is
then calculated.
Based on the obtained transmittance, the concentration of the solution can
then be determined using the Beer-Lambert law, in which,the distance the light
travels through the material is l, E is the molar absorbtivity of the absorber,
and c is the concentration of absorbed species in the material
Applications:
Quantitative determination of solutions of transition metal ions, biomolecules
and highly conjugated organic compounds.
FLUORIMETRY
Definition:
Fluorescence is a property where light is absorbed and remitted within a few
nanoseconds (approx. 10ns) at a lower energy (=higher wavelength)
Bioluminescence is biological chemiluminescence, a property where light is
generated by a chemical reaction of an enzyme on a substrate.
Phosphorescence is a property of materials to absorb light and emit the energy
several milliseconds or more lately (due to forbidden transitions to the ground
state of a triplet state, while fluorescence occurs in exited singlet states)
Principle:
The principle behind fluorescence is that the fluorescent moiety contains
electrons which can absorb a photon, and briefly enter an excited state before
either dispersing the energy non-radiatively or emitting it as a photon, but with
a lower energy, i.e. at larger wavelength (wavelength and energy are inversely
proportional).
The difference in wavelengths is called the Stokes shift.
The time taken to emit the photon is called a lifetime (τ).

Fluorophores can be attached to protein to specific functional groups, such as
amino groups (e.g. via succinimide, Isothiocyanate or hydrazine), carboxyl
groups (e.g. via Carbodiimide), thiol (e.g. via maleimide or acetyl bromide),
azide (e.g. via click chemistry) or non-specificately (Glutaraldehyde) or non-
covalently (e.g. via hydrophobicity etc).
These fluorophores are small molecules, protein or quantum dots.
Applications:

1. To determine the specific functional groups in enzyme, protein etc.
In immunological assays etc.
FLAME PHOTOMETRY
Definition:
A photoelectric flame photometer is a device used in inorganic chemical
analysis to determine the concentration of certain metal ions, among them
sodium, potassium, lithium, and calcium.
Principle:
In principle, it is a controlled flame test with the intensity of the flame colour
quantified by photoelectric circuitry.
The sample is introduced to the flame at a constant rate. Filters select which
colours the photometer detects and exclude the influence of other ions.
Before use, the device requires calibration with a series of standard solutions of
the ion to be tested.
Instrumentation: 1) Sample solution sprayed or
aspirated as fine mist into flame.
Conversion of sample solution into
an aerosol by atomiser (scent
spray) principle.
2) Heat of the flame vaporizes sample
constituents.
3) By heat of the flame + action of the reducing gas (fuel), molecules & ions of
the sample species are decomposed and reduced to give ATOMS. eg Na+ + e-
--> Na
4) Heat of the flame causes excitation of some atoms into higher electronic
states.
5) Excited atoms revert to ground state by emission of light energy, hγ, of
characteristic wavelength; measured by detector

6) Atoms in the vapour state give LINE SPECTRA (Not band spectra, because
no covalent bonds hence no vibrational sub-levels to cause broadening).
7) Coloured glass filter usually able to isolate the line of analyte element if well
separated from other emission lines.
E.g. To measure sodium and potassium separately in samples containing both
Emission of
Na ||
K ||
___________________________________
400 500 600 700 800 (nm)
MASS SPECTROMETRY
Definition
Mass spectrometry (MS) is an analytical technique for the determination of the
elemental composition of a sample or molecule.
It is also used for elucidating the chemical structures of molecules, such as
peptides and other chemical compounds
Principle:
The MS principle consists of ionizing chemical compounds to generate charged
molecules or molecule fragments and measurement of their mass-to-charge
ratios
Procedure:
A sample is loaded onto the MS instrument, and undergoes vaporization.
The components of the sample are ionized by one of a variety of methods (e.g.,
by impacting them with an electron beam), which results in the formation of
positively charged particles (ions)
The positive ions are then accelerated by a magnetic field
Computation of the mass-to-charge ratio of the particles based on the details of
motion of the ions as they transit through electromagnetic fields, and
Detection of the ions, which in step 4 were sorted according to m/z.

Example:
Consider a sample of sodium chloride (table salt).
In the ion source, the sample is vaporized (turned into gas) and ionized
(transformed into electrically charged particles) into sodium (Na+) and chloride
(Cl-) ions.
Sodium atoms and ions are monoisotopic, with a mass of about 23 amu.
Chloride atoms and ions come in two isotopes with masses of approximately 35
amu (at a natural abundance of about 75 percent) and approximately 37 amu
(at a natural
The analyzer part of the spectrometer contains electric and magnetic fields,
which exert forces on ions traveling through these fields.
The speed of a charged particle may be increased or decreased while passing
through the electric field, and its direction may be altered by the magnetic field.
The magnitude of the deflection of the moving ion's trajectory depends on its
mass-to-charge ratio.
Lighter ions get deflected by the magnetic force more than heavier ions.
The streams of sorted ions pass from the analyzer to the detector, which
records the relative abundance of each ion type.

This information is used to determine the chemical element composition of the
original sample (i.e. that both sodium and chlorine are present in the sample)
and the isotopic composition of its constituents (the ratio of 35Cl to 37Cl).
Applications:
1. Isotope dating and tracking
2. Trace gas analysis
3. Pharmacokinetics
4. Protein characterization
5. Space exploration.
6. Respired gas monitor.
DISTILLATION:
Distillation has been used widely to separate volatile components from
nonvolatile compounds. The underlying mechanism of distillation is the
differences in volatility between individual components. With sufficient heat
applied, a gas phase is formed from the liquid solution. The liquid product is
subsequently condensed from the gas phase by removal of the heat. Therefore,
heat is used as the separating agent during distillation. Feed material to the
distillation apparatus can be liquid and/or vapor, and the final product may
consist of liquid and vapor.
A typical apparatus for simple distillation used in chemistry laboratories is one
in which the still pot can be heated with a water, steam, or oil bath. When
liquids tend to decompose or react with oxygen during the course of
distillation, the working pressure can be reduced to lower the boiling points of
the substances and hence the temperature of the distillation process.
In general, distillation can be carried out either with or without reflux involved.
For the case of single-stage differential distillation, the liquid mixture is heated
to form a vapor that is in equilibrium with the residual liquid. The vapor is
then condensed and removed from the system without any liquid allowed to
return to the still pot. This vapor is richer in the more volatile component than
the liquid removed as the bottom product at the end of the process.

