Principles Of Column Chromatography

Column chromatography is a biochemical technique used to separate compounds based on their physical and chemical properties.

It has two main components — the solid stationary phase or the matrix and the liquid mobile phase or the solvent.

The matrix-packed column is loaded with a sample, such as a protein mixture. The solvent is then used to carry the sample through the column.

Inside the column, the matrix acts as a molecular mesh filtering the proteins based on their size or interaction with the matrix. The larger proteins tend to travel faster than the smaller proteins, resulting in a size-based separation.

Additionally, proteins in a sample may interact with the matrix. Weak interactions allow proteins to pass quickly, while strong interactions retain proteins within the column.

A gradual change in the pH or ionic strength of the solvent alters the proteins’ interactions with the matrix and helps to elute proteins into separate fractions.