Multiple step gradient analysis in stationary phase optimised selectivity LC for the analysis of complex mixtures

Stationary phase optimised selectivity liquid chromatography (SOSLC) is an approach to tune a given LC separation by combining different stationary phases in a multi- segment column set-up. The presently available SOSLC optimisation procedure and algorithm are, however, only applicable to isocratic conditions. This is a severe limitation for the analysis of mixtures composed of components covering a broad hydrophobicity range. A strategy is described to circumvent this limitation. The components of a mixture are divided into different groups according to hydrophobicity as elucidated by a gradient analysis on a C ₁₈ reversed-phase column. Each group separation is then individually optimised with a specific isocratic mobile phase composition using the original SOSLC strategy. The mobile phase composition thereby only differs in the percentage of organic modifier between the various groups. Finally, a combination of stationary phases that guarantees sufficient selectivity for all the groups is selected and the separation is performed by a multiple step gradient, whereby each level consists of the mobile phase composition applied for the SOSLC optimisation of the individual groups. The multi step gradient approach is demonstrated through the analysis of a mixture of 27 steroids covering a wide range of hydrophobicity.