Computer-assisted selection of mobile phase composition in reversed-phase liquid chromatography : Definition of the optimisation search area

Abstract

In order to reduce the time required for successful implementation of computer-assisted methods for the selection of the optimal mobile phase composition in reversed-phase liquid chromatography, it is desirable to limit the range of the mobile phases that are to be considered. This is conventionally achieved by defining an optimisation search area bounded by isoeluotropic binary mobile phases of water with methanol, acetonitrile or tetrahydrofuran. This process requires the use of equations (or “transfer rules”) relating the eluotropic strengths of these three organic modifiers. By reference to the separation of seven amines by reversed-phase ion-interaction chromatography, published transfer rules are shown to be unreliable as a means of calculating isoeluotropic mobile phases. New transfer rules were derived for a small group of structurally related amines, and use of these rules to define the optimisation search area gave improved performance of the optimisation process. Nevertheless, these new transfer rules also showed considerable error in predicting isoeluotropic mobile phases. It is concluded that the optimisation search area is best established empirically, using transfer rules initially to estimate isoeluotropic mobile phase compositions, followed by experimentation to confirm that the mobile phases selected are isoeluotropic. When this approach was applied- to the isocratic separation of ten fat-soluble vitamins, excellent results were obtained.