Chromatography report

1. PRACTICE SCHOOL
CHROMATOGRAPHY
SUPERVISED BY:- SUBMITTED BY:-
Mr.Anup Pramanik Vikas Singh
Asst.Prof B.Pharm. Sem-VII (2021-22)
Enrollment No:-2018/5790
REGIONAL COLLEGE OF PHARMACY
ISI-17,RIICO Institutional Area,Sitapura,Jaipur-302022
Affilated to:-
RAJESTHAN UNIVERSITY OF HEALTH SCIENCES,
JAIPUR

2. CERTIFICATE
This is certify that the project entitled “Chromatography” submitted
by Vikas Singh of B.Pharma.Sem-VII(2021-22) has completed his
project as prescribed by Rajesthan University of health
sciences,Jaipur,for the award of the degree of Bachelor of pharmacy
is a record of own work carried out by her under my guidance.
APPROVED BY:- SUPERVISED BY:-
Dr.Tarachand Mr.Anup Pramanik (Assi.Prof)
Principal Regional college of Pharmacy
Regional College of Pharmacy ISI-17,RIICO Instutional Area
ISI-17,RIICO Instutional Area Sitapura,Jaipur(Raj)
Sitapura,Jaipur(Raj.)

3. ACKNOWLEDGEMENT
I have taken efforts in this project.However, it would not have been
possible without the kind support and help of many individuals. I
would like to extend my sincere thanks to all of them.
I take immense pleasure in thinking Dr.Tarachand, principal,Regional
college of Pharmacy,Jaipur having permitted me to carry out this
project work.I am highly indebted to Mr.Anup pramanik Assist.
Professor for their guidance and constant supervision as well as for
providing necessary information regarding the project and for their
support in completing the project and for giving me such attention and
time.
I would like to express my gratitude towards my belived parents for
their blessings and friends for their kind co-operation and
encouragements which help me in completion of this project.
My thanks and appreciations also go to my clssmates i.e. Mr.Pritam
Singh ,Rahul Samota etc. in developing the project who have willingly
helped me out with their abilitie
Vikas Singh

4. TABLE OF CONTENT
S.NO CHAPTER NAME PAGE NO
CERTIFICATE
ACKNOWLEDGEMENT
1 INTRODUCTION OF CHROMATOGRAPHY 1 – 3
2 TYPES OF CHROMATOGRAPHY 4 – 5
3 LIQUID CHROMATOGRAPHY 6 – 13
4 GAS CHROMATOGRAPHY 14 – 16
5 THIN LAYER CHROMATOGRAPHY 17 – 19
CONCLUSION 20
REFERENCES 21 - 23

5. CHAPTER-1
Chromatography
Introduction
Some materials appear homogenous, but are actually a combination
of substances. For example, green plants contain a mixture of
different pigments. In addition, the black ink in the pens that are used
in this experiment is a mixture of different colored materials. In many
instances, we can separate these materials by dissolving them in an
appropriate liquid and allowing them to move through an absorbent
matrix, like paper.
Chromatography is a method used by scientists for separating organic
and inorganic compounds so that they can be analyzed and studied.
By analyzing a compound, a scientist can figure out what makes up
that compound. Chromatography is a great physical method for
observing mixtures and solvents.
The word chromatography means "color writing" which is a way that
a chemist can test liquid mixtures. While studying the coloring
materials in plant life, a Russian botanist invented chromatography in
1903. His name was M.S. Tswett.
Chromatography is such an important technique that two nobel
prizes have been awarded to chromatographers. Over 60% of
chemical analysis worldwide is currently done with chromatography
or a variation thereon.
Chromatography is used in many different ways. Some people use
chromatography to find out what is in a solid or a liquid. It is also used
to determine what unknown substances are. The Police, F.B.I., and
other detectives use chromatography when trying to solve a crime. It
is also used to determine the presence of cocaine in urine, alcohol in
blood, PCB's in fish, and lead in water. Chromatography is used by
many different people in many different ways. Chromatography is
based on differential migration. The solutes in a mobile phase go
through a stationary phase. Solutes with a greater affinity for the

