Factors affecting the separation and loading capacity of proteins in preparative gradient elution high-performance liquid chromatography.

The optimum conditions for the purification of proteins by gradient elution in reversed-phase liquid chromatography were studied, with emphasis on the column length. Because of the strong dependence of the retention of proteins on the mobile phase composition, very short columns can be used successfully to perform analytical separations. A similar conclusion is extended to preparative separations. Columns with different lengths and diameters were used. The dependence of the loading capacity for touching band separation on the column length, diameter and volume was studied, in addition to the regeneration time between successive runs, the starting mobile phase composition and the necessary column efficiency.