However, when products of much higher purity are desired, part of the
condensate has to be brought into contact with the vapor on its way to the
condenser and recycled to the still pot. This procedure can be repeated for
many times to increase the degree of separation in the original mixture. Such a
process is normally called "rectification.
Diagram:
1: Heat source, 2: Still pot, 3: Still head, 4:
Thermometer/Boiling point temperature, 5:
Condenser, 6: Cooling water in, 7: Cooling
water out, 8: Distillate/receiving flask, 9:
Vacuum/gas inlet, 10: Still receiver, 11: Heat
control, 12: Stirrer speed control, 13:
Stirrer/heat plate, 14: Heating (Oil/sand)
bath, 15: Stirrer bar/anti-bumping granules,
16: Cooling bath.
Applications of distillation:
Distillation has long been used as the separation process in the chemical and
petroleum industries because of its reliability, simplicity, and low-capital cost.
It is employed to separate benzene from toluene, methanol or ethanol from
water, acetone from acetic acid, and many multicomponent mixtures.
Fractionation of crude oil and the production of deuterium also rely on
distillation.
LIQUID – LIQUID EXTRACTION
Introduction:
It is a very popular technique. It is used for non-semi volatile compounds.
It uses the basic mechanism of partitioning the sample between 2 immiscible
phases-
Aqueous phase- sample matrix
Organic phase- organic solvent
It makes use of the basic principle that like dissolves like:
A(aq) →A(org)

Principle:
Feed phase contains a component, i, which is to be removed. Addition of a
second phase (solvent phase) which is immiscible with feed phase but
component i is soluble in both phases. Some of component i (solute) is
transferred from the feed phase to the solvent phase. After extraction the feed
and solvent phases are called the raffinate (R) and extract (E) phases
respectively.
Normally one of the two phases is an organic phase while the other is an
aqueous phase. Under equilibrium conditions the distribution of solute i over
the two phases is determined by the distribution law. After the extraction the
two phases can be separated because of their immiscibility. Component i is
then separated from the extract phase by a technique such as distillation and
the solvent is regenerated. Further extractions may be carried out to remove
more component i. Liquid liquid extraction can also be used to remove a
component from an organic phase by adding an aqueous phase.
Instrumentation::
Application: Separation of biological sample
Solvent lighter than water Solvent heavier than water

CENTRIFUGATION
Basic principles of centrifugation:
A particle, whether it is a precipitate, a macromolecule, or a cell organelle, is
subjected to a centrifugal force when it is rotated at a high rate of speed.
The centrifugal force, F, is defined by
The force on a sedimenting particle increases with the velocity of the rotation
and the distance of the particle from the axis of rotation. A more common
measurement of F, in terms of the earth’s gravitation force, g, is relative
centrifugal force, RCF, defined by equation

The fractional coefficient, f, depends on the size and shape of the particle, as
well as the viscosity of the solvent. The frictional force increases with the
velocity of the particle until a constant velocity is reached. At this point, the
two forces are balanced
The rate of sedimentation, sometimes called sedimentation velocity, v, is
defined by
The term is most often defined under standard conditions, 20OC and water as
the medium, and denoted by s20,W. the s value is a physical characteristic used
to classify biological macromolecules and cell organells. Sedimentation
coefficients are in the range 1 10-13 to 10,000 10-13 second.
Instrumentation for centrifugation:
The basic centrifuge consists of two components, an electric motor with drive
shaft to spin the sample and a rotor to hold tubes or other containers of the
sample.
A wide variety of centrifuges is available, ranging from a low speed centrifuge
used for routine pelleting of relatively heavy particles to sophisticated
instruments that include accessories for making analytical measurements
during centrifugation.
Centrifuges are three types
1) The low-speed or clinical centrifuge
2) The high-speed centrifuge

3) The ultra centrifuge
Low-speed centrifuge:
Most laboratories have a standard low-speed centrifuge used for routine
sedimentation of relatively heavy particles.
The common centrifuge has a maximum speed in the range of 4000 to
5000rpm, with RCF values up to 3000 X g.
These instruments usually operate at room temperature with no means of
temperature control of the samples.
Two types of rotors, fixed angle and swinging bucket.
Centrifuge tubes that contain 12 or 50 ml of sample are commonly used.
Low-speed centrifuges are especially useful for the rapid sedimentation of
coarse precipitates or red blood cells. The sample is centrifuged until the
particles are tightly packed into a pellet at the bottom of the tube. The upper,
liquid portion, the supernatant, is then separated by decantation.
High-speed centrifuges:
For more sophisticated biochemical applications, higher speeds and
temperature control of the rotor chamber are essential. The operator of this
instrument can carefully control speed and temperature, which is especially
important for carrying out reproducible centrifugations of temperature-

sensitive biological samples. Rotors chambers in most instrument are
maintained at or near 4OC.
Three types of rotors are available for high-speed centrifugation, the fixed-
angle, the swinging-bucket, and the vertical rotor.
Fixed-angle rotors are especially useful for differential pelleting of particles. In
swinging-bucket rotors the sample tubes move to a position perpendicular to
the axis of rotation during centrifugation. These are used most often for density
gradient centrifugation. In the vertical rotor the sample tubes remain in an
upright position. These rotors are used often for gradient centrifugation.
The preparation of biological samples almost always requires the use of a high-
speed centrifuge.
Cell debris after cell homogenization
Ammonium sulfate precipitates of proteins
Microorganisms
Cellular organelles such as chloroplasts, mitochondria, and nuclei.
THE ULTRACENTRIFUGE:
The ultracentrifuge is an instrument used to measure sedimentation
coefficients. The above Figure shows an ultracentrifuge with its component
parts. The sample is held in a centrifugal cell in a titanium or aluminum rotor,
which is rotated by an electric motor at speeds up to 70,000 revolutions per

minute (rpm). The rotor assembly, including the sample cell, is kept inside an
armored chamber to protect the users against any explosive accident. In order
to avoid excessive heating that may be caused by friction with air; the rotor
chamber is evacuated and refrigerated. In order to avoid collision of the
particles in the sample with the cell wall, the sample cell is shaped like the
sectors of a circle drawn around the axis of rotation. Due to the high speeds,
the balance of weight on the rotor is critical. A flexible shaft is used which can
tolerate a mismatch of up to 0.5 g. A centrifuge can be either a preparative
instrument or an analytical one. In a preparative centrifuge the sample is spun
for a fixed length of time to separate mixtures or purify samples. In an
analytical centrifuge, the movement of the boundary during sedimentation can
be monitored using optical devices like Schlieren optics, Rayleigh interference
or absorbance, and the period and rate of centrifugation can be accordingly
adjusted. The Schlieren optical system is based on the fact that light does not
deviate as long as it moves through a solution of uniform concentration but
undergoes refraction in a solution with varying densities. The change in
refractive index with change in concentration is recorded and is used in the
determination of the sedimentation coefficient.
Molecular weight determination can be carried out by the sedimentation
velocity technique or the sedimentation equilibrium method. In the
sedimentation velocity method the ultracentrifuge is operated at high speeds
and the movement of the sedimentation boundary is recorded as a function of
time. This is a measure of rate of sedimentation. Rayleigh’s interference method
is used to make this record, since the Schlieren optical system is not very
sensitive to small changes in concentration. The Rayleigh interference system
uses a double sector cell, one for the solvent and the other for the solution. By
measuring the displacement of interference fringes, the system measures the
difference in refractive index between the reference solvent and the solution. In
the equilibrium method the sample is centrifuged till equilibrium is reached
between sedimentation and the diffusive movement of the particles, i.e. there is

no net movement of solute particles in the cell and the concentration gradient
remains stable
SALT FRACTIONATION
Salting out is the most common method used to precipitate a target protein.
Addition of a neutral salt, such as ammonium sulfate, compresses the
solvation layer and increases protein-protein interactions. As the salt
concentration of a solution is increased, more of the bulk water becomes
associated with the ions. As a result, less water is available to partake in the
solvation layer around the protein, which exposes hydrophobic patches on the
protein surface. Proteins may then exhibit hydrophobic interactions, aggregate
and precipitate from solution.
DIALYSIS
Principle:
Dialysis works on the principles of the diffusion of solutes and ultrafiltration of
fluid across a semi-permeable membrane. Diffusion describes a property of
substances in water. Substances in water tend to move from an area of high
concentration to an area of low concentration.
Application:

