Abstract
An experimental system has been developed which is suitable for the collection of data for use in the theoretical modelling of preparative reversed-phase high performance liquid chromatography (RP-HPLC). The system developed uses commercially available media and columns to achieve isocratic separation of bovine and porcine insulin; peptides that differ from each other by only two amino acids in the A chain. This mimics systems designed to resolve peptides or proteins which are structurally very similar. The resolution between the two peptides was found to be critically dependent on the concentration of acetonitrile in the mobile phase. The similarity of chromatograms obtained in experiments with columns of constant length but with diameters ranging between 0.46 cm and 5.08cm indicated that the separation could be scaled-up to process scale without alteration in separation performance and thus was suitable as a model system to test the validity of theoretical modelling approaches to optimization and scale-up. These studies also highlighted a number of factors of importance when RP-HPLC is conducted at process scale.
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