Adsorption and separation of RNA and protein on imidazolium-type ionic liquids-based chromatographic media: influence of counterions

Imidazolium-based ionic liquids (ILs) as ligand have shown great potentials in biomacromolecule separation by chemically coupling the cationic functional group of the ILs to solid supports. Whether and how the counterions affect the adsorption of biomacromolecule, however remains unclear. In this work, GigaMIm chromatographic media was prepared by coupling an ionizable 1-methylimidazole (MIm) to giga-porous microspheres and paired with four different counterions including Cl − , H 2 PO 4 − , benzoate ion (BZA − ), and 4-hydroxybenzenesulfonate (HBS − ). The types of the counterions on GigaMIm was found to significantly influence the adsorption kinetics and isotherms of yeast RNA and BSA, as well as the chromatographic separation of RNA-BSA mixture. Compared to the media paired with inorganic ions (Cl − and H 2 PO 4 − ), those paired with benzene ring-containing counterions (HBS − and BZA − ) exhibited slightly slower pore diffusion kinetics and lower adsorption capacities for both RNA and BSA, but provided better separation resolution for their mixture. Molecular docking suggested that enhanced hydrogen bonding between MIm⁺/HBS⁻ or MIm⁺/BZA⁻ and biomolecules contributed to improved resolution. The counterion-tuning strategy was further validated by separation of eGFP mRNA from in vitro transcription system. Compared to GigaMIm-Cl, purification using GigaMIm-HBS obtained higher mRNA recovery (38.42% vs. 30.94%) with high mRNA purity and lower T7 polymerase residual (0.16% vs. 0.21%). These findings demonstrate that modulating counterions in ILs-based media may offer a promising strategy for optimizing nucleic acid and protein separation.