Supervisor: Submitted by

Assignment of
SUPERVISOR: SUBMITTED BY:

Prof. Asmita Gajbhiye Arpit Khare
Dr. Adarsh Sahu Y19150012
Mr. Debarshi Kar Mahapatra B.Pharm (7th Sem)
Department of Pharmaceutical Sciences,
Dr. Harisingh Gour Sagar University
(A Central University)
CERTIFICATE
I would like to express my special thanks of gratitude to Prof. Asmita
Gajbhiye , who gave me the golden opportunity to do this assignment
of Instrumental Methods of Analysis on Affinity chromatography
(Applications). I came to know about so many new things .I would
also like to thank my parents and friends who helped me a lot in
completing this assignment within the limited time frame.
Arpit Khare
Y19150012 B.pharm , VII semester
ACKNOWLEDGEMENT
This is to certify that Arpit Khare, student of B.pharm ,VII

semester, Department of Pharmaceutical sciences, DHSGSU
has successfully completed the assignment on the Affinity
chromatography (Applications) under the guidance of
respected subject teachers during the year 2022-23.
Signature of teacher
APPLICATIONS OF
AFFINITY
CHROMATOGRAPHY
a) Immunoglobin purification (antibody
immobilisation)
b) Recombinant tagged proteins,
c) Protein a, g and l purification,
d) Biotin and biotinylated molecules
purification,
e) Affinity purification of albumin and
macroglobulin contamination,
f) Lectin affinity chromatography, and
g) Reversed phase chromatography
IMMUNOGLOBIN PURIFICATION (ANTIBODY IMMOBILISATION)
i. Antibodies can be immobilised by covalent and adsorption methods.
ii. Random covalent immobilisation methods are used for linking the antibodies to the solid
support via their free amine groups using cyanogen bromide, N-hydroxysuccinimide, N,N'
carbonyldiimidazole, tresyl chloride, or tosyl chloride.
iii. These are random immobilisation methods, so the antibody binding sites may get blocked
due to improper orientation, multi-site attachment, or steric hindrance.
RECOMBINANT TAGGED PROTEINS
1. Purification of proteins becomes easier and simpler if the desired protein is tagged with a
known sequence.
2. The properties of fusion tags allow the tagged proteins to be easily employed in the
laboratory.
3. The well-characterised tag-ligand chemistry allows single step affinity purification of

tagged molecules using immobilised versions of their corresponding affinity ligands.
PROTEIN A, G AND L PURIFICATION
a) These are native or recombinant proteins of microbial origin. They bind to specific
immunoglobulins, including immunoglobulin G (IgG, representing 80% of serum
immunoglobulins).
b) The proteins A and G are combined and used for purifying mammalian IgG samples.
c) The protein L binds to the kappa light chain of immunoglobulins and these chains
exist in other immunoglobulins also (i.e., IgG, IgM, IgA, and IgE), thus protein L is
used for purifying antibodies of different classes.
BIOTIN AND BIOTINYLATED MOLECULES PURIFICATION
I. By incorporating a biotin tag into a biomolecule, it is used for purifying the biomolecule
using a streptavidin or avidin affinity support.
II. This can be done by inserting a biotinylation sequence into a recombinant protein.
III. Biotin has an advantage as an affinity tag that due to its small size it produces minimum
effect on the activity of a large biomolecule.
IV. Biotinylated biomolecules can be purified by immobilising streptavidin on a support

material, and using streptavidin for extracting the biotinylated molecules out of solution.
AFFINITY PURIFICATION OF ALBUMIN AND
MACROGLOBULIN CONTAMINATION
 Affinity purification is used for cleaning up and removing excess of albumin

and α2 -macroglobulin contamination from samples.
 It is necessary to remove them as they can interrupt with the analysis (e.g.,
mass spectrometry and immunoprecipitation).
 In this method, the dye ligand is covalently bound to sepharose via

