Optimization strategies using response surface methodologies in high performance liquid chromatography

Development of effective chromatographic separation involves judicious deciding of the selection of optimal experimental conditions that can provide an adequate resolution at a reasonable run time for the separation of interested components. The isomers of 2-methyl-6-nitroaniline and 2-methyl-4-nitroaniline were selected as the model compounds for the application of optimization strategies by high performance liquid chromatography. Response surface methodologies based on three level, three variable designs such as the Box-Behnken design, central composite face centered, central composite circumscribed design, and full factorial design were comparatively used for the optimization with respect to column temperature, flow rate, and the percentage of eluent. Statistical interpretation of the variables on different responses such as resolution and retention time of the last component was performed. The optimum conditions of these variables were predicted by using a second order polynomial model fitted to the results obtained by applying four designs. The response surface plots using three experimental designs revealed a separation optimum with a 25C column temperature, a flow rate of 1.0mL/min, and ACN percentage of 70%. The significance of the statistical designs was confirmed by the generally good agreement obtained between predicted responses and actual experimental data. We have concluded that experimental designs offer a rapid means of optimizing several variables and provide an efficient test for the robustness of the analytical method.