affinity chromatography

1. A REVIEW ON AFFINITY CHROMATOGRAPHY
• BY
• OKOYE, Shedrack Chukwuebuka
• 2013/1/46797BH
• Supervisor: PROF. H.O. AKANYA

2. INTRODUCTION
• This is the process used in separating mixtures by the virtue of
differences in its permeability or absorbency.
• This is done by passing the mixture with in a solution, suspension or
vapor through a medium in which the compounds move at different
rates.
• These fluids in which it is dissolved is known as the mobile phase.
• The separation is as a result of travelling at different speeds on the
basis of differential partitioning between the mobile and stationery
phase.

3. TYPES OF CHROMATOGRAHY
• Paper Chromatography
• Thin-layer Chromatography
• Ion Exchange Chromatography
• Liquid Chromatography
• Gas Chromatography
• Chromatofocusing
• Molecular exclusion
• Adsorption
• Affinity chromatography

4. AFFINTY CHROMATOGRAPHY
• It has been in existence for the past 50 years
• Affinity chromatography involves the use traditional purification
methods on the basis of pH, Ionic strength and temperature.
• Affinity chromatography is based on molecular recognition of a target
molecule by another molecule bound to a column.

5. Matrix: for ligand attachment. Matrix should be chemically and physically inert.
Spacer arm: used to improve binding between ligand and target molecule by overco
ming any effects of steric hindrance.
Ligand: molecule that binds reversibly to a specific target molecule or group of target
molecules.

6. PRINCIPLES
• The purification of protein is based on the reversible interactions
between the protein to be purified and the affinity ligand.
• The protein has inherent recognition site to which the ligand binds to
in a specific and reversible manner.
• To release and elute the bound molecules, a desorption step is usually
performed either specifically using a competitive ligand or
• Nonspecifically by changing the media atmosphere (ionic strength, pH
or polarity). As the elution is performed, the purified protein is
collected in a concentrated form.

8. STAGES IN AFFINITY CHROMATOGRAPHY

9. factors to consider in choosing a support material
• Chemical inertness: - This requires that the support binds only the
molecule of interest.
• Chemical Stability: - The matrix must be resistant to possible
degradation that may be caused by enzymes
• Mechanical Stability: - The material must be able to withstand
pressures without compressing during separations
• Pore Size: - This should be at least five (5) times the diameter of the
molecule to be purified

10. Applications
• To study drug-protein binding interactions.
• Immunoglobulin purification.
• Recombinant tagged proteins

11. Advantages
• Affinity chromatography is a fairly achievable technique because of th
e great selectivity of the glucose residues and the target protein, givin
g purified product with a high yield of recovery.
• It can be a one step process in many cases.
• The technique can be used for substances of low concentration.
• Rapid separation is achieved while avoiding contamination.

12. Disadvantages
• The interaction of proteins of interest and ligand has to be determine
d carefully. This process required expensive materials, time, and small
amount of protein that can be processed at once.