6. mobile phase will spend more time in this phase than the solutes that
prefer the stationary phase. As the solutes move through the
stationary phase they separate. This is called chromatographic
development.
Working
In all chromatography there is a mobile phase and a stationary phase.
The stationary phase is the phase that doesn't move and the mobile
phase is the phase that does move. The mobile phase moves through
the stationary phase picking up the compounds to be tested. As the
mobile phase continues to travel through the stationary phase it takes
the compounds with it. At different points in the stationary phase the
different components of the compound are going to be absorbed and
are going to stop moving with the mobile phase. This is how the results
of any chromatography are gotten, from the point at which the
different components of the compound stop moving and separate
from the other components.
In paper and thin-layer chromatography the mobile phase is the
solvent. The stationary phase in paper chromatography is the strip or
piece of paper that is placed in the solvent. In thin-layer
chromatography the stationary phase is the thin-layer cell. Both these
kinds of chromatography use capillary action to move the solvent
through the stationary phase.
What is the Retention Factor, Rf ?
The retention factor, Rf, is a quantitative indication of how far a
particular compound travels in a particular solvent. The Rf value is a
good indicator of whether an unknown compound and a known
compound are similar, if not identical. If the Rf value for the unknown
compound is close or the same as the Rf value for the known
compound then the two compounds are most likely similar or
identical.
The retention factor, Rf, is defined as

7. Rf = distance the solute (D1) moves divided by the distance traveled
by the solvent front (D2)
Rf = D1 / D2
Where
D1 = distance that color traveled, measured from center of the band
of color to the point where the food color was applied
D2 = total distance that solvent traveled

8. CHAPTER-2
The Different Types of Chromatography
There are four main types of chromatography. These are Liquid
Chromatography, Gas Chromatography, Thin-Layer Chromatography
and Paper Chromatography.
Liquid Chromatography is used in the world to test water samples to
look for pollution in lakes and rivers. It is used to analyze metal ions
and organic compounds in solutions. Liquid chromatography uses
liquids which may incorporate hydrophilic, insoluble molecules.
Gas Chromatography is used in airports to detect bombs and is used
is forensics in many different ways. It is used to analyze fibers on a
persons body and also analyze blood found at a crime scene. In gas
chromatography helium is used to move a gaseous mixture through a
column of absorbent material.
Thin-layer Chromatography uses an absorbent material on flat glass
or plastic plates. This is a simple and rapid method to check the purity
of an organic compound. It is used to detect pesticide or insecticide
residuesin food. Thin-layer chromatography is also used in forensics
to analyze the dye composition of fibers.
Paper Chromatography is one of the most common types of
chromatography. It uses a strip of paper as the stationary phase.

9. Types of
Chromatography
Application in the
Real Word
Why and what is it
Liquid
Chromatography
test water samples to
look for pollution,
Used to analyze metal
ions and organic
compounds in
solutions. It uses
liquids which may
incorporate
hydrophilic, insoluble
molecules.
Gas Chromatography detect bombs in
airports, identify and
quantify such drugs as
alcohol, used in
forensics to compare
fibers found on a
victim
Used to analyze
volatile gases. Helium
is used to move the
gaseous mixture
through a column of
absorbent material.
Thin-Layer
Chromatography
detecting pesticide or
insecticide residues in
food, also used in
forensics to analyze
the dye composition of
fibers
Uses an absorbent
material on flat glass
plates. This is a simple
and rapid method to
check the purity of the
organic compound.
Paper
Chromatography
separating amino acids
and anions, RNA
fingerprinting,
separating and testing
histamines, antibiotics
The most common
type of
chromatography. The
paper is the stationary
phase. This uses
capillary action to pull
the solutes up through
the paper and separate
the solutes.

10. CHAPTER-3
Liquid chromatography
Liquid chromatography is a type of chromatography where
the mobile phase is a liquid, while the stationary phase can be
solid or liquid.
According to the interaction between phases, which will define
the method of sample separation, there are several types of
liquid chromatography:
 Ion exchange chromatography.
 Affinity chromatography.
 Size exclusion chromatography.
Fig. General path flow in liquid chromatography.

