<p>We previously developed a high-performance liquid chromatography (HPLC) system employing phase-separation multiphase flow (PSMF) as the eluent, referred to as the phase-separation mode in HPLC. However, direct visualization of the flow behavior within the HPLC column had not yet been achieved. In this study, we directly visualized the PSMF behavior in a silica bead-packed microchannel using fluorescence microscopy. A two-phase separation mixed solution composed of 1-butyl-3-methylimidazolium chloride [(C₄mim)Cl, an ionic liquid], K₂HPO₄, and water, with Eosin Y as a fluorescent dye, was introduced into a microchannel (200&#xa0;μm wide, 40&#xa0;μm deep) packed with 10&#xa0;μm silica beads. Phase separation was induced by cooling from 40 to 25&#xa0;°C, resulting in an ionic liquid-rich phase containing Eosin Y and a K₂HPO₄-rich phase. Fluorescence microscopy equipped with a CMOS color camera enabled visualization of the flow distribution. When the ionic liquid-rich solution was used, non-fluorescent (black) circular regions approximately 10&#xa0;μm in diameter—corresponding to the silica beads—were observed, surrounded by green fluorescent areas resulting from the distribution of Eosin Y in the ionic liquid phase. These observations indicate that the ionic liquid-rich phase predominantly flows away from the surfaces of the beads. Conversely, when the K₂HPO₄-rich solution was used, green fluorescent regