AFFINITY

CHROMATOGRAPHY
T E C H N I Q U E S I N B I O C H E M I S T RY
 AFFNITY CHRMOATOGRAPGHY

• Biological substances, such as enzymes and substrates or

antigens and antibodies, have the property of selectively
recognizing each other to form complexes.
• Affinity chromatography separates proteins on the basis of a
reversible interaction between a protein and a specific ligand
coupled to a chromatography matrix.
• Affinity chromatography is a separation and purification
method that exploits this biological affinity.
 SPECIFICITY

• it is the only technique that enables the purification of a

biomolecule on the basis of its biological function or individual
chemical structure.
• In a single step, affinity purification can offer immense time-
saving over less selective multistep procedures.
• The concentrating effect enables large volumes to be processed.
• Target molecules can be purified from complex biological
mixtures.
• small amounts of biological material can be purified from high
levels of contaminating substances.
 PRINCIPLE

• In principle, affinity chromatography can effectively extract a

target component from a complex matrix that contains
multiple components just using simple operations.

• The technique offers high selectivity, hence high resolution,

and usually high capacity for the protein(s) of interest.
 PROCEDURE FOR AFFINITY
CHROMATOGRAPHY
• The procedure comprises the following four steps:
1. Column equilibration:
The column is filled with a packing onto which a suitable ligand is immobilized.
The column is then equilibrated with the solvent that will be used for sample
adsorption.
2. Sample addition and adsorption of target substance:
The sample is introduced into the column and the target component adsorbed
onto the packing.
3. Column washing:
Washing is conducted to eliminate components not adsorbed onto the packing
from the column.
4. Elution of target components:
A solvent is introduced into the column to elute the target component adsorbed on
the packing to recover the target component. The procedure then reverts to step.
 LIGAND SELECTION AND
IMMOBILIZATION ON THE PACKING
• The selection of the ligand and packing matrix and method to
immobilize the ligand must be carefully considered before
conducting actual affinity chromatography.
• Selecting the Matrix Commonly used matrices are agarose and
hydrophilic synthesized macromolecules (polyvinyl alcohol,
polyacrylate, etc.) beads.
 ELUTION

• pH elution
A change in pH alters the degree of ionization of charged groups
on the ligand and/or the bound protein. This change may affect
the binding sites directly, reducing their affinity, or cause
indirect changes in affinity by alterations in conformation.
• Ionic strength elution
The exact mechanism for elution by changes in ionic strength
will depend upon the specific interaction between the ligand and
target protein. This is a mild elution using a buffer with
increased ionic strength (usually NaCl), applied as a linear
gradient or in steps.
• Competitive elution
Selective eluents are often used to separate substances on a
group specific medium or when the binding affinity of the
ligand/target protein interaction is relatively high. The eluting
agent competes either for binding to the target protein or for
binding to the ligand.
• Reduced polarity of eluent
Conditions are used to lower the polarity of the eluent promote
elution without inactivating the eluted substances. Dioxane (up
to 10%) or ethylene glycol (up to 50%) are typical of this type of
eluent.
THE END