11. Ion exchange chromatography:-
Ion exchange chromatography is a type of liquid
chromatography where the molecules in the sample are
separated according to their charge.
It is commonly used to separate charged biological molecules
such as proteins, peptides, amino acids, or nucleotides. For
instance, the amino acids in the proteins contain both positively
and negatively charged chemical groups. Proteins may carry a
net positive or negative charge or even no charge depending on
the pH of their environment.
The pH where the protein has no charge is called an isoelectric
point. Therefore, the choice of buffer pH determines the net
charge of the protein of interest. If the buffer pH is greater than
the isoelectric point of the protein of interest, the protein will
carry a net negative charge. On the other hand, a pH lower than
the isoelectric point will make the protein carry a net positive
charge.
Depending on the net charge of the molecules to be separated,
the stationary phase should be selected accordingly. Also, since
samples are mixtures of different compounds with different
charges, a buffer gradient is normally used to elute each different
compound separately.

12. Fig. Example of ion exchange chromatography.
Affinity Chromatography
 Chromatography is an important biophysical technique that enables
the separation, identification, and purification of the components of
a mixture for qualitative and quantitative analysis.
 It is a separation technique in which a mobile phase carrying a
mixture is caused to move in contact with a selectively absorbent
stationary phase.
 Affinity chromatography is a type of liquid chromatography for the
separation, purification or specific analysis of sample components.
 It utilizes the reversible biological interaction or molecular
recognition called affinity which refers to the attracting forced
exerted in different degrees between atoms which cause them to
remain in combination.
Example: Enzyme with an inhibitor, antigen with an antibody, etc.
 It was discovered by Pedro Cuatrecasas and Meir Wilcheck.

13. Principle of Affinity Chromatography
 The stationary phase consists of a support medium, on which the
substrate (ligand) is bound covalently, in such a way that the
reactive groups that are essential for binding of the target molecule
are exposed.
 As the crude mixture of the substances is passed through the
chromatography column, substances with binding site for the
immobilized substrate bind to the stationary phase, while all other
substances is eluted in the void volume of the column.
 Once the other substances are eluted, the bound target molecules
can be eluted by methods such as including a competing ligand in
the mobile phase or changing the pH, ionic strength or polarity
conditions.
Steps in Affinity Chromatography
 Affinity medium is equilibrated in binding buffer.
 Sample is applied under conditions that favor specific binding of the
target molecule(s) to a complementary binding substance (the
ligand). Target substances bind specifically, but reversibly, to the
ligand and unbound material washes through the column.

14.  Elution is performed specifically, using a competitive ligand, or non-
specifically, by changing the pH, ionic strength or polarity. Target
protein is collected in a purified, concentrated form.
 Affinity medium is re-equilibrated with binding buffer.
These events can be summarized into the following three major steps:
1. Preparation of Column
 The column is loaded with solid support such as sepharose, agarose,
cellulose etc.
 Ligand is selected according to the desired isolate.
 Spacer arm is attached between the ligand and solid support.
2. Loading of Sample
 Solution containing a mixture of substances is poured into the elution
column and allowed to run at a controlled rate.
3. Elution of Ligand-Molecule Complex
 Target substance is recovered by changing conditions to favor elution
of the bound molecules.
Applications of Affinity Chromatography
 Affinity chromatography is one of the most useful methods for the
separation and purification of specific products.
 It is essentially a sample purification technique, used primarily for
biological molecules such as proteins.
Its major application includes:
 Separation of mixture of compounds.
 Removal of impurities or in purification process.
 In enzyme assays
 Detection of substrates
 Investigation of binding sites of enzymes
 In in vitro antigen-antibody reactions
Size Exclusion Chromatography
Size exclusion chromatography (SEC) is a chromatographic
separation procedure that separated analyte molecules according to their
size or geometry and some cases by their molecular weight. SEC principle is
used mainly for the separation of macromolecules like proteins, enzymes,
antibodies, nucleic acid

15. s (DNA and RNA), and industrial polymers.
The size exclusion chromatography principle is based on the exclusion of
analyte molecules through a column containing porous beads.
When we used an aqueous solution for the transportation of samples
through the column, the procedure is called gel-filtration or gel permeation
chromatography.
Size exclusion chromatography column
The size exclusion chromatography column contains a porous matrix of
spherical particles or beads. Generally, SEC chromatographic columns are
composed of dextran polymers (Sephadex), agarose (Sepharose), or
polyacrylamide (Sephacryl or BioGel P).