chlorotriazine ring.
 Albumin binds non-specifically by electrostatic and/or hydrophobic

interactions with the aromatic anionic ligand.
LECTIN AFFINITY CHROMATOGRAPHY
 Lectins are carbohydrate-binding proteins containing two or more

carbohydrate binding sites.
 In lectin affinity chromatography, protein is bound to an

immobilised lectin via its sugar moieties (N linked or O-linked).
 After the glycosylated protein binds to the affinity support, the

unbound contaminants are washed off, and the purified protein is
eluted.
 A wide range of lectins are commercially available in

immobilised form
REVERSED PHASE CHROMATOGRAPHY
 This technique involves affinity interaction between a biomolecule (e.g.,

proteins, peptides, and nucleic acids) dissolved in a solvent (mobile phase)
having some hydrophobicity and an immobilised hydrophobic ligand
(stationary phase).
 Reversed phase chromatography is suitably used for separating non-
volatile molecules.
 The macromolecules (e.g., protein or peptides) are adsorbed on the
hydrophobic column surface.
 Elution is carried out using a mobile phase, which is a combination of
water and organic solvents.
 Macromolecules remain bound to the hydrophobic column surface till the
organic phase concentration is sufficiently high to elute the
macromolecules from the hydrophobic surface.
 In reversed phase chromatography, the highly polar macromolecules are
eluted first and the non-polar ones are eluted later, i.e., more the polarity
(hydrophilic) of a solute, faster is the elution and vice versa.
OTHER APPLICATIONS
1. This technique has great impact on molecular biology,

biochemistry, biotechnology.
2.Affinity chromatography is used as a powerful tool for
biologically active molecules purification process like proteins.
3. Affinity chromatography can be used whenever a suitable ligand
is available for protein of interest.
4. This technique initially used for purification of enzymes but now
it is used for various purposes like purification of nucleotides,
nucleic acid, immunoglobin, membrane receptors etc.
REFERENCES
 Malathi S, Patil P.V, Kumar S; “Instrumental Method Of Analysis”; Published By

Thakur Publication Pvt. Ltd. Lucknow; 2019th Edition; 290-93.
 Mahadik K.R, Satyanarayanan L; “A Text Book Of Instrumental Method Of Analysis”;

Published By Nirali Prakashan; 2019th Edition; 18.4.
 Sirisha P, Etal; “A Overview Of Affinity Chromatography”; Indo American Journal Of

Pharmaceutical Research; 2018; 1462-71.
 Hage D.S; “Affinity Chromatography: A Review Of Clinical Application”; University

Of Nebraska; 1999; 593-615.

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Supervisor: Submitted by

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Assignment of

SUPERVISOR: SUBMITTED BY:

This is to certify that Arpit Khare, student of B.pharm ,VII

i. Antibodies can be immobilised by covalent and adsorption methods.

3. The well-characterised tag-ligand chemistry allows single step affinity purification of

IV. Biotinylated biomolecules can be purified by immobilising streptavidin on a support

 Affinity purification is used for cleaning up and removing excess of albumin

 In this method, the dye ligand is covalently bound to sepharose via

 Albumin binds non-specifically by electrostatic and/or hydrophobic

 Lectins are carbohydrate-binding proteins containing two or more

 In lectin affinity chromatography, protein is bound to an

 After the glycosylated protein binds to the affinity support, the

 A wide range of lectins are commercially available in

 This technique involves affinity interaction between a biomolecule (e.g.,

1. This technique has great impact on molecular biology,

 Malathi S, Patil P.V, Kumar S; “Instrumental Method Of Analysis”; Published By

 Mahadik K.R, Satyanarayanan L; “A Text Book Of Instrumental Method Of Analysis”;

 Sirisha P, Etal; “A Overview Of Affinity Chromatography”; Indo American Journal Of

 Hage D.S; “Affinity Chromatography: A Review Of Clinical Application”; University

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