Blood flows by one side of a semi-permeable membrane, and a dialysate, or
special dialysis fluid, flows by the opposite side. A semipermeable membrane is
a thin layer of material that contains holes of various sizes, or pores. Smaller
solutes and fluid pass through the membrane, but the membrane blocks the
passage of larger substances (for example, red blood cells, large proteins). This
replicates the filtering process that takes place in the kidneys, when the blood
enters the kidneys and the larger substances are separated from the smaller
ones in the glomerulus
Types:
The two main types of dialysis, hemodialysis and Peritoneal dialysis, remove
wastes and excess water from the blood in different ways.
Hemodialysis removes wastes and water by circulating blood outside the body
through an external filter, called a dialyzer, that contains a semipermeable
membrane
In peritoneal dialysis, wastes and water are removed from the blood inside the
body using the peritoneal membrane of the peritoneum as a natural
semipermeable membrane.
University questions:
1. Write brief notes on the following questions
2. Beer-Lambert’s Law (3) {2003}
3. Dialysis. (3) {2004}
4. Electro osmotic flow. (3) {2005}
5. Density gradient centrifugation. (3) {2007}
6. ELISA. (3) {2007}, {2010}
7. UV absorption spectra of amino acids. (3) {2008}
8. RIA. (3) {2009}
9. Sedimentation co-efficient. (3) {2010}
10. Distillation (3) {2012}
11. Adsorption (3) {2012}
12. Discuss the methods of fractionation of cell organelles. (5) {2004}

13. Discuss the principle and method of estimation of proteins by Lowry’s method.
(5) {2004}
14. Compare the merits and demerits of the methods of colorimetric estimation of
proteins. (5) {2005}
15. Describe colorimetric estimation of reducing sugars. (5) {2007}
16. Describe the methods and applications of radio-immunoassay. (5) {2007}
17. Write a note on Beer-Lambert’s law and its applications. (5) {2008}
18. Write the methods for analysis of nucleic acids. (5) {2009}
19. Write an account on mass spectroscopy and its applications. (5) {2009}
20. Write the methods for analysis of proteins. (5) {2010}
21. Describe flame photometer. (5) {2010, 2012}
22. Give a detailed account of the quantification methods of proteins and nucleic
acids. (15) {2003}
23. Elucidate the methods for the qualification of carbohydrates and lipids. (15)
{2003}
24. Write critical notes on Density gradient centrifugation. (15) {2003}
25. Explain the methods for quantification of lipids and nucleic acids. (15) {2004}
26. Describe the methods for separation of cell organelles. (15) {2005}
27. Describe the principle and applications of atomic absorption spectroscopy. (15)
{2007}, {2010}
28. Explain the technique of X-ray crystallography and its applications. (15) {2008}
29. Explain the principle, instrumentation and applications of spectrophotometry.
(15) {2009}
30. Explain different spectrophotometric techniques and their applications. (15)
{2012}

Unit – 2: Chromatographic Techniques:
Principles and applications of gel filtration- ion exchange
chromatography, thin layer chromatography- affinity chromatography,
gas liquid chromatography, high performance liquid chromatography
(HPLC).
PRINCIPLES AND APPLICATIONS OF CHROMATOGRAPHY
The partition principle: the partition chromatography
When a solute is allowed to equilibrate itself between two equal volumes of two
immiscible liquids, the ratio of the concentration of the solute in the two
phases at equilibrium at a given temperature is called the partition coefficient.
A mixture of substances with different partition coefficients can be
quantitatively separated by a technique known as countercurrent distribution.
In true partition chromatography, the only factor which influences the
movement of a compound as the solvent travels along the stationary phase is
the relative solubility of that compound in the two phases.
Adsorption chromatography:
Substances differ in their adsorption-desorption behaviour between a moving
solvent (a liquid or a gas) and a stationary solid phase. This behaviour of a
substance can be exploited to achieve its separation. Adsorption is a surface
phenomenon which signifies a higher concentration at an interface as
compared to that present in the surrounding medium.
It usually denotes interactions involving hydrogen bonding and weaker
electrostatic forces of the substance with the absorbent. The solute molecule
which interacts more with the adsorbent, which is also the stationary phase, is
retarded more while less interacting solute molecules are retarded less. In this
way a separation of sample components is achieved.
Ion exchange chromatography:
This procedure was first developed by W. Cohn and may be defined as the
reversible exchange of ions in solution with ions electro statically bound to

some sort of insoluble support medium. The ion exchanger consists of an inert
support medium coupled covalently to positive (anion exchanger) or negative
(cation exchanger) functional groups. To these covalently bound functional
groups are bound, through electrostatic attraction, oppositely charged ions
which will be exchanged with like charged ions in the sample. Thus, if anion
exchange chromatography is performed, negatively charged sample
components will interact more with the stationary phase and will be exchanged
for like charged ions already bound to the matrix.
Molecular size: gel filtration chromatography:
This technique exploits the molecular size as the basis of separation. The
support medium, a gel, consists of porous beads where pore size is strictly
controlled. Macromolecules smaller than the pores get entrapped in the pores
(and move slowly), while those bigger than the pores travel unhindered through
the column (and elute out faster than the smaller molecules). Thus the main
interaction between the solute and the stationary phase is with respect to the
size and this is ultimately the basis of separation.
Affinity chromatography:
The technique utilizes the specificity of an enzyme for its substrate or
substrate analogue for the enzyme’s separation. A substrate analogue is
coupled to the gel matrix and the cellular suspension is allowed to percolate
through. The enzyme which is specific for the substrate analogue binds to the
gel becoming immobile while all other components move down and out. The
technique has a very high resolution power.
GEL FILTRATION CHROMATOGRAPHY
Introduction:
The separation of molecules on the basis of their molecular size and shape
utilizes the molecular sieve properties of a variety of porous materials.
The most commonly used of such materials are a group of polymeric organic
compounds, which possess a three dimensional network of pores, which confer
gel properties upon them.

