AFFINITY

CHROMATOGRAPH
Y Chaithanya V Sagar
AM.BT.U3BIT21113
S5 BSc BT-B
 PRINCIPLE
The fundamental idea behind affinity chromatography is to take use of the specific binding
affinity that exists between an immobilized target molecule (like an important protein) and a
ligand (like an enzyme substrate, an antibody, or an antigen). Other non-specific molecules pass
through or are washed away while the target molecule attaches securely to the immobilized
ligand. The target molecule is eluted after purification, often by altering buffer conditions to
break the particular binding relationship.

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 STEPS
INVOLVED
Preparing the Matrix:
The production of a chromatography column filled with a solid support matrix is the first
step in affinity chromatography. Agarose, sepharose, and other porous materials that may
be readily functionalized with the required ligand are common matrices.

Immobilisation of Ligands:
The ligand, which binds to the target molecule precisely, is covalently bonded to the
matrix. During the purification process, this step ensures that the ligand remains stable
and ready for binding.

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Loading of Samples:
The sample containing the biomolecule combination is put to the column. Non-specific
molecules pass through or are washed away by a buffer while the target molecule
attaches to the immobilised ligand.

Washing:
By washing the column with a buffer solution, unbound or weakly bound molecules
are eliminated. This procedure aids in the removal of impurities and improves the
purity of the target molecule.Elution:By altering the buffer conditions, the precise
interaction between the target molecule and the immobilised ligand is broken. This can
include adjusting the pH, ionic strength, or employing competitive binding agents. The
target molecule is thereby liberated from the column.

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Elution:
By altering the buffer conditions, the precise interaction between the target molecule
and the immobilised ligand is broken. This can include adjusting the pH, ionic strength,
or employing competitive binding agents. As a result, the target molecule exits the
column.

Fraction Collection:
The pure target molecule-containing eluted fractions are collected. The purity and
concentration of these fractions can be determined further.Affinity chromatography is a
highly selective and effective technology for filtering biomolecules that are
distinguished by their distinct biological characteristics. It's commonly utilised in the
research, biotechnology, and pharmaceutical sectors for things like protein purification,
antibody purification, and isolating particular DNA or RNA sequences.

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Thank You