Impact of selector regioisomerism on mixed-mode retention and enantioresolution: Comparative evaluation of 3-chloro-4-methyl and 4-chloro-3-methyl substituted cellulose chiral stationary phases

This study examines how selector regioisomerism affects retention and enantioselective performance on cellulose-based chiral stationary phases (CSPs) under aqueous-organic mixed-mode conditions. Two regioisomeric selectors, cellulose tris(3‑chloro-4-methylphenylcarbamate) (Cell2) and cellulose tris(4‑chloro-3-methylphenylcarbamate) (Cell4), were evaluated using a library of 75 structurally diverse chiral compounds over a broad range of acetonitrile/water mobile phases. Both CSPs showed U-shaped retention profiles, consistent with a transition from reversed-phase retention to HILIC-like behavior in the organic-rich region. Quantitative analysis using the empirical HILIC induction factor (HF) and stoichiometric-displacement modeling indicated that, while global median induction was comparable, Cell2 induced a significantly stronger retention amplification for specific basic families at high acetonitrile contents. In contrast, Linear Solvent Strength analysis in the water-rich region suggested that the intrinsic hydrophobic contributions of both phases are statistically comparable for the subset of analytes exhibiting classical reversed-phase behavior. In enantioselective terms, Cell2 displayed the broader screening scope, particularly for flexible amine-containing analytes, whereas Cell4 provided higher resolving power for selected rigid and aromatic scaffolds. Remarkably, despite strong thermodynamic selectivity in the organic-rich region, practical maximum enantioresolution was systematically achieved within the highly aqueous RPLC domain for almost all chemical families, driven by favorable kinetic residence times. PCA-based descriptor mapping associated the Cell2 advantage in HF with analytes rich in hydrogen-bond-donor and hydroxyl functionalities, whereas the Cell4 advantage in resolution was associated with sterically demanding and more aromatic structures. Overall, the results support a practical data-driven structure-retention-resolution framework for selecting between these two regioisomeric polysaccharide CSPs according to analyte class and mobile-phase composition.