Salt-dependent elution of uncharged aromatic solutes in ion-exchange chromatography

Ion-exchange chromatography and multimodal ion-exchange chromatography are widely used for the separation of small molecules, peptides and proteins. Salts generally attenuate the electrostatic interactions between charged moieties of solutes and those of resins through electrostatic screening. However, little is known about how salts affect the interaction between the uncharged moieties of the solutes, such as aromatic moieties, and the charged moieties of the resins. In this study, we used alkyl gallates as model aromatic solutes to investigate the interaction mechanism of aromatic moieties with multimodal and conventional ion-exchange resins. Interestingly, alkyl gallates retained by these resins were readily eluted from the columns by the addition of 0.01-1 M NaCl, even though the alkyl gallates used contained no charged group. Molecular dynamics (MD) simulations were performed to understand the mechanism of these interactions. The MD simulation with a conventional force field showed that 1 M NaCl enhances the binding of an alkyl gallate molecule to the ligand, which contradicts the experimental results. Thus, we modified the force field to express a cation-π interaction between sodium ions and aromatic moieties, which successfully reproduced the experimental results at 1 M, suggesting that the cation-π interaction between sodium ions and aromatic moieties plays a crucial role in reducing the binding affinity of alkyl gallates for the ligands. These results provide new information indicating that aromatic moieties, including the aromatic residues of proteins and nucleobases of nucleic acids, favorably interact with multimodal and conventional ion-exchange resins and that cations, such as sodium ions, contribute to attenuating the binding of aromatic moieties to the ligands.