Research into the application of deep eutectic solvents (DESs) in capillary electrophoresis (CE) is still in its infancy. Even so, a controversy has already arisen over the “true role” of DESs in CE. This is because the DESs employed previously were all hydrophilic, and it is therefore reasonable to speculate that they may be virtually completely dissociated into its initial components (hydrogen-bond acceptor (HBA) and hydrogen-bond donor (HBD)) in aqueous CE buffers. Since there seems to be no essential difference in CE performance between adding a DES and adding its two constituents separately (HBA and HBD) in a running buffer, the significance of using DESs in these CE systems has not been clearly established. In this context, this study established the first DES-based CE system in which a DES really worked as a single entity. To achieve this goal a hydrophobic DES (HDES), (−)-menthol:octanoic acid, was employed to act as a pseudo-stationary phase (PSP) in electrokinetic chromatography (EKC) mode. We found that the HDES is capable of forming stable aggregates (micro-droplets) which can act as a PSP in the running buffer. The novel HDES-type PSP exhibited impressive selectivities toward several groups of analogues, homologues, and isomers via hydrophobic mechanism. It can be used either solely for direct EKC separation, or in combination with traditional PSPs (e.g., cyclodextrins). As a proof-of-concept study, we have demonstrated that the synergistic effect generated in an “HDES + Hydroxypropyl-γ-cyclodextrin” dual-PSP system allowed an excellent separation of a simulated complex mixture consisting of 13 aromatic acids.

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