Correlation between chromatographic behavior and microfluidic phase distribution in the phase-separation mode of HPLC

We previously proposed the phase-separation mode in high-performance liquid chromatography (HPLC), in which phase-separation multiphase flow (PSMF) is used as the mobile phase. Although chromatographic behavior in this mode has been examined, its relationship with microfluidic phase organization inside packed structures has not been directly clarified. In this study, we correlated chromatographic behavior with fluorescence visualization of PSMF in a silica bead-packed microchannel. A water/acetonitrile/NaCl mixture was used as the eluent, and six solvent compositions ranging from water-rich to organic-rich conditions were examined at 40 °C and 0 °C. At 40 °C, the eluents remained homogeneous, whereas phase separation occurred at 0 °C. In water-rich systems under phase-separation conditions, hydrophilic 2,6-naphthalenedisulfonic acid eluted earlier than hydrophobic 1-naphthol, whereas the opposite tendency was observed in organic-rich systems. Fluorescence microscopy revealed that the phase with the larger volume fraction flowed mainly through regions away from the silica bead surfaces, while the minor phase was localized near the surfaces and moved more slowly. This phase-volume-dependent localization explained the observed elution order and retention behavior. The results demonstrate that separation in the phase-separation mode is governed by the combined effects of analyte distribution between the two coexisting liquid phases and their different flow velocities within the packed structure. This study provides direct experimental support for the separation mechanism of the phase-separation mode in HPLC.