DISPLACEMENT EFFECTS IN PREPARATIVE GRADIENT HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SEPARATIONS

Experiments were carried out which were designed to elucidate the influence of gradient parameters on the strong displacement effects which have been observed in the preparative chromatography of proteins. Unexpected loading phenomena were observed in which a proportion of the model protein load was not taken up by the column, even though the saturation capacity of the column was not exceeded. This proportion was a function of the mobile phase composition at which the protein was loaded. Low solvent strengths were seen to be essential to allow high preparative sample loads. It was shown that the saturation capacity of the column changed little with gradient parameters and also that the saturation capacities calculated on the assumption of approximate Langmuir isotherm behaviour were in error. Mass overloaded separations of binary mixtures of proteins all demonstrated the existence of displacements between the solutes, even when a substantial amount of the second-eluting protein was not adsorbed on initial loading. It was demonstrated that the gradient slope had little effect on the separation because of the very sharp displacement zones observed. Steeper gradients allowed a higher production rate without incurring a decrease in purity or recovery. Guidelines for the design of reversed-phase gradients for the purification of proteins are given with a view to maximising solute-solute displacements. Such gradients will allow the maximum production rates of purified product.