Effect of organic solvent modifier and ion-pair reagent (tetrabutylammonium salt) on the chromatographic behaviour of protected peptides in reversed-phase high-performance liquid chromatography

Abstract

The effect of organic solvent modifiers (methanol and acetonitrile) on the retention of synthetic peptides with protective groups in high-performance liquid chromatography (HPLC) was examined. In the HPLC of totally protected peptides, an increase in the organic solvent content in the eluent up to 90% did not result in abnormal changes to the retention mechanism, which did occur in the separation of unprotected peptides. The chromatographic behaviour of partially protected peptides with free amino groups varied on addition of methanol or acetonitrile to the eluent; a parabolic dependence of log k′ on acetonitrile concentration with a minimum at ca. 80% acetonitrile was obtained, whereas an increase in the methanol concentration in the eluent up to 90% led to a gradual reduction in the k′ values of the peptides studied. The effect of counter ions (Br − and HSO − 4) of tetrabutylammonium salts as mobile phase components on the retention of protected peptides is discussed.