MIXED-MODE HYDROPHILIC AND IONIC INTERACTION CHROMATOGRAPHY RIVALS REVERSED-PHASE LIQUID-CHROMATOGRAPHY FOR THE SEPARATION OF PEPTIDES

Peptide separations based upon mixed-mode hydrophilic and ionic interactions with a strong cation-exchange column have been investigated. The peptide separations were generally achieved by utilizing a linear increasing salt (sodium perchlorate) gradient in the presence of acetonitrile (20-90%, v/v) at pH 7. The presence of acetonitrile in the mobile phase promotes hydrophilic interactions with the hydrophilic stationary phase, these hydrophilic interactions becoming increasingly important to the separation process as the acetonitrile concentration is increased. At acetonitrile concentrations of 20-50% (v/v) in the mobile phase, the peptides utilized in this study were eluted in order of increasing net positive charge, indicating that ionic interactions were dominating the separation process. Peptides with the same net positive charge were also well resolved by an hydrophilic interaction mechanism, being eluted in order of increasing hydrophilicity (decreasing hydrophobicity). At higher acetonitrile concentrations (70-90%, v/v), column selectivity was changed dramatically, with hydrophilic interactions now dominating the separation process. Under these conditions, specific peptides may be eluted earlier or later than less highly charged peptides, depending upon their hydrophilic/hydrophobic character. This mixed-mode methodology was compared to reversed-phase liquid chromatography of the peptides at pH 2 and pH 7. The results of this comparison suggested that mixed-mode hydrophilic-ion-exchange chromatography on a strong cation-exchange column rivals reversed-phase liquid chromatography for peptide separations.