Influence of operating parameters on the preparative gradient elution chromatography of insulins.

The mass-overloaded separation of bovine and porcine insulins has been studied in the reversed-phase gradient elution mode. Strong solute-solute displacement effects have been found, which are related to the efficiency of the column used. Low flow-rates and small particle diameters maximise the displacements, as well as improve the resolution between the parent insulin and its desamido contaminant. The gradient slope did not substantially affect the separation between the parent insulins, but, due to the relative "S" values of the solutes, an increase in gradient slope improved the separation of the parent insulin from its desamido compound. An optimum pore size of 150 A was found for the insulins. Experiments to optimise the loadability were not carried out, but a recovery of 90% at a purity of 99.5% was obtained with a loading of 12 mg/g of porcine insulin.