Tandem separation schemes for preparative high-performance liquid chromatography of proteins

Preparative chromatography of protein mixtures was carried out by tandem separation schemes involving frontal chromatography followed by stepwise desorption or displacement. In this way, with columns and instruments generally employed in analytical high-performance liquid chromatography, proteins could be purified in quantities similar to those typically separated by a preparative-scale system. A mixture of beta-lactoglobulin A and B was loaded onto an anion-exchange column, and, in the process, a large fraction of the less-retained beta-lactoglobulin B was recovered in pure form. The column was then flushed with the carrier, and subsequent desorption of the substances bound on the stationary phase was carried out by single-step desorption, two-step desorption, or displacement. With this mixture, the last two methods yielded approximately the same results in terms of the amount of product obtained per unit column volume. Whereas stepwise desorption is a simpler technique than displacement, the latter is required for the separation of components having similar adsorption behavior. In another set of experiments, a protein mixture obtained by heat treatment of human growth hormone was fractionated on a reversed-phase column. After loading the column by frontal chromatography, which separated a large fraction of the main product from the other components retained by the column, four desorption steps were applied to recover the individual components. These separation schemes offer an approach to preparative chromatography of proteins that is superior to conventional linear elution in terms of column load capacity, low mobile phase consumption, simultaneous separation and concentration, as well as enrichment of trace components.