Use of mixed-mode, high-performance liquid chromatography for the separation of peptide and protein mixtures

The packing material recently introduced for use in a radial compression chamber, with a partially flexible cartridge, has a low C18-coating (5% w/s) combined with an absence of secondary capping. This report demonstrates that such a support, which contains significant concentrations of both free silanol and hydrocarbon groups, can allow a mixed-mode separation to occur via adsorption and reversed-phase separation mechanisms. In any given separation, the predominant mechanism depends both on the nature of the sample and the mobile phase. For efficient peptide and protein separations, it was necessary to suppress most silanol group interactions by the use of a mobile phase which contained a high concentration of an amine phosphate, e.g., 0.17 M triethylammonium phosphate, pH 3.2. In addition, it was necessary to deactivate further the silanol groups by an initial column wash of at least 20 column volumes of methanol. Samples which contained strongly basic groups, for example the guanidino group of arginine, can still exhibit poor separation efficiencies on such a support. These problems were largely overcome, however, with the use of isopropanol as an organic modifier. If these precautions were followed, the packing material gave excellent selectivities in the separation of closely related materials, as well as allowing increased sample capacities. These observations will be supported by an examination of the chromatographic properties of a range of small peptides, the C-apolipoprotein mixture present in human very-low-density lipoproteins and the purification of an 8-mg sample of a synthetic pentadecapeptide in a single chromatographic run.