16. Size exclusion chromatography principle
The size exclusion chromatography (SEC) principle is based on high-
performance liquid chromatography (HPLC) techniques which contain
porous particles in the column to separate molecules by their size or
molecular weight. SEC principle is used mostly to separate biological
molecules or determine molecular weight distributions of proteins and
peptides. It is also used to separate a long list of water soluble industrial
polymers.
SEC technique is used mainly for the analysis of large molecules such as
proteins, peptides, or industrial polymers.
Size exclusion chromatography works by trapping smaller molecules in the
pores of the chromatographic column.
 Molecules that are larger than the pores are unable to defuse on
the beads. Therefore, they are eluate first.
 Smaller molecules penetrate deep inside the pores and they are
eluate last.
Size exclusion chromatography procedure
The size exclusion chromatography procedure has been divided into the
following categories,
 Gel permeation or gel filtration procedure
 Ion exclusion procedure

17. Gel permeation or gel filtration procedure
Gel filtration or gel termination procedure fractionates substances
according to their molecular size. It is based upon the inclusion and
exclusion of solutes through a stationary phase containing a heterosporous
crosslinked polymeric gel.
Ion exclusion procedure
Ion exclusion is a procedure for separating ionic materials from nonionic
materials by differences in the distribution of two types of solutes between
an ion exchange resin phase and a true aqueous solution.
Applications
Applications of size exclusion chromatography give great advantages in the
separation or purification of sugars, polypeptides, viruses, proteins,
enzymes, hormones, antibodies, nucleic acids (DNA and RNA), etc.
The main application of SEC is to separate or analysis of proteins and other
water-soluble polymers from their mixture. It is also used to examine the
stability and properties of natural organic matter present in water.

18. CHAPTER-4
What Is Gas Chromatography?
Gas Chromatography or Gas Liquid Chromatography is a technique
applied for separation, identification and quantification of components of
a mixture of organic compounds by selective partitioning between the
stationary phase and mobile phase inside a column followed by
sequential elution of separated components. The technique is suitable
for separation of compounds having following characteristics :
 High volatility
 Thermal stability
 Low molecular weights
Gas
Chromatograph
Purpose of gas chromatography
The main purpose of the gas chromatography technique is to separate
the compounds that possess:
 High volatility
 Low molecular weights
 Thermal stability.

19. Sample injector
The injector is a heated block where the sample is injected. Through the
carrier gas stream, the sample is spontaneously vaporized and led to the
column.
With the help of a gas-tight syringe, the liquid sample mixtures are
injected whereas, with the help of automated injection valves, the
gaseous mixtures are injected.
Column
This is filled with the stationary phase or its walls are covered with a
liquid adsorbent. This is done for selective absorbance and retention of
the sample components.
Commonly used: Packed columns and Capillary columns (More popular)
Component of a Column – Oven
The column is enclosed by a column oven which is responsible for
maintaining a constant temperature during isothermal operation. This
temperature when temperature programming is needed can be
increased in a controlled way for acquiring effective separation of
mixture components possessing different volatilities.
Detector
This is employed for the identification and quantification of components.
Here, the regions of individual peaks created relate to their
concentrations and their retention times are representative of their
identity.
Common examples: Flame ionization detector, Thermal conductivity
detector (TCD), and Electron capture detector (ECD).
Data system
It is a set of dedicated software that provides control over many
important operational parameters like injection sequence, wash cycles,
over-temperature control, the flow rate of gases, detector wavelength,
etc. Simultaneously, the data station calculates and displays the
parameters.

