Multi-column preparative reversed-phase sample displacement chromatography of peptides.

Preparative reversed-phase sample displacement chromatography (SDC) of peptides was examined utilizing a multi-column approach. The effects of various SDC run parameters (flow-rate, run time and sample load) on the distribution of a single purified peptide and a mixture of three synthetic peptides was examined. The peptides in the mixture were closely related in hydrophobicity and mixed in a 1:4:1 ratio designed to mimic a typical preparative separation problem frequently encountered in crude synthetic peptide mixtures, that is, where there exist both hydrophobic and hydrophilic synthetic impurities close to the product of interest. Based on the results of these model systems, a SDC protocol was applied to the preparative purification of a crude synthetic peptide. The multi-column SDC approach provides rapid separations that are easy to employ because isocratic elution is utilized both in the separation process and in elution of the column segments. There is minimal fraction analysis, minimal use of organic solvents and increased utilization of the stationary phase such that the method involves considerably lower costs than traditional gradient-elution chromatography.