This study investigates the impact of peptide primary structure and the characteristics of two different types of immobilised hydrophobic ligands on the open tubular electro-chromatographic migration behaviour of a family of related synthetic peptide analogues. The results further document the important role that mixed mode electrophoretic/chromatographic processes play in determining peptide selectivity in OTCEC systems. Procedures to fine-tune selectivity of closely related peptides in these systems have been established with buffer electrolytes of different composition in terms of the organic solvent content and different pH conditions.