Considerations on advanced ion pairing co-gradients for ion-pair reversed phase liquid chromatography separations of oligonucleotides

This study introduces a novel methodology for ion-pair reversed-phase liquid chromatography (IP-RPLC) of oligonucleotides (ONs), in which gradients of both the organic co-solvent and ion-pair (IP) reagent concentrations are applied independently or in combination. Building upon prior work on weak-to-strong IP gradients, this approach decouples the co-solvent and IP transitions or IP concentration effects, offering a more flexible framework for optimizing selectivity. Using a quaternary pumping system, both linear and segmented gradients were tested for two or three variables (i.e. co-solvent gradient steepness, weak IP gradient steepness or strong IP gradient steepness) simultaneously. A semi-empirical model was proposed to describe retention behavior as a function of co-solvent and IP reagent concentrations, enabling retention time prediction across complex gradient programs. Experimental results demonstrate significant improvements in resolution and selectivity compared to traditional single IP reagent and co-solvent gradient methods. These findings highlight the advantages of multi-variable gradient strategies in ON separations. The method is practical, easy to implement, and well-suited for modern analytical workflows in therapeutic ONs development.