Implementation of generalised retention models in RPLC using serially coupled columns: a study based on lemon balm...

Generalised (or global) models (GEMs) are gaining attention in reversed-phase liquid chromatography for their ability to predict retention times with accuracy comparable to conventional individual solute models (ISMs). By incorporating solute-specific parameters alongside common system descriptors that account for the combined effects of the column and solvent, GEMs offer a comprehensive approach to retention modelling. However, because system parameters remain fixed across all solutes, GEMs are not sufficiently accurate to predict changes in elution order in single columns.

This work investigates and confirms the hypothesis that GEMs can anticipate peak reversals when hybrid coupled column setups are used instead of single columns. When two or more columns with distinct selectivity patterns are serially coupled using zero-dead-volume connectors, solutes undergo relative retention shifts as they transition between columns. These shifts, exceeding those observed in single columns, may lead to pronounced changes in selectivity. This added complexity allows GEMs to effectively predict major selectivity shifts, including peak reversals. As a case study, the separation of selected constituents in lemon balm extracts was examined using an experimental design featuring bilinear gradients. A set of 16 peaks from the chromatograms of lemon balm extracts, tracked across three columns with distinct retention mechanisms (conventional C18, phenyl, and cyanohexyl), under different gradient conditions, was selected to construct GEMs characterising the three chromatographic systems. The predictive accuracy of GEMs was systematically compared against ISMs, with a focus on evaluating selectivity changes across the three columns.

The results demonstrate that combining columns with different retention behaviours not only amplifies the impact of selectivity changes, but also expands the practical applicability of GEMs. The use of GEMs in serially coupled column systems may contribute to streamline method development for medicinal plant analysis.