20. Gas chromatography applications
Since the discovery of the gas chromatographic system, the areas of
Gas chromatography applications is ever-increasing which includes:
 Pharmaceutical industry
 Research
 Medical and Forensic
 Environmental monitoring (both inside laboratories, and natural
water bodies)
 Petroleum refining and petrochemicals
 Edible oils
 Flavors, beverages, and the food industry
 Fragrance industry (Cosmetics)
 Polymers and plastics

21. CHAPTER-5
Thin Layer Chromatography
Introduction
Thin Layer Chromatography is a technique used to isolate non-volatile
mixtures. The experiment is conducted on a sheet of aluminium foil,
plastic, or glass which is coated with a thin layer of adsorbent material.
The material usually used is aluminium oxide, cellulose, or silica gel.
On completion of the separation, each component appears as spots
separated vertically. Each spot has a retention factor (Rf) expressed as:
Rf = dist. travelled by sample / dist. travelled by solvent
The factors affecting retardation factor are the solvent system, amount of
material spotted, adsorbent and temperature. TLC is one of the fastest,
least expensive, simplest and easiest chromatography technique.
Thin Layer Chromatography Procedure

22. Before starting with the Thin Layer Chromatography Experiment, let us
understand the different components required to conduct the procedure
along with the phases involved.
1. Thin Layer Chromatography Plates – ready-made plates are used
which are chemically inert and stable. The stationary phase is
applied on its surface in the form of a thin layer. The stationary
phase on the plate has a fine particle size and also has a uniform
thickness.
2. Thin Layer Chromatography Chamber – Chamber is used to
develop plates. It is responsible to keep a steady environment
inside which will help in developing spots. Also, it prevents the
solvent evaporation and keeps the entire process dust-free.
3. Thin Layer Chromatography Mobile phase – Mobile phase is the
one that moves and consists of a solvent mixture or a solvent. This
phase should be particulate-free. The higher the quality of purity
the development of spots is better.
4. Thin Layer Chromatography Filter Paper – It has to be placed
inside the chamber. It is moistened in the mobile phase.
Thin Layer Chromatography Applications
 The qualitative testing of Various medicines such as sedatives,
local anaesthetics, anticonvulsant tranquilisers, analgesics,
antihistamines, steroids, hypnotics is done by TLC.
 TLC is extremely useful in Biochemical analysis such as
separation or isolation of biochemical metabolites from its blood
plasma, urine, body fluids, serum, etc.
 Thin layer chromatography can be used to identify natural products
like essential oils or volatile oil, fixed oil, glycosides, waxes,
alkaloids, etc.
 It is widely used in separating multicomponent pharmaceutical
formulations.
 It is used for the purification of samples and direct comparison is
done between the sample and the authentic sample.
 It is used in the food industry, to separate and identify
colours, sweetening agent, and preservatives
 It is used in the cosmetic industry.
 It is used to study if a reaction is complete.

23. Advantages of Thin-Layer Chromatography
 A simple method of component separation.
 Fewer types of equipment are used in this technique. As the
components elute rapidly, the separation is achieved in a very
short time.
 It is possible to visualize all elements of UV light.
 By this process, the non-volatile compounds can be isolated.
 It is also possible to separate the microlitre volume of the sample
through TLC.
 Easy isolation and recovery of the components of complex
mixtures.
Disadvantages Of Thin Layer Chromatography:
 Thin Layer Chromatography plates do not have longer stationary
phase.
 When compared to other chromatographic techniques the length of
separation is limited.
 The results generated from TLC are difficult to reproduce.
 The detection limit is high and therefore if you want a lower
detection limit, you cannot use TLC.
 It is only a qualitative analysis technique and not quantitative.