The technique has been called by different names like gel permeation
chromatography, exclusion chromatography and molecular sieve
chromatography.
The term gel filtration is used to describe the separation of molecules of varying
molecules size utilizing gel materials.
Principle:
A column of gel particles or porous glass granules is in equilibrium with a
suitable solvent for the molecules to be separated.
Large molecules which are completely excluded from the pores will pass
through the interstitial spaces.
Smaller molecules will be distributed between the solvent inside and outside
the molecular sieve and will pass through the column at a slower rate.
Thus the distribution of a solute in a column of a swollen gel is determined by
the total volume of solvent, both inside and outside the gel particles.
Quantitative aspects of the technique:
For a given tube of gel, the
distribution of a particular substance
between the inner and outer solvent
is defined by a distribution
coefficient.
If the solute is large and completely
excluded from the solvent within the
gel, the distribution coefficient Kd = 0.
Where as if the solvent is sufficiently
small to gain complete accessibility to
the inner solvent, the distribution
coefficient Kd = 1.

Due to the variation is pore size for a given gel, there is some inner solvent
which will be available some which will not be available to solutes of
intermediate size, hence the Kd values vary between 0 and 1.
It is this complete variation of Kd between their two limits which makes
possible separation of solutes within a narrow molecular size range of a given
gel.
Effluent volume:
The effluent volume Ve of a given solute depends on the volume external to gel
particles, V0 (The void volume) on the distribution coefficient (Kd) and on
volume inside gel matrix itself, Vi,
Thus Ve = V0 + Kd Vi
V0 = volume of solvent occupies by the interstitial space present between the
gel volume.
Materials:
Various gels used.
1) Cross linked dextans (Sephadex)
2) Agarose (Sepharose, Bio gel)
3) Polyacrylamide (Bio gel)
4) Polyacryloyloyl morphine (Enzocry gel)
5) Polystyrenes (Bio-beads)
Water region:
1) Gel chromatography media are most supplied in dehydrated form and are
swollen in a solvent, usually water, before use.
2) The weight of water taken up by 1g of dry (weight) gel id known as the water
region.
3) E.g. for sephadex G-50, this value is 5.0 + 0.3g or 5.0 – 0.3g.
4) This value does not include the water surrounding the gel particles.
Bed volume:
1) Most commercial suppliers provide in addition to water region, a bed volume
value.
2) This is the first volume taken up by 1g of dry gel when swollen in water.

3) E.g. for G-50 the bed volume is G-11 ml 1g of dry gel.
Void volume:
1) This is the total space surrounding the gel particles in a packed volume.
2) This value is determined by measured the volume of solvent requires to elute a
solute that is completely excluded from the gel matrix.
Applications:
Gel permeation chromatography is chiefly used for the purpose of separation of
biological molecules leading to their ultimate purification. Proteins, enzymes,
hormones, antibodies, nucleic acids, polysaccharides, and even viruses have
been separated in various experiments which have used different types of gels
or glass granules.
One of the common separation problems in biochemistry is the removal of salts
and small molecules from macromolecules. This can be easily performed using
gel filtration since the distribution coefficients of salt molecules will be largely
different from those of macromolecules.
ION EXCHANGE CHROMATOGRAPHY
Introduction:
Ion exchange chromatography is the process by which a mixture of similar
charged ions can be separated by using an ion exchange resin which
exchanges ions according to their relative affinities. Many biological materials
for e.g. amino acids and proteins have ionisable groups i.e. a net positive or
negative charge can be utilized in separating mixtures of such compounds.
The principle of separation is by reversible exchange of functional group of ions
present in the solution and those present in the ion exchange resin. It is the
attraction between oppositely charged particles. Ion exchange separations are
carried out usually in columns packed with an ion exchanger. The ion
exchanger is an inert, insoluble support medium. Ion exchangers can be
divided into two groups: anion exchangers and Cation exchangers.

Cation exchangers possess negatively charged groups and these will attract
positively charged molecules. Anion exchangers have positively charged groups
which will attract negatively charged molecules.
Basic process of ion exchange:
CATION EXCHANGER:
RSO3- ---- Na+ + NH3+ R
RSO3- ----- NH3+ R + Na+
Exchanger Counter Charged
molecule Bounded
Exchanged
Ion to be exchanged
molecular ion ion
Anion exchanger:
(R)4 N+ ------- Cl- + -OOCR (R)4 N+ ------- -OOCR + Cl-
The more highly charged the molecule to be exchanged, the tighter it binds to
the exchange and less readily it is displaced by other ions. Diffusion of the
exchanged ion through the exchange to the surface. Selective adsorption by the
eluant and diffusion of the molecule in to the external solution. The selective
desorption of the bound molecule is achieved by changes in pH or ionic
concentration or by affinity elution.
Types of ion exchange resins:
Ion exchangers may be classified according to the chemical nature.
a) Strong cation exchange resin
b) Weak cation exchange resin
c) Strong anion exchange resin
d) Weak anion exchange resin

Two main groups of materials
are used to prepare ion exchange
resins, polystyrene and cellulose.
Polystyrene resins are prepared
by polymerization reaction of
styrene and divinyl benzene.
The number of cross linkage is
determined by the ratio of divinyl
benzene to styrene. A higher
concentration of divinyl benzene
produces higher cross linkages.
Increasing the cross linkage
increases the rigidity, reduces
swelling, reduces porosity and
reduces the solubility of the
polymeric structure.
Resins substituted with sultonic acid groups are strong cationic exchangers.
Weakly acidic (weak cationic exchangers) can be prepared by attracting
carboxylated groups to the aromatic.
Polystyrene resins are very useful for separating small molecular weight
compounds. Cellulose resins have much greater permeability to macro
molecular polyelectrolyte.
S.NO TYPE POLYMER FUNCTIONAL GROUP EXAMPLES
1 Weakly
Acidic(Cation
exchanger)
Polyacrylic acid -COO- Amberlite
IRC 50

Cellulose
(or)Dextran
-CH2COO- CM
Sephadex
Agarose -CH2COO- CM
Sephalose
2 Strongly
Acidic(cation
exchanger)
Polystyrene -SO3- Amberlite
FR120 Dowex
50
Cellulose(or)
Dextran
-CH2CH2CH2SO3 SP—
Sephadex
3 Weakly
basic(Anion
exchanger)
Polystyrene -CH2+NHR2 Amberlite IR
45
Cellulose(or)
Dextran
-
CH2CH2+NH(CH2CH3)2
DEAE
Sephadex
4 Strongly
basic(Anion
exchanger)
Polystyrene -CH2+N(CH3)3 Bio-Rad 1
Dowex 1
Cellulose(or)
Dextran
CH2 CH2+N(CH2CH3)2
CH2CH(OH)CH3
QAI-
Sephadex
Preparation of the exchange medium:
There are three steps in exchanger preparation.
Swelling the medium. This is known as precycling.
Removal of very small particles of the exchanger.
Finally the exchanger has to be equilibrated with the suitable conversions.
(HCL if H+ is the counter ion).
CHOICE OF BUFFER:
The choice of buffers which maintain the pH of the column is indicated by the
compounds to be separated and the type of ion exchange being carried out
(anionic or cationic). Anionic exchange chromatography should be carried out

with cationic buffers. Cationic exchange chromatography should be carried out
with anionic buffers. The pH of the buffer should impart the same charge to the
sample ions as is present on the counter ion.
Buffer pH range
Ammonium acetate 4 – 6
Ammonium formate 3 – 5
Pyridinium formate 3 – 6
Procedure:
The choice of the ion exchanger depends upon the stability of the sample
components, their molecular weight and the specific requirements of the
separation.
The amount of sample which can be applied to the column is dependent upon
the size of the column and the capacity of the exchanger.
Gradient elution is far more common than isocratic elution with the anion
exchanger pH gradient decreases and the ionic strength increase.
The separation of amino acids is usually achieved by using a strong acid cation
exchanger. It can be easily done using an automatic amino acid analyzer.
FOR ION EXCHANGE CHROMATOGRAPHY:
UV monitor
Recorder
Fraction collector
Chosen ion exchanger Chosen starting buffer
Swell gel if necessary and packed in suitable column
Set up equipment

