Enhanced calculation of optimal gradient programs in reversed-phase liquid chromatography.

The resolution of a mixture of 16 beta-blockers under gradient elution was optimised using both isocratic and gradient training sets, with a reversed-phase column and acetonitrile-water eluents. Error theory was applied to measure the information extracted from different gradient experimental designs. This allows checking the expected accuracy when gradient predictions exceed the initial solvent concentrations tested in the training set. This work applies the results on modelling found in a previous study [J. Chromatogr. A 1018 (2003) 169] where the performance of several retention models was compared. Enhanced retention predictions were applied to the optimisation of gradient programs involving three factors (gradient slope, initial solvent composition and gradient curvature), using the peak purity criterion as resolution assessment. Peak shape parameters required in peak purity evaluation were modelled by adapting previous developments in isocratic mode. The mixture, which required prohibitive analysis times under isocratic elution, was almost baseline resolved in less than 35 min with linear gradients. Curvilinear gradients did not enhance this result significantly.