New approach to optimize HPLC separations of acid-base compounds with elution order involved, by using combined three-band resolution maps.

Some of the optimization methods in reversed phase high-performance liquid chromatography (RP-HPLC) are based on resolution of the critical band pair. Mobile phase composition is changed systematically to establish those conditions giving an acceptable resolution for such a critical band pair, but sometimes the critical pair may change with the separation conditions, which obliges to identify it for each of those conditions. In the case of ionizable compounds, more than two bands may be involved in resolution, showing--in some cases--changes in the elution order when the mobile phase composition was modified. In this paper, an alternative way that does not identify the critical pair after changing experimental conditions is proposed. The relative separation of the three bands involved in two alternating critical band pairs is evaluated as a sort of conjugate or combined resolution, represented as contour maps vs. two variables (content of organic modifier and pH). These maps are obtained from data of chromatograms made under different separation conditions; these conditions were generated by experimental design and data was mathematically processed with a computer program. Analytes of three families that have acid-base properties, triazines, phenoxyacids, and phenols, were used for this purpose. The chromatographic behavior when elution order reversion of ionizable compounds exists is studied.