A review on affinity chromatography.ppt shady

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. A ffinity 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 overcoming 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 s pecifically 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.

7. STAGES IN AFFINITY CHROMATOGRAPHY

8. 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

9. Applications T o study drug-protein binding interactions . Immunoglobulin purification. Recombinant tagged proteins

10. Advantages  Affinity chromatography is a fairly achievable technique because of the great selectivity of the glucose residues and the target protein, giving 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 .

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