How HPLC Columns Work

After the sample enters the flow path, the mobile phase carries the sample to the column, where the separation occurs.

Columns can function in ambient air but are generally thermostatted and housed within a temperature-controlled column compartment. Proper column temperature control is essential to conserving retention time precision, selectivity, and separation efficiency.

A general rule to remember is that as the column temperature increases, analyte retention decreases, leading to faster separation.

HPLC columns contain a stationary phase bonded to a support material, usually porous silica particles, to provide a large surface area. The stationary phase provides the basis for separating sample components.

Stationary phase chemistry dictates the affinity of the sample components to stick or retain on the column as the mobile phase moves the sample through the column. As a result, the sample components traverse the column and elute at different rates.

For example, you can visualize a mixture (A) containing multiple components represented as a grey band introduced at the front, or head, of the column.

As the mobile phase moves the mixture through the column, the red component (B) retains more strongly than the purple. As a result, the purple and blue components move through the column faster and are the first ‘bands’ to elute from the column (C). The green, yellow, and red bands retain longer and elute later (D).