24. Conclusion
Chromatography can purify basically any soluble or volatile substance
if the right adsorbent material, carrier fluid, and operating conditions are
used. • A mixture of different components enters a chromatography
process, and the different components are flushed through the system at
different rates. These differential rates of the mixture as it moves over
adsorptive materials provide separation • Even very similar components,
such as proteins that may only vary by a single amino acid, can be
separated with chromatography • Because chromatography is usually
not performed under serious conditions it can be used to separate
delicate products
chromatography has become a popular technique. While there are many
pharmaceuticals that are being manufactured using SMB, the interest is
growing for its use in other industries and research.  Higher purity,
lower cost, and production edge can make SMB more attractive than
single column chromatography.  Most new developments being made
in SMB focus on optimizing the technique through sensors and valve
switching. These advancements are often safeguarded trade secrets
My hypothesis turned out to be correct for the most part. The non-
primary colors each split up into different colors when I added water,
and the red split up into red and pink. The other primary color, yellow,
didn’t separate into different colors.This happened because the marker
inks are made up of different color molecules. The water separated them.
Some inks are made of a single type of molecule. Others are made up of
a mixture of molecules. Using paper chromatography, I was able to
separate the colors and see all the colors they were made of.

25. Refrences
 1”Chromatographic Techniques for Preparative Separation with
Moderate Resolution." Still, W. C.; Kahn, M.; Mitra, A. J. Org.
Chem. 1978, 43 (14),
 2. HISTORY OF AFFINITY CHROMATOGRAPHY • 1930s, first
developed by A.Wilhelm Tiselius-a swedish biochemist, won the Nobel
Prize in 1948. • Used to study enzymes and other proteins. • Relies on
the affinity of various biochemical compounds with specific properties.
GOKULAKRISHNAN CHROMATOGRAPHY 2
 3. AFFINITY CHROMATOGRAPHY: Discovered by Pedro and Meir
Wilcheck. Affinity chromatography is a type of chromatography that
makes use of a specific affinity between a substance to be isolated and
a molecule that it can specifically bind. GOKULAKRISHNAN
CHROMATOGRAPHY 3
 4. PRINCIPLE Based on specific affinity between substance to be
isolated and a molecule that it can specifically bind(a ligand). M + L ML
Macromolecule Ligand (attached Complex to matrices)
GOKULAKRISHNAN CHROMATOGRAPHY 4
 5. EXAMPLES  Antigen -Antibody  Antibody- Antigen  Substrate -
Enzyme  DNA- Histon  Hormone -Binding Protein/Receptor
GOKULAKRISHNAN CHROMATOGRAPHY 5
 6. SPECIFICITY OF AFFINITY CHROMATOGRAPHY Specificity is
based on three aspect of affinity: Matrix: for ligand attachment. Spacer
arm: used to bind ligand to matrix. Ligand: molecule that binds reversibly
to a specific target molecule(site of interaction). GOKULAKRISHNAN
CHROMATOGRAPHY 6
 7. PROCEDURE  The Sample is injected into the equilibrated affinity
chromatography column.  Only the substance with affinity for the ligand
are retained on the column.  The substance with no affinity to the ligand
will elute off.  The substances retained in the column can be eluted off
by changing the pH of salt or organic solvent concentration of the
eluent.GOKULAKRISHNAN CHROMATOGRAPHY 7
 8. GOKULAKRISHNAN CHROMATOGRAPHY 8
 9. MATRIX The matrix is an inert support to which a ligand can be directly
or indirectly coupled. It has some peculiar qualities like:-  Does not itself
adsorb molecules to a significant amount.  Ligand must be coupled
without altering its binding properties.  Stability under a wide range of

