What happens to the proteins as they pass through the chromatography column?

As proteins pass through the chromatography column, they primarily move downward through the spaces between the beads. This movement is facilitated by the flow of the mobile phase, which carries the proteins along with it. The column is typically packed with beads that provide a stationary phase, which can interact with the proteins in various ways depending on the type of chromatography being used (such as size exclusion, affinity, or ion exchange).

In size exclusion chromatography, for instance, smaller proteins enter the pores of the beads and are temporarily delayed, while larger proteins are excluded from these pores, allowing them to elute more quickly. In affinity chromatography, specific interactions between the proteins and the substances attached to the beads cause certain proteins to bind to the stationary phase while others pass through. However, in both cases, the primary action involves the proteins moving through the interstitial spaces in the column, demonstrating their differing affinities or sizes based on the designed application of the chromatography. Thus, this movement is a key aspect of the purification process, allowing for effective separation of proteins based on their physical or chemical properties.