Application of phase optimized liquid chromatography to oligopeptide separations

Stationary phase optimized liquid chromatography (POPLC) has been applied to the separation of oligopeptides. The retention factors and theoretical plate numbers of 13 peptides were determined on five different stationary phases. Based on these values, an optimal stationary phase composition of 250 mm total length consisting of 3 segments of 20 mm octadecyl silica, 10 mm phenyl silica and 220 mm embedded polar octadecyl silica was calculated by the optimizer software. Good agreement between the calculated and experimental chromatograms was observed. In order to achieve short analysis time and baseline separation of all peptides gradient elution and optimization of the column temperature were performed. The optimized mobile phase conditions consisted of (A) acetonitrile and (B) 0.025 M aqueous sodium phosphate buffer, pH 3.0, operated at 10% A (0–12 min) followed by 10–50% A (12–32 min) at a flow rate of 0.5 mL min−1 and column temperature of 35 °C. Using these conditions all peptides could be separated in less than 30 min with good resolution and peak symmetry.