Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.

Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification.

Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and the stationary fluid phase (stable phase) is separating from each other while moving with the aid of a mobile phase. The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into the mobile phase, and leave the system faster.