How does column chromatography work?

Column chromatography works on the principle of selective adsorption. The process involves passing a chemical mixture through a column containing an adsorbent material and a solvent. There are two phases involved in column chromatography – a solid stationary phase, which may be cellulose, silica gel, glass or alumina, and a liquid mobile phase.

In this process, the adsorbent material is slowly added to a chromatography column that contains the solvent mixture. Molecules in the mixture migrate at different rates depending on their polarity and strength of interaction with the stationary phase. The target molecules finally flow through the column after eluting with an appropriate buffer. Fractions from the column are collected into a series of test tubes and each fraction is analyzed for the presence of the target protein using a range of techniques, depending on whether the compound is colored or colorless.