Introduction of Affinity Chromatography

Introduction

• Affinity Chromatography is a liquid chromatography technique that separates molecules based on specific and reversible interactions between a molecule in a mixture and a counter-molecule bound to a stationary phase.

• It is extensively used in biochemistry and pharmaceuticals to purify and concentrate specific molecules from complex mixtures, such as isolating a protein from a cell lysate.

Affinity chromatography: Introduction & Priniple

Principle

• Affinity chromatography relies on the selective and reversible binding of a target molecule (ligand) to a complementary molecule (counter-ligand) immobilized on the stationary phase.

• Key interactions include:

• Enzyme-Substrate

• Antigen-Antibody

• Receptor-Ligand

During the process:

1. Binding: The target molecule binds to the immobilized ligand.

2. Non-binding: Molecules with weak or no affinity pass through.

3. Elution: The target molecule is released by altering conditions (e.g., pH, ionic strength) or introducing a competing ligand.

Advantages

1. High Specificity: Selective interaction ensures high specificity for the target molecule.

2. High Purity: Achieves significant purity in a single step.

3. Versatility: Broad applicability with various immobilized ligands.

4. Scalability: Suitable for both small-scale and industrial-scale processes.

Disadvantages

1. Ligand Leakage: Ligands may leach from the column, leading to contamination.

2. Time-Consuming: Binding equilibrium can slow down the process.

3. Expense: High costs associated with ligands and setup.

4. Non-specific Binding: Can lead to co-purification of undesired molecules.