Affinity chromatography is a laboratory technique for separating mixtures, particularly used for biological molecules like proteins, by exploiting specific interactions with immobilized ligands. Developed in the 1930s, it allows for high specificity and purity by binding target molecules to a stationary phase, while unbound components are washed away. Its applications span genetic engineering, vaccine production, and therapeutic uses, though it has disadvantages including high costs and potential ligand leakage.

1. AFFINITY
CHROMATOGRAPHY
Sweta Yadav

2. CHROMATOGRAPHY
Chromatography is the collective term for a set
of laboratory techniques for the separation of
mixtures.

3. CHROMATOGRAPHY
It is a physical method of separation in which the
components to be separated are distributed between
two phases, one of which is stationary (immobilize
phase) while the other (the mobile phase) moves in a
definite direction

4. • Can separate complex mixtures composed of many
very similar components.
• Chromatography is often coupled with analytical
instruments to complete analysis.
• A single chromatographic analysis can isolate, identify
, and quantitate multiple components of mixtures

5. The term comes from Greek
χρώμα:chroma means color and
γραφειν:graphein means write

6. INVENTED BY
⚫1903 – Tswett, a
Russian botanist
coined the term
chromatography.

7. TERMINOLOGY
An immobilized phase is a stationary phase which is
immobilized on the support particles, or on the inner wall of the
column tubing.
The mobile phase is the phase which moves in a definite
direction. It may be a liquid (LC), a gas (GC), or a supercritical
fluid (SFC).
A better definition:The mobile phase consists of the sample being
separated and the solvent that moves the sample through the
column.

8. TERMINOLOGY
The analyte is the substance that is to be separated
during chromatography.
The sample is the matter analysed in
chromatography. It may consist of a single component
or it may be a mixture of components.

9. ⚫Elution
- washing of the mixture
⚫Eluent
- additional solvents used for elution
⚫Residency
- time spent on column

10. Types of Chromatography
Techniques

11. Properties of Chromatography
1) Immiscible stationary and mobile phases
2) An arrangement where a mixture is deposited at one
end of the Stationary phase
3) Flow of the mobile phase towards the other end of
the stationary Phase
4) Different rates of partitioning for each component
5) Means for visualizing the separation of each
component

12. AFFINITY CHROMATOGRAPHY

13. Affinity History
• 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
• Affinity Chromatography is essentially a sample purification
technique, used primarily for biological molecules such as
proteins.

14. • It is a method of separating a mixture of proteins or nucleic acids
(molecules) by specific interactions of those molecules with a
component known as a ligand, which is immobilized on a
support. If a solution of, say, a mixture of proteins is passed over
(through) the column, one of the proteins binds to the ligand on
the basis of specificity and high affinity (they fit together like a
lock and key).
• The other proteins in the solution wash through the column
because they were not able to bind to the ligand.

16. The principle of affinity chromatography
1)Inject a sample into an initially equilibrated affinity chromatography
column.
2) Only the substances with affinity for the ligand are retained in the
column.
3) Other substances with no affinity for the ligand are eluted from the
column.
4) The substances retained in the column can be eluted from the column
by changing pH or salt or organic solvent concentration of the eluent.

17. 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)

18. Chromatographic Media
• A matrix in its use here is a substance, usually in bead form to which a
specific ligand is covalently bound.
Properties:
• 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

21. Immobilized Ligand
• 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
effector may be used as an immobilized ligand.

22. Examples
● Antigen Antibody
● Antibody Antigen
● Substrate Enzyme
● DNA Histon
● Hormone Binding
Protein/Receptor

23. Attachment of Ligand to Matrix
Several procedures have been developed for the covalent
attachment of the ligand to the stationary phase. All procedures
for gel modification proceed in two separate
Chemical steps:
• Activation of the functional groups on the matrix and
• Joining of the ligand to the functional group on the matrix.

25. Pre-packed columns
• Hi-Trap Heparin HP (High performance)
• Column size: 5 × 1 mm 1 × 5 mm 5 × 5 mm
• Average particle diameter : 34μm
• Maximum operating flow rate: 4 ml/min 20 ml/min.

26. Storage of pre-packed columns
• At 2-8 °C in an upright position with both caps in place.
• Thio-mersal may be added for long term storage.
• DO NOT FREEZE
• Application areas : purification, isolation or removal of the
following substances: Anti-thrombin III and other coagulation
factors, lipoproteins, lipases, protein synthesis factors

27. 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

28. Applications
• Used in Genetic Engineering
• - nucleic acid purification
• Production of Vaccines
• - antibody purification from blood serum
• And Basic Metabolic Research
• - protein or enzyme purification from cell
free extracts

29. Industrial Applications
• Affinity chromatography is widely used in the pharmaceutical
industry to purify and extract molecules of interest from
complex mixtures.
• These molecules tend to be enzymes, proteins or amino acids,
but other biological species can be selectively retained.
• Once isolated, these biological species can be selectively
amplified to produce larger quantities, although at large
concentrations.

30. Therapeutic & clinical application:
• Hyper-lipidemia : here the sample is made to pass through
coloumn containing antibody & plasma LDL so, it can easily be
separated out by eluting with glycine hydrochloride buffer
(pH 3).
• Others :
• Pregnancy test
• Allergy test
• Immuno assay
• Kinetic studies
• Qualitative measurment of substrate.

31. ADVANTAGES OF AFFINITY CHROMATOGRAPHY
1) Extremely high specificity
2) High degrees of purity can be obtained
3) The process is reproducible
4) The binding sites of biological molecules can be simply
investigated

32. DISADVANTAGES OF AFFINITY CHROMATOGRAPHY
1) Expensive ligands
2) Leakage of ligand
3) Degradation of the solid support
4) Limited lifetime
5) Non-specific adsorption
6) Relatively low productivity

33. REFERENCES
[1]http://en.wikipedia.org/wiki/Affinity_chromatography
[2]www.apsu.edu/reedr/.../Affinity%20Chromatography%201.ppt
[3] www.rpi.edu/dept/chem-eng/WWW/faculty/.../Lecture%2001.pdf -
[4]www.chemistryinnovation.co.uk/.../Technology%20Area
%20Affinity%20Chromatography.pdf -

34. THANKS…