Pump
Gradient mixer
.
Applications of ion exchange chromatography:
Separation of amino acids
Separation of nucleic acids and nucleotides
Separation of carbohydrates
Separation of lipids
Separation of organic substances
Equilibrate (2-3 volumes of buffer)
Applied sample (Sample equilibrium if necessary)
Washed away unbound substances
Separation optimized
Eluted bound substances
Desalted
Gel regenerated
Separation analysed

THIN LAYER CHROMATOGRAHY (TLC)
Principle:
TLC can be carried out either by adsorption or partition principle. A thin layer
of stationary phase is formed on a suitable flat surface such as glass, foil or
plastic plate.
Since the layer is so thin, the movement of mobile phase across the layer
generally by simple capillary action is rapid their been little resistance to flow.
As the mobile phase moves across the layer from one edge to the oppositely it
transfer any “analytes” placed on the layer at a rate determined by distribution.
Coefficient between the stationary and mobile phase.
The principle of the distribution process may be based on that of adsorption,
partition, ion exchange chromatography of exclusion charity.
The “analytes” movement ceases either when the mobile phase (solvent front)
reaches the end of the layer (or) when the plate is removed from the mobile
phase reservoir.
The movement of the analyzer is expressed by its retardation factor (Rf).
Distance travelled by analyte from origin
Rf =
Distance moved by solvent front from origin
Distribution coefficient:
The basis of all forms of chromatography is the partition (or) Distribution
coefficient (Kd) which describes the way in which a compound distributes itself
between two immiscible phases.
For two such immiscible phases A and B, the value for this coefficient is a
constant at a given temperature and is given by the following expression.
Concentration in A
Kd=
Concentration in B

Instrumentation:
solvent front
C
B
A
origin
Thin layer preparation:
A slurry of the stationary phase applied to a glass, plastic (or) foil plate.
The layer is prepared uniformly by means of a plate spreader starting at one
end of the plate and moving towards the other end.
The thickness of the layer is based on the nature of separation
Analytical separation = 0.25 mm thick
Preparative separation = 2 mm thick
When the stationary phase is to be used for adsorption chromatography, there
arises a problem. Since the adsorbent do not adhere satisfactory to the glass
plates.
Activation of adsorbents:
Once the slurry layer has been prepared, the plates are dried.
In order to obtain very active layers silica gel and alumina plates can be heated
to 150 degree for about 4 hrs.
Coating materials:
Coating materials often used are silica gel, alumina, kieselguhr and cellulose
powder.
Sample application:
The sample is applied to the plate 2.0-2.5 cm from the edge by means of
micropipette (or) micro syringe.

Development tank:
In TLC the plate is placed in the development chamber at an angle of 45
degree.
The bottom of the chamber is covered up to nearly from by the solvent.
The top is covered with a lid.
It is in TLC that the development chamber is perfectly saturated with solvent
vapour.
This is essential so as to avoid integral solvent evaporation losses from the
development plate which can be lead to various errors resulting in lack of
reproducibility.
Two component solvent systems:
Examples:
Chloroform (9:1)
Benzene, methanol (95:5)
Cyclohexane, ethyl acetate (1:1)
Development methods:
Generally ascending technique in which the solvent is allowed to the height of
about 15-18 on the 20 cm
This is quick process that requires 20-40 mins.
At the end of this time, the plate is removed from the developing tank.
The solvent front is marked on the plate and is finally allowed to dry.
Analyte detection:
Examination of the plate under UV will show the position of UV absorbing or
fluorescent compounds.
Subjecting the plate to iodine vapour is useful if unsaturated compounds are
being investigated.
Spraying of the plates with specific coloring reagents will stain certain
compounds.
E.g. Ninhydrin will locate AA’s and peptides.
Estimation:
a) On-plate quantification may be achieved by means of densitometry.

b) Off-plate quantification may be carried out by scrapping off the spot.
Applications:
1) It is used for the analysis of components of food stuffs.
2) TLC has been used for separating cationic, anionic and also some organic
derivatives of the metals.
3) TLC has been used for checking of the separation procedures and purification
process.
4) Finally, TLC finds its application in the quantitative analysis of various organic
and inorganic materials.
AFFINITY CHROMATOGRAPHY
Introduction:
Affinity chromatography exploits the capacity of bio-molecules for specific non-
covalent binding of their molecules called ligand. This chromatography is
carefully used for the separation of various biological molecules.
Principle:
Affinity chromatography is theoretically capable of giving absolute purification,
even from complex mixture in a single process. The technique requires that the
material to be isolated is capable of binding reversibly to a specific ligand that
is attached to a insoluble mixture.
K+1
M + L ↔ ML complex
K-1

Group specific ligand commonly used in affinity chromatography:
MACRO MOLECULE/CELL LIGAND
Avidin Biocytin
Thrombin Benzamidine
Coagulation factor Heparin
Poly(A) messenger RNA Poly(V) or Poly dT
Glycoproteins, Glycolipids Concanavalin A
Fat cells Insulin, Concanavalin A
Affinity chromatography requires concentration in following things:
They are
The type of matrix used.
Selection of the ligand, its nature and the means of covalently binding it to the
matrix.
The conditions applied to bind and dissociate (elute) the macromolecule from
the column.
Supporting matrix:
a) The matrix should be inert.
b) It should posses good flow properties.
c) It should be chemically and mechanically stable.
d) It should contain large number of suitable chemical groups for ligand
attachment.
e) The most commonly use are agarose, polyacrylamide and controlled porosity
glass beads.
Ligand selection:
The ligand to be bound should possess functional groups that can be modified
to form covalent linkage with the supporting matrix.
Ligand attachment:
Covalent coupling of the ligand to the supporting matrix involve.
a) Activation of the matrix functional groups.
b) Covalent attachment of the ligand to these activated groups.

The most common method of activation of polysaccharides supports (agarose)
involves treatment with cyanogens bromide at alkali pH (11.0)
The arm:
If a ligand is directly attached to
activated groups of the support, the
macromolecule might encounter
steric restrictions. It is used to
introduce a spacer arm between the
activated groups of the support and
the ligand. This spacer is known as
the arm.
Alternative methods for activation of polysaccharide supports:
METHOD ACTIVE
FUNCTIONALITY FOR
LIGAND COUPLING
REACTION WITH TYPE
OF LIGAND
Epoxides Reaction with amines
and other nucleotide.
Periodate Reacts with amines or
hydrazines.