26. experimental conditions such as high and low pH, detergent and
dissociating conditions.  The most useful matrix materials are agarose
and polyacrylamide GOKULAKRISHNAN CHROMATOGRAPHY 9
 10. SUPPORT MATERIALS USED IN AFFINITY CHROMATOGRAPHY
GOKULAKRISHNAN CHROMATOGRAPHY 10 SUPPORT MATERIALS
TRADE NAME Agarose Sepharose 2B,4B,6B Cross-linked dextran
Sephadex Polyacrylamide Bio-gel-P Cross linked cellulose Matrex
Cellufine
 11. LIGAND: The ligand is the molecule that binds reversibly to a specific
molecule or group of molecules ,enabling purification by affinity
chromatography. The selection of the ligand for affinity chromatography
is influenced by two factors: Ligand must exhibit specific and reversible
binding affinity for the target substance(s) It must have chemically
modifiable groups that allow it to be attached to the matrix without
destroying binding activity. GOKULAKRISHNAN CHROMATOGRAPHY
11
 12. COMMENLY USED LIGANDS GOKULAKRISHNAN
CHROMATOGRAPHY 12 LIGAND AFFINITY Concanavalin A
Glycoproteins and polysaccharides Calmodulin Calmodulin binding
enzymes Avidin Biotin containing enzyme Heparin Lipoproteins, lipases ,
coagulation factors, DNA polymerases , steroid receptor proteins serine
protease inhibiter Proteins A and G Immunoglobins
 13. TYPES  Lectin Affinity Chromatography  Immuno Affinity
chromatography  Metal Chelate Chromatography  Dye Ligand
Chromatography  Covalent chromatography GOKULAKRISHNAN
CHROMATOGRAPHY 13
 14. LECTIN AFFINITY CHROMATOGRAPHY  Used for purification of
glycoproteins particularly membrane receptor proteins.  Lectins are a
group of proteins produced by plants & animals, which have the ability to
bind carbohydrates and glycoproteins.  Used to separate mixtures of
cells by taking advantage of the saccharide components of their outer
membrane.  Commonly used lectins are: ConcanavalinA, Soyabean
lectin,etc. GOKULAKRISHNAN CHROMATOGRAPHY 14
 15. IMMUNO AFFINITY CHROMATOGRAPHY  Exploited in the
isolation & purification of a range of proteins including antigens,
membrane proteins of viral origin.  Used for purification of antibodies.
Ligands used is Protein A and protein B. METAL CHELATE
CHROMATOGRAPHY  Special form of chromatography in which an
immobilised metal ions such as Cu 2 + ,Zn2 +,Mn2 +,Ni2 + etc. are used.

27.  Used for purification of proteins containing imidazole groups or indole
groups.  Commonly metal ions are immobilised by attachment to an
imino-diacetate or tris(carboxymethyl)ethylenediamine substituted.
GOKULAKRISHNAN CHROMATOGRAPHY 15
 16. DYE LIGAND CHROMATOGRAPHY  Uses a number of triazine
dyes as ligands. Most widely used dye is Cibracron Blue. F3G-A. Used
for purification of lipoproteins, interferons, coagulation factors etc.
COVALENT CHROMATOGRAPHY  Developed specifically to separate
thiol containing proteins.  Most commonly used ligand is a disulphide 2’-
pyridyl group.  Used for purification of a number of proteins but its use
is limited by its cost and rather difficult regeneration stage.
GOKULAKRISHNAN CHROMATOGRAPHY 16
 17. AFFINITY CHROMATOGRAPHY Can be used,  Purify and
concentrate a substance from a mixture into a buffering solution. 
Reduce the amount of a substance in a mixture.  Discern what biological
compounds bind to a particular substance, such as drugs.  Purify and
concentrate an enzyme solution. GOKULAKRISHNAN
CHROMATOGRAPHY 17
 18. APPLICATIONS Used in Genetic Engineeringnucleic acid
purification Production of Vaccinesantibody purification from blood
serum Basic Metabolic Researchprotein or enzyme purification from cell
free extracts. GOKULAKRISHNAN CHROMATOGRAPHY 18
 19. ADVANTAGES OF AFFINITY CHROMATOGRAPHY  Extremely
high specificity.  High degrees of purity can be obtained.  The process
is very reproducible.  The binding sites of biological molecules can be
simply investigated. GOKULAKRISHNAN CHROMATOGRAPHY 19
 20. DISADVANTAGES OF AFFINITY CHROMATOGRAPHY 
Expensive ligands  Leakage of ligand  Degradation of the solid support
 Limited lifetime  Non-specific adsorption  Relatively low
productivityGOKULAKRISHNAN CHROMATOGRAPHY 20
 21. REFERENCE  Hand book of Affinity chromatography, principles and
Method from GE Healthcare  Practical Biochemistry, Principles and
techniques by Keith Wilson and John Walker, Cambridge University
Press  Affinity Chromatography.