Simultaneous selectivity optimization of mobile and stationary phases in reversed-phase liquid chromatography for isocratic separations of phenylthiohydantoin amino acid derivatives

A mixture-design statistical technique has been used to optimize simultaneously the selectivity of both mobile and the stationary phases for the isocratic high-performance liquid chromatographic separation of phenylthiohydantoin derivatives of the 20 common amino acids. This approach permits the fine tuning of selectivity to achieve the rapid separation of this relatively complex mixture with maximum resolution between the various components. An optimum isocratic reversed-phase separation has been achieved in 13 min with a minimum resolution of 1.1 for all component peak pairs. The system uses an optimum combination of four mobile phase solvents and three stationary-phase packings (C sub(8)-, CN-, phenethyl-modified silica) to obtain various separation goals.