Chromatography procedure:
1) The gel beads are swollen before loading in to a column.
2) The buffer which encourages adsorption of the desired molecule on the gel
surface is used.
3) The buffer which encourages adsorption must be supplemented with any co-
factors (e.g. metal ions) required for ligand macromolecule interaction.
4) The buffer should also possess a high ionic strength so as to minimize non-
specific adsorption on to charged groups in the ligand.
5) The sample is applied at the top of the column and the buffer flow started.
6) Once the macromolecule is bound, the column is eluted with buffer to remove
non-specifically bound unwanted macromolecules.
7) The purified bound component may now be eluted by taking either specific or
non-specific elution.
Specific elution:
Specific or affinity elution is carried out
a) By addition of compounds for which the macromolecule has more affinity.
b) By addition of compounds for which ligand has more affinity that it has for
desired macromolecule.
Applications:
1) Affinity chromatography has used to purify a large variety of macromolecules
such as enzymes, immunoglobulins, membrane receptors, nucleic acids and
even polysaccharides.

2) Whole cell have been purified using this technique cells separated include fat
cells T and B lymphocytes spleen cells, lymph node cells, oocytes etc.
3) Metal chelate affinity chromatography is used for proteins which have similar
molecular weights and even iso-electric points.
4) Use of immobilized enzymes is very important in affinity chromatography.
GAS LIQUID CHROMATOGRAPHY
Introduction:
Gas chromatography consists of gas solid chromatography and gas liquid
chromatography. In both types gas is used as mobile phase and either solid or
liquid is used as stationary phase. In gas solid chromatography the principle of
separation is adsorption. Gas solid chromatography is used only in case where
there is less solubility of solutes in stationary phase.
Gas liquid chromatography:
The basis for the separation of the compound in gas liquid chromatography is
the difference in partition coefficient of volatilized compound in the liquid
stationary phase.
Principle of separation:
The principle of separation in gas liquid chromatography is partition.
Gas is used as mobile phase liquid, which is coated on to a solid support, is
used as stationary phase. The mixture of compound to be separated is
converted to vapour and mixed with gaseous mobile phase. The component
which is more soluble in the stationary phase travel slower and eluted later.
Partition coefficient is the ratio of solubility of substance distributed between
two immiscible liquids at constant temperatures.
The diagrammatic representation of the principle of gas chromatography
separation it is on the basis of their partition coefficient.
Sample consisting of three components is introduced in the column. The
components indicated by the first arrow interact more with liquid phase and
less with gas phase. The third arrow indicates that the components interact

more with gas phase and less with liquid phase. The component indicated by
the second arrow interacts equally with both gas and liquid phase.
PROCEDURE:
The stationary phase is used in GLC is liquid phase. The liquid phase is
displaced over a surface of an inert solid support. The solid support which is
coated on to the inside surface of a long column is inert to the sample
component and does not react with it any way. A gas stream termed as carrier
gas flows continuously through the column at a flow rate, which is control.
The essential component of a gas chromatography is
Carrier gas tank
Flow regulator (or) pressure regulator
Injection device
Columns
Temperature control devices
Detectors
Recorder
The inert carrier gas stored in gas tank passes through the pressure regulator
into the sample injection chamber from where it carries sample on to the
column.
Sample separation in the column:
In the column the sample component becomes distributed between the liquid
and the gas phases. These compounds therefore travel more strongly than the
carrier gas because they are being retarded by virtues of their interaction with
the liquid phase. The retarding effect is different for different components.

Procedure:
The final passage of the gas is through the flow meter in the atmosphere. Since
the procedure is usually carried at high temperature, a thermo stated oven is
provided for the column injector and the detector. Criteria for compounds to be
analysed by gas chromatography.
a) Volatility:
Unless a compound is volatile it cannot be mixed with melting point. Hence
volatility is important.
b) Thermo stability:
All the compounds will not be in form of vapour. They will be present as a solid
as well as liquid sample. Hence to convert them to a vapour form, they have to
be heated to a high temperature.
Requirements of a chromatography:
a) The gas must be chemically inert and pure.
b) It must be cheap and easily available.
c) It must possess less risk of expulsion.
d) A low –density gas might give a faster separation.
The choice of the gas usually depends on the requirements of the detector and
also the availability of the gas. Most commonly used gases are nitrogen and
argon but helium, hydrogen, carbon dioxide are also used.
Column:
Column is one of the important part of the gas chromatography which decides
the separation efficiency column are made up of glass or stainless steel.
Solid support:
An ideal should be chemically inert although wet table by the liquid phase, so
that it will be spread in a thin layer of uniform thickness. Commonly used
supports are derived from diatomaceous earth and Teflon.
Liquid phase:
A good separation will occur only when the sample dissolves well in the liquid
stationary phase. The separation occurs only in the liquid phase.

Stationary phase Typical example Tmax ( . C )
Silicon Steroids
Rubber Alkaloids 350. C
Sample preparation and introduction:
If the sample are non-polar or have a very low polarity thus weight not needed
any pre-treatment.
However if the sample possesses such polar functional groups as –OH, -COOH,
-NH2 etc.
Detectors:
The detectors the presence of the individual components as they leave the
column. Therefore most commonly used detectors are
a) Flame ionization detectors
b) Electron captures detectors
c) Thermionic emission detectors
Retention time of quantitative analysis:
Recorder:
Recorder is used to record the responses obtained from detectors after
application.
Retention time (Rt):
Retention time is the difference in time between the point of injection and
appearance of peak maxima.
Retention volume:

Retention volume is the volume of the carrier gas required to elute 50% of the
component from the column.
Retention volume = Retention time X flow rate
Efficiency:
Efficiency of column is expressed by the number of theoretical plates
(theoretical plate is an imaginary) or hypothetical unit of a column where
equilibrium has been established between stationary phase and mobile phase.
16Rt2
n =
W2
APPLICATIONS OF GAS LIQUID CHROMATOGRAPHY:
Gas chromatography is used for the separation of components of tobacco,
smoke, atmosphere, pollutions, solvents; plant extracts essential oils volatile
vegetables oil and organic acids etc.
Gas chromatography is widely used in the field of solution chemistry including
study of polymer Lewis acid; base precipitates liquid crystals and gas liquid
interfluid adsorption have found gas chromatography to be a tool of important.
Gas chromatography is one of most widely used produces to study reactions
rates, energies of mechanisms.
Gas chromatography is widely used to analyze such molecular precipitates as
a) vapor pressure b) heat of vaporization c) molecular d) bind angle deformation
etc.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Introduction:
Liquid chromatography was defined in the early 1900s by the work of the
Russian botanist, Mikhail S. Tswett. His studies focused on separating
compounds [leaf pigments], extracted from plants using a solvent, in a column
packed with particles.
Tswett coined the name chromatography [from the Greek words chroma,
meaning color, and graph, meaning writing—literally, color writing] to describe
his colorful experiment.

