Effect and Mechanism of Deep Eutectic Solvents as Mobile-Phase Additives on Retention Behavior of G-Quadruplexes in Reversed-Phase High-Performance Liquid Chromatography

Separating complex biomolecules by liquid chromatography has always been challenging. In this work, deep eutectic solvents (DESs) were employed as mobile-phase additives to separate different G-quadruplex (G4) conformations using reversed-phase high-performance liquid chromatography (RP-HPLC). We investigated how different types and concentrations of DESs affected the retention behavior of G4s. The addition of DESs was found to significantly improve the peak shapes and resolution. At low concentrations, DESs enhanced the retention time of G4s, whereas higher concentrations led to a reduced retention. By combining circular dichroism analysis, we identified the G4 conformations corresponding to distinct chromatographic peaks and revealed that parallel G4 was more hydrophobic than nonparallel G4. In DESs, both hydrogen-bond donors and hydrogen-bond acceptors were shown to influence G4 retention behavior, with hydrogen-bond acceptors playing a dominant role. Using SERS and molecular docking, we further investigated the interaction mode between DESs and G4s to gain deeper insights into the retention mechanism. It showed that DESs interact with G4s through groove binding, supported by hydrogen bonding, cation-π, and electrostatic interactions, as well as electrostatic interaction toward the phosphate backbone, while DESs could also shield the silanol groups of the C18 stationary phase, thereby modulating the retention behavior of G4s. This work demonstrated that DESs, as promising green solvents, could effectively improve G4 separation in RP-HPLC. Notably, this is the first application of DESs in the liquid chromatographic separation of complex biological macromolecules.