B pharm 7th sem industrial method of analysis

1. Assignment on: Affinity Chromatography
Submitted to:
Prof. (Dr.) P. Malairajan
Head Of Department
Submitted By:
Name: Netra Prasad Neupane
Bachelor of pharmacy (7th sem)
Id No. 17BPH080
Subject: instrumental method of analysis (701P)
Sam Higginbottom University of Agriculture,
Technology and Sciences, Prayagraj, UP
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2. Acknowledgment
In preparation of my assignment, I had to take the
help and guidance of some respected seniors and
professors, who deserve my deepest gratitude. As the
completion of this assignment gave me much
pleasure. I would like to show my gratitude to Prof.
(Dr.) P. Malairajan (HOD, SHIATS.FHS,SHUATS)
Course Instructor. He gave me wonderful opportunity
to prepare assignment on “AFFINITY
CHROMATOGRAPHY” topics. I would also like to
expand my gratitude to all those who have directly
and indirectly guided me in writing this assignment.
Many people, especially my parents and classmates
have made valuable comment suggestions on my
assignment which gave me an inspiration to improve2

3. Tablet of contents
S.N CONTENTS
1. Introduction and definition to affinity chromatography
2. Common term use in affinity chromatography, Principle of
affinity chromatography
3. Components of affinity chromatography
4. Step involves in affinity chromatography
5. Application, advantage and disadvantage
6. Conclusion and References
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4. Introduction to affinity
chromatography
 Affinity chromatography separates proteins on the
basis of a reversible interaction between a protein
(or group of proteins) and a specific ligand
coupled to a chromatography matrix.
 The technique offers high selectivity, hence high
resolution, and usually high capacity for the
protein(s) of interest. Purification can be in the
order of several thousand-fold and recoveries of
active material are generally very high.
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5. Definition of affinity chromatography
 Affinity chromatography is unique in purification
technology since it is the only technique that
enables the purification of a biomolecule on the
basis of its biological function or individual
chemical structure.
 The technique can be used to separate active
bimolecule from denatured or functionally
different forms, to isolate pure substances
present at low concentration in large volumes of
crude sample and also to remove specific
contaminants.
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6. Common terms in affinity chromatography
 Matrix: for ligand attachment. Matrix should be
chemically and physically inert.
 Spacer arm: used to improve binding between
ligand and target molecule by overcoming any
effects of steric hindrance.
 Ligand: molecule that binds reversibly to a
specific target molecule or group of target
molecules.
 Binding: buffer conditions are optimized to
ensure that the target molecules interact
effectively with the ligand and are retained by the
affinity medium as all other molecules wash
through the column.6

7.  Elution: buffer conditions are changed to
reverse (weaken) the interaction between the
target molecules and the ligand so that the target
molecules can be eluted from the column.
 Wash: buffer conditions that wash unbound
substances from the column without eluting the
target molecules or that re-equilibrate the column
back to the starting conditions (in most cases the
binding buffer is used as a wash buffer).
 Ligand coupling: covalent attachment of a
ligand to a suitable pre-activated matrix to create
an affinity medium.
 Pre-activated matrices: matrices which have
been chemically modified to facilitate the coupling
of specific types of ligand.7

8. 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 mobile8

9. Components of Affinity Chromatography
1) Matrix
 The matrix is an inert support to which a ligand can be directly or
indirectly coupled.
 In order to for the matrix to be effective it must have certain
characters:
 Matrix should be chemically and physically inert.
 It must be insoluble in solvents and buffers employed in the
process
 It must be chemically and mechanically stable.
 It must be easily coupled to a ligand or spacer arm onto which
the ligand can be attached.
 It must exhibit good flow properties and have a relatively large
surface area for attachment.
 The most useful matrix materials are agarose and
polyacrylamide.9

10. 2) Spacer arm
 It is used to improve binding between ligand and
target molecule by overcoming any effects of steric
hindrance.
3) Ligand
 It refers to the molecule that binds reversibly to a
specific target molecule.
 The ligand can be selected only after the nature of the
macromolecule to be isolated is known.
 When a hormone receptor protein is to be purified by
affinity chromatography, the hormone itself is an ideal
candidate for the ligand.
 For antibody isolation, an antigen or hapten may be
used as ligand.
 If an enzyme is to be purified, a substrate analog,
inhibitor, cofactor, or effectors may be used as a the
immobilized ligand.10

11. 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.
 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.
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12. 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 Complex12

13. Applications and uses of affinity
chromatography.
1) Immunoglobulin purification (antibody
immobilization)
2) Recombinant tagged proteins.
3) Protein A, G, and L purification
4) Biotin and biotinylated molecules purification
5) Affinity purification of albumin and macroglobulin
contamination.
6) Separation of mixture of compounds.
7) Detection of Single Nucleotide polymorphisms and
mutations in nucleic acids
8) In enzyme assays
9) Detection of substrates
10) Investigation of binding sites of enzymes
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14. Advantages of Affinity
Chromatography
 High specificity
 Target molecules can be obtained in a highly pure
state
 Single step purification
 The matrix can be reused rapidly.
 The matrix is a solid, can be easily washed and dried.
 Give purified product with high yield.
 Affinity chromatography can also be used to remove
specific contaminants, such as proteases.
Disadvantage
 Time consuming method.
 More amounts of solvents are required which may
be expensive.
 Intense labour
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15. Conclusion : In above, details study about affinity
chromatography, Common term use in affinity
chromatography, Principle of affinity chromatography ,
Components of affinity chromatography , Step involves in
affinity chromatography, Application, advantage and
disadvantage has been done.
References :
1) https://microbenotes.com/affinity-chromatography/
(accessed date 02/09/2020)
2) http://cdn.intechopen.com/pdfs/33046/InTech
Affinity_chromatography_principles_and_applications.
pdf (accessed date 02/09/2020)
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