Smaller particle sizes are required to improve separation power. However,
smaller particles have greater resistance to flow, so higher pressures are
needed to create the desired solvent flow rate.
Pumps and columns designed to withstand high pressure are necessary. When
moderate to high pressure is used to flow the solvent through the
chromatographic column, the technique is called HPLC.
Definition:
High performance liquid chromatography is defined as a separation of mixtures
of compounds due to differences in their distribution equilibrium, between the
stationary phase and the mobile phase, in the presence of high pressure.
The term HPLC (High Pressure Liquid Chromatography) was coined by Prof.
Csaba Horvath in 1970.
With advent, new HPLC instruments could incorporate improved injectors,
detectors, and columns, with advances in performance. Therefore it was
renamed as High PERFORMANCE Liquid Chromatography.
Components:
The components of a basic high-performance liquid chromatography [HPLC]
system are shown in the following diagram:
Components of HPLC:
a) Solvent reservoir:
A reservoir holds the solvent [called the mobile phase, because it moves].
b) Sample injector:
An injector [sample manager or autosampler] is able to introduce [inject] the
sample into the continuously flowing mobile phase stream that carries the
sample into the HPLC column.
5-500µl of sample can be introduced by the sample injectors.
c) Pumps-:
A high-pressure pump [solvent delivery system or solvent manager] is used to
generate and maintain a specified flow rate of mobile phase.
100-400 atm. pressure is desired to maintain flow rate of 0.5-2 ml/min.
d) Analytical column:

The column contains the chromatographic packing material needed to effect
the separation.
This packing material is called the stationary phase because it is held in place
by the column hardware.
Column length ranges from 10-30cm with a diameter of 2-5mm.
The column is packed with standard 5µm packing.
e) Detector(s):
The detector contains a flow cell that sees [detects] each separated compound
band against a background of mobile phase.
Types of detectors:
Absorbance (UV with Filters, UV with Monochromators)
IR Absorbance
Fluorescence
Refractive-Index
Evaporative Light Scattering Detector (ELSD)

Electrochemical
Mass-Spectrometric
Photo-Diode Array
f) Data recorder:
It detects the presence of a compound and sends its corresponding electrical
signal to a computer data station.
The recorder helps analyze and interpret the data.
g) Fraction collector:
A fraction detector is a device that allows regular or specified samples to be
taken from a column eluate and stored in a retrievable form.
TYPES OF HPLC:
Normal phase HPLC
Reverse phase HPLC
Ion exchange HPLC
Chiral HPLC
Affinity HPLC
Size exclusion HPLC
Mode Normal
phase
Reversed
phase
Ion exchange Chiral Affinity Size
exclusion
Stationary
phases
chemistry
Polar
hydrophilic
Non-polar
lipophilic
Ion-bonding Chiral
recognitio
n
Bioaffinity Sieving by
size
Typical
stationary
phases
Silica,
alumina
Alkylated
silica,
mostly C-18
Ionic
functional
groups on
silica or
polymer
Chiral
groups on
silica
surfaces
Either
substrates
or
biomolecule
s
Gel type
Polymers
Typical
mobile
phases
Hexane,
isopropanol
, methelene
chloride
Water,
methanol,
acetonitril,
buffers
Water, buffers,
acid, base
Two
modes:
aqueous
& non-
aqueous
Water,
buffers
Two modes:
aqueous &
non-
aqueous
Typical
solutes
Fats and
oils
almost all
organic
compounds
Any ion,
charged
compounds
Enantiom
ers, small
& large
molecules
Biomolecule
s or their
substrates
Polymers:
synthetic or
biological
Advantages of HPLC:
1) It is a very fast separation method.

2) It gives high resolution and high accuracy.
3) It is useful for resolving optically active compounds using chiral stationary
phases.
4) The technique is applicable to very small amounts of samples.
5) All kinds of soluble solid samples and liquid samples can be analysed.
6) HPLC is useful for continuous monitoring of the column effluent.
7) HPLC provides repetitive and reproducible analysis using the same column.
8) It is used for the separation of ionic and non-ionic species such as various
organic and inorganic substances.
9) It has wider choice of stationary and mobile phases in comparison to other
chromatographic techniques.
10) It can be used for the separation of closely related compounds as well as the
purification of compounds.
Disadvantages of HPLC:
1) It cannot be used for the analysis of volatile compounds such as hydrocarbons.
2) It cannot be used for the analyses of industrial products such as alloys,
polymers, etc.
3) High purity solvents are required because any impurity may affect the
separation and resolution.
4) It requires extensive training in order to operate the instrument and optimize
the conditions.
5) Sample preparation is often required.
Applications:
FIELD TYPICAL MIXTURES
1. Pharmaceuticals -antibiotics, sedatives, steroids,
analgesics
2. Biochemical’s -Amino acids, lipids, carbohydrates,
proteins
3. Food products -Antioxidants, additives, aflatoxins
4. Industrial chemicals -Dyes, surfactants, propellants,
aromatics
5. Clinical medicines -Drug metabolites, urine extracts,
estrogens
6. Polymers -Molecular weight determination

7. Forensic chemistry -Drugs, poisons, narcotics, blood
alcohol
8. Pollutants -Pesticides, herbicides, phenols
9. Quality control -Purification from mixture
University questions:
Write brief notes on the following questions
1. Adsorption Chromatography. (3) {2003}
2. TLC. (3) {2003}
3. Void volume. (3) {2005}
4. Partition co-efficient (3) {2012}
5. Cation exchanger (3) {2012}
6. Explain the principle involved in ion-exchange chromatography. (5) {2003}
7. Discuss the principle of affinity chromatography. (5) {2004}
8. Discuss the principle and advantages of gas liquid chromatography. (5) {2005}
9. Describe the principle of metal chelate affinity chromatography. (5) {2005}
10. Describe the principle and applications of gel filtration. (5) {2012}
11. State the principle and discuss the applications of Chromatography. (15)
{2003}, {2009}
12. Describe the isolation of lipids and their analysis by thin-layer
chromatography. (15) {2005}
13. Explain the principle and applications of column chromatography. (15) {2007}
14. Explain the principle and application of HPLC and affinity chromatography.
(15) {2012}

Unit: 3 Electrophoresis
Principles and applications of moving boundary electrophoresis, zone
electrophoresis, gel electrophoresis-PAGE and SDS PAGE agarose gel
electrophoresis, isoelectric focusing and 2D Gel electrophoresis, pulsed
field electrophoresis.
PRINCIPLES AND APPLICATIONS OF ELECTROPHORESIS
Introduction:
Majority of the polymers of biological interest are electrically charged.
The electric charge differences among them can be used to separate and
analyze mixtures of biopolymers.
Electrophoresis is usually carried out to determine the number, amount, and
mobility of components in a given sample or to separate them.
Definition:
Electrophoresis is the migration of charged particles or molecules in a medium
under the influence of an applied electric field.
Principle:
The separation is based upon the mobility of charged macromolecules under
the influence of an electric field.
Mobility depends on the magnitude of its charge, its molecular weight, and its
tertiary or quaternary structure.
Movement of charged species in electrophoresis
Factors affecting electrophoretic mobility:
Charge
Size

