The present model of mass-overloaded high-performance liquid chromatographic separation has been extended to the case of gradient elution. It is shown that the separation of two compounds by gradient elution varies with mass overload in the same way as in isocratic elution. If gradient conditions are selected to be equivalent to those in isocratic elution [for small samples, value of k (gradient) equal k′ (isocratic)], resolution is the same in either gradient or isocratic runs when the sample size is the same. Therefore gradient separations in a mass-overload mode can be predicted by means of model simulations for corresponding isocratic systems (where ko k). Additional relationships are derived for use in optimizing gradient separations in a mass-overload mode.
