Preparative purification of peptides by reversed-phase chromatography. Sample displacement mode versus gradient elution mode

Abstract

In this study, we describe a novel method of preparative liquid chromatography, applicable to analytical columns and instrumentation, where the reversed-phase column is operated in sample displacement mode (SDM). This technique takes advantage of the different relative hydrophobicities of components of a sample mixture, so that when a column is optimally loaded with an aqueous solution of the sample mixture, there is competition among the sample components for the adsorption sites on the hydrophobic stationary phase. The more hydrophobic components compete more successfully for these sites than more hydrophilic components, which are displaced and immediately eluted from the column. Thus, the major separation takes place in water. Subsequent treatment with an aqueous organic eluent is only required to wash retained components off the column and takes no part in the major separation process. A two-column (precolumn and main column, in series) SDM strategy was applied to the preparative purification of a peptide product from neighbouring hydrophilic and hydrophobic impurities. Hydrophobic impurities were isolated on a shorter precolumn trap while hydrophilic impurities were displaced from the main column in the aqueous mobile phase (0.05% aq. trifluoroacetic acid), leaving the main column filled with homogeneous product. Gradient elution was then used to remove the peptide product from the main column. The researcher can regulate the size of the precolumn trap depending on the amount of hydrophobic impurities in a particular sample, or the size of the main column depending on the amount of product desired. The simplicity and flexibility of the SDM approach to preparative-scale purification enabled rapid separation of a single peptide component from a complex multicomponent mixture and should prove to be valuable for researchers in the peptide/protein field.