Effect of mass loadability, protein concentration and n-alkyl chain length on the reversed-phase high-performance liquid chromatographic behaviour of bovine serum albumin and bovine follicular fluid inhibin.

The chromatographic behaviour of bovine serum albumin and partially purified preparations of the 58 kilodalton form of bovine follicular fluid inhibin has been investigated using two different n-alkylsilica stationary phases. In particular, the effects of mass loadability, protein concentration and loading conditions on the relative retention, peak width and recovery of these proteins have been studied over a dynamic range up to 100 mg protein per g packing material per injection. The influence of variable amounts of more abundant contaminating proteins such as bovine serum albumin in crude inhibin preparations on the chromatographic purification of trace quantities of the 58 kilodalton inhibin protein has also been examined. Based on these observations, recommendations are offered for the selection of protein loading conditions with n-alkylsilica stationary phases, particularly for the trace recovery and purification of hydrophobic proteins similar to inhibin where self-aggregation, adsorption to glass or plastic surfaces, and aberrant chromatographic behaviour on size exclusion or adsorptive chromatographic supports may occur.