The influence of pH on retention and migration of peptides in systems with octadecyl silica-based adsorbent by high-performance thin-layer chromatography and pressurized planar electrochromatography techniques

Abstract

In our paper we investigate the influence of pH of the mobile phase buffer, on retention of peptides in both: normal phase (NP) and reversed phase (RP) high-performance thin-layer chromatography systems (HPTLC), using C18 silica-based adsorbent. We also compare the influence of pH on retention and/or migration distance of peptides in separation systems of HPTLC and PPEC (pressurized planar electrochromatography) techniques. Our results show, that the change of pH of the mobile phase buffer can be used for two-dimensional HPTLC separation of peptides, but much stronger change of separation selectivity can be obtained by overall inversion of separation system type (NP/RP), as we have described before (Gwarda et al., 2016). In PPEC influence of the mobile phase pH on selectivity of peptide separation is very clear and strictly different than that in HPTLC (including overall inversion of direction of solutes migration), what results from the significant share of electrophoretic effect in overall mechanism of separation. Migration of peptides in two opposite directions results in better selectivity of separation by PPEC. In separation systems of both HPTLC and PPEC techniques, the change of pH clearly affects the efficiency of the separation system, so it can be used for optimization of separation conditions. Our results also show, that some modifications of PPEC equipment and materials could be useful for application of this technique in the field of peptide analysis.

Keywords: Mobile phase pH; Peptide separation; Pressurized planar electrochromatography; Separation selectivity; Thin-layer chromatography/high-performance thin-layer chromatography.