·
Affinity use
a chromatography required specific ligand that has been immobilized chemically
to an insoluble matrix to adsorb reversibly a single molecule species (affine
components) from a mixture of solutes.
·
It exploits the unique property of extremely
specific reversible biological interaction to achieve separation and
purification.
The
separation exploits “Lock n Key” binding that prevalent in a biological system.
Two types of ligands are there
Monospecific
ligand
and
Group-specific ligand
Monospecific:-
·
Mono specific ligands bind only to one specific
antibody/ antigen
·
Disadvantages:-
·
It has a high effectively so special ligand matrix
combination is require for every substance to be purified.
·
Bind more strongly and require harsh elutents than
group specific.
·
Eg. Steroid hormone, vitamins, certain enzyme
inhibitor.
Group specific:
·
It binds to specific group on target species.
·
Important for the elucidation of the molecule.
·
It is possible to immobilized the ligand via
different chemical groups of the ligand.one can identify those structures that
are essential for binding by means of their adsorption behaviour.
·
Eg. Dyes
Choice of Ligand or properties Of
Ligand:
·
Specifically form a reversible complex with the substance
to be purified.
·
A chemically modified group through which the
covalent linkage to matrix can occur should be present.
·
Form stable complex on binding to substance to be
purified.
·
Should be able to withstand harsh condition used
during process.
·
Easy to dissociate the complex by simple change in
medium without by adversely affecting the component.
·
Consistently available and economical.
Example of ligand
Following
are the example of the ligand which attract the particular target molecule
sample
Enzyme:
subtract analogue
Lectin:
polysaccharide
Hormone:
receptor
Nucleic
acid : Complementary base
sequence
Metal ion: Poly
fusion protein
Protein A and G : Immunoglobulin
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