Affinity chromatography was first developed in the 1930s by Swedish biochemist Arne Tiselius. It relies on the reversible binding of biochemical compounds to separate and purify proteins and other molecules. Specificity is based on the matrix, spacer arm, and ligand. The sample is injected and molecules that bind to the ligand are retained on the column while others pass through. The retained molecules can then be eluted by changing pH or solvent concentration. Affinity chromatography is used to purify antibodies, enzymes, nucleic acids, and other biomolecules.

2. Affinity History
• 1930’s first developed by A.Wilhem 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

3. Examples
• Antigen Antibody
• Antibody Antigen
• Substrate Enzyme
• DNA Histon
• Hormone Binding Protein/
Receptor

4. SPECIFICITY OF AFFINITY
CHROMATOGRAPHY
• Specificity is based on 3 aspects.
MATRIX: for ligand attachment
SPACER ARM: use to bind ligand to
matrix
LIGAND: molecule that binds
revesibly to a target
molecule (site of interaction)

6. So Now What….???
• 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 substance retained in the column can be
eluted off by changing the pH or organic
solvents concentration of the eluent.

7. Matrix…
• The matrix simply provides a structure to
increase the surface area to which the
molecule can bind
• The matrix must be activated for the ligand to
bind to it but still to retain it’s own activation
towards target molecules

8. Matrix
• Amino, hydroxyl, carbonyl & thio groups
located with the matrix serve as ligand binding
sites.
• Matrix are made up of agarose & other
polysaccharides
• The matrix also must be able to withstand the
decontamination process of rinsing with
sodium hydroxide or urea

9. Ligand
• The ligand binds only to the desired molecule
within the solution
• The ligand attaches to the matrix which is made
up of an inert substance
• The ligand should only interact with the desired
molecule & from a temporary bond
• The ligand/ molecule complex will remain in the
column, eluting everything else off
• The ligand/ molecule complex dissociates
changing the pH

10. Antibody affinity
(Immuno-affinity Chromatography)
• Used to purify antibody against a specific antigen
Eg: Immunoglobulins
Purification of IgG, IgG 1 fragments & subclasses have the
high affinity of protein A & protein G for the Fc region
of polyclonal & monoclonal IgG-type antibodies

11. Affinity Chromatography
Can be used;
• Purify & concentrate a substance from a
mixture into a buffering solution
• Reduce the amount of substance in a mixture
• Discern what biological compounds bind to a
particular substance, such as drugs
• Purify & concentrate an enzyme solution

12. 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