Shape
Molecular weight
Types of electrophoresis:
a) Moving boundary electrophoresis
b) Zone electrophoresis
c) Agarose gel electrophoresis
d) PAGE
e) Iso-electric Focusing
f) 2-D electrophoresis
g) Pulse field electrophoresis
MOVING BOUNDARY ELECTROPHORESIS:
Introduction:
Moving-boundary electrophoresis was the first electrophoretic technique
introduced and was first developed by A. Tiselius of Sweden in the 1930s.
It is also called as Free Electrophoresis. In free electrophoresis, the migration of
boundaries can be observed.
Schematic diagram of separation of protein mixture (x, y, z) by moving
boundary electrophoresis
i. Shows- the initial position after the apparatus has been filled with protein and
buffer solutions.

ii. Shows- the final position after separation.
• All proteins have negative charge at the pH of the buffer. x has the highest
mobility and z has the lowest mobility.
• Note that only some amount of x and z have separated completely.
Method:
The separation is carried out usually in ‘U’ tube with the sample being layered
under a buffer solution.
The analyte solution is initially located at the bottom of the U. [It has a high
concentration of a non-ionizing component such as sucrose that increases its
density and minimizes initial mixing at the boundaries of the dilute buffers
present in the anodic and cathodic compartments.]
When the electric field is applied, migration of the components occurs in both
directions, according to their charges, and the boundaries between analyte
solution and buffer solutions move towards the electrodes as the separation
proceeds.
This is monitored with refractive index detectors positioned near the upper
ends of the cell.
ZONE ELECTROPHORESIS:
Zone electrophoresis is the name given to the separation technique employing
stabilizing media such as gels.
It is also known as electrophoresis in stabilized media.
The zone separation uses stabilizing media (paper or, gels such as
polyacrylamide, agarose etc.)
In this technique, the components of the analyte mixture separate completely
to form discrete zones, or bands.
The analyte solution is applied to the medium as a spot or band, and the
electric field causes the initial band to separate into component bands through
migration.
Each zone consists of a single component which can be easily isolated.

The zones have higher density than the medium, but the use of stabilizing
system does not allow the zones to disperse and spoil the separation, as is the
case with free electrophoresis.
Uses:
Analysis of complex mixtures of biomolecules,
Etermination of molecular weight and purity of isolated proteins and nucleic
acids
Diagnostic tests.
A comparison between free electrophoresis and zone electrophoresis:
X and Y are two
components resolved by the two
techniques.
• If the aim is separation and not a study of electrical properties, zone
electrophoresis is much more useful tool than free electrophoresis.
AGAROSE-GEL ELECTROPHORESIS:
• This technique involves the separation of molecules based on their size, in
addition to the electrical charge.
• Agarose gel electrophoresis is used for the separation of high molecular
weight proteins and DNA.
• Separation in agarose gels is achieved because of resistance to their
movement caused by the gel matrix.

The movement of large
molecules is slow in gel
electrophoresis whereas
smaller molecules pass
easily.
Consequently the mobility of
DNA molecules during gel
electrophoresis will depend
on size, the smallest
molecules moving fastest.
Gels containing 0.3% agarose will separate double-stranded DNA
molecules of between 5 and 60 kb size, whereas 2% gels are used for
samples of between 0.1 and 3 kb.
Many laboratories routinely use 0.8% gels, which are suitable for
separating DNA molecules in the range 0.5 to10 kb.

POLYACRYLAMIDE GEL ELECTROPHORESIS:
• The most commonly used components to synthesize the matrix are
acrylamide monomer, N, N’- methylenebisacrylamide (bis), ammonium
persulphate and tetramethylenediamine (TEMED).
• TEMED acts as a catalyst of gel formation because of its ability to exist in
free radial arm.
Ammonium persulphate when dissolved in water generates free radicals

SDS PAGE
The use of gels such as starch, polyacrylamide, agarose and agarose-
acrylamide as supporting media in electrophoretic techniques as enhanced
resolution ,particularly for proteins and amino acids, due to the combination of
reduced diffusion by the gel network and the separating action of gel
chromatography.
Electrophoresis in acrylamide gels is frequently referred to as PAGE
(polyacrylamide gel electrophoresis).
The components used in PAGE are acrylamide, bisacrylamide and TEMED
(tetra methyl ethylene diamine).
Cross linked polyacrylamide gels are formed from the polymerization of
acrylamide monomer in the presence of smaller amounts of bisacrylamides.
Acrylamide monomer is polymerized in a head to tail fashion into a long chain;
occasionally a bisacrylamide molecule joins this polymer and introduces a
second site for chain elongation. By this way cross linked matrix of fairly well
defined structure is formed.
The pore size in the gel can be varied by the changing the concentrations of
both the acrylamide and bisacrylamide.
Low percentage gels (3%) have large pore size and are used in the
electrophoresis of protein. High percentage gels (10-20%), due to their smaller
pore size, introduce a sieving effect and that becomes the basis for the
separation of proteins according to their size.
Two different experimental arrangements are basically used for conducting
PAGE. They are column gel and slab gels .Two types of column PAGE
techniques are routinely used.
Native gel electrophoresis and SDS-PAGE.
Use of solublisers in PAGE:
Several proteins of biological importence contain more than one polypeptide
chain. These proteins are referred to as oligomeric proteins. The structure of
this protein is stabilized by hydrogen bonding, disulphide bridges or by
hydrophobic interaction.These proteins migrate as a single band during gel

electrophoresis.The subunits of these proteins can be separated from each
other by a class of substances known as solubilizers.
E.g: urea,B-mercaptoethanol and SODIUM DODECYL SULPHATE, CH3-(CH
2)10-CH2OSO3 -Na+.
SDS-PAGE:
When PAGE is performed in the presence of sodium dodecyl sulphate CH3-(CH
2)10- CH2OSO3 Na +, it is known as SDS-PAGE.It is the most widely used
method for analysing protein mixtures qualitatively .It is particularly useful for
monitoring protein purification. since the method is based on the separation of
proteins according to size ,it is also used to determine the relative molecular
weight of protein subunits .
Method:
As the first step, the sample to be separated is boiled for 5 mins in a sample
buffer containing B-mercaptoethanol and SDS. This treatment completely
denatures each protein present in the sample and imparts negative charge to
the polypeptide chains.
The sample buffer also contains an ionisable tracking dye which allows
monitoring the electrophoretic run.
The gel consists of two parts namely, the main separating gel and a shorter
stacking gel (about 1cm long).The main separating gel is first poured into the
glass tube and allowed to set.
Then 1 ml of stacking gel is poured on top of the separating gel. The stacking
gel has a very large pore size (4% acrylamide) which allows the proteins to move
freely and concentrate over the separating gel under the influence of the
electric field.
The separating gel used is a 15 per cent polyacrylamide gel. Now proteins
continue their movement toward the anode. Since proteins have the same
charge per unit length, all proteins travel with the same mobility.
However, as they pass through the separating gel the proteins separate, owing
to the molecular Sieving properties of the gel.

Bth 201 enzymology and biochemical techniques

Bth 201 enzymology and biochemical techniques

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