Retention behaviour of peptides in capillary electrochromatography using an embedded ammonium in dodecacyl stationary phase

Abstract

The potential of a silica stationary phase bearing an embedded cationic quaternary amine in dodecacyl chain, to separate peptides by capillary electrochromatography (CEC) has been evaluated. The ability of this stationary phase, to generate a consistent anodic electroosmotic flow was first evaluated. This flow was found to be independent of pH over a wide range (2–12), of the acetonitrile percentage in the electrolyte. The stability of the stationary phase evaluated through the electroosmotic flow variations was demonstrated at extreme pH values (2.5 and 9.1). A careful examination of the influence of mobile phase conditions (acetonitrile percentage, salt concentration and nature of buffer) on the electrochromatographic retention and electrophoretic migration behaviour of different standard peptides was carried out. In acidic conditions, the electrokinetic contribution appears to be predominant compared to the chromatographic one. Several types of chromatographic interactions, reversed-phase partitioning and anion exchange, were involved in the CEC of peptides, whereas repulsive electrostatic interaction could be considered as negligible. This stationary phase affords different selectivity compared to that observed on a C 18 stationary phase. Finally, the method was applied to the peptide mapping of β-lactoglobulin and human growth hormone under unpressurized and isocratic elution.