Biochemical Techniques and Instrumentation

2.4.1. Principle

Separation and purification of a molecule is based on a reversible interaction between the molecule and a specific ligand that is immobilized to a insoluble chromatography matrix.

Molecule (M) + Ligand (L) « ML complex

A sample containing a specific molecule of interest is passed through a column containing immobilized ligand. The specific molecule will then bind to the ligand to form a ML complex. This biological interaction between ligand and specific molecule occurs due to electrostatic or hydrophobic interactions, van der Waals' forces or hydrogen bonding. The molecules that are not specifically bound get washed away with the buffer. The molecules that are specifically bound are recovered from the ligand by reversing the interaction.