Investigation into reversed phase chromatography peptide separation systems part I: Development of a protocol for...

A protocol was defined which utilised peptides as probes for the characterisation of reversed phase chromatography peptide separation systems. These peptide probes successfully distinguished between differing stationary phases through the probe's hydrophobic, electrostatic, hydrogen bonding and aromatic interactions with the stationary phase, in addition, to more subtle interactions such as the phase's ability to separate racemic or isomeric probes. The dominating forces responsible for the chromatographic selectivity of peptides appear to be hydrophobic as well as electrostatic and polar in nature. This highlights the need for other types of stationary phase ligands with possibly mixed mode functionalities / electrostatic / polar interactions for peptide separations rather than the hydrophobic ligands which dominate small molecule separations. Selectivity differences are observed between phases, but it appears that it is the accessibility differences between these phases which play a crucial role in peptide separations i.e. accessibility to silanols, the hydrophobic acetonitrile / ligand layer or a thin adsorbed water layer on the silica surface.