Phase-separation mode in HPLC: Theoretical prediction and experimental evaluation of elution times

A novel high-performance liquid chromatography (HPLC) approach, termed the phase-separation mode, was developed by employing a phase-separation multiphase flow (PSMF) as the mobile phase. Unlike conventional modes, this system exploits two coexisting liquid phases that generate distinct liquid-liquid interfaces in packed columns. In this study, we established a theoretical framework for predicting analyte elution times in the phase-separation mode and validated them experimentally. A theoretical equation was derived on the basis of the proposed separation mechanism, incorporating capacity factors between the stationary and mobile phases as well as between the two mobile phases, in addition to their respective hold-up times. The model was applied using an octadecylsilyl (ODS)-modified silica column and biphasic water/acetonitrile/ethyl acetate mixtures as eluents, with naphthalene derivatives serving as analytes. The calculated elution times showed good agreement with experimental values in most solvent systems. Some deviations were observed when the minor phase might behave as a non-continuous liquid domain near the particle surface. These deviations were not simple errors but provided supplementary mechanistic insights we discussed. The present work establishes a mechanistic and theoretical basis for the design and evaluation of chromatographic separations in the phase-separation mode using PSMF.