Optimization of selected chromatographic responses using a designed experiment at the fine-tuning stage in reversed-phase high-performance liquid chromatographic method development

This study evaluated the applicability of a designed experiment at the fine-tuning stage in reversed-phase high-performance liquid chromatographic (HPLC) method development. Using acetaminophen, theophylline, and caffeine as model drugs, a 3(2) factorial design was used to optimize selected chromatographic responses. The effects of the ratio of water to acetonitrile (%v/v) in the mobile phase and mobile phase flow rate on the theoretical plate number of acetaminophen peak, capacity factor of acetaminophen, resolution of acetaminophen and theophylline peaks, and the time for the elution of last peak (run time) were determined. Polynomial equations were derived to evaluate the quantitative relationships between the experimental factors and responses. A solution space was found by overlaying contour plots. Results indicated that, once the mobile phase that provides reasonably good retention and resolution has been identified, the strategy of using a designed experiment is advantageous over the conventional one-jactor-at-a time approach since it would enable the analyst to optimize important responses, including run time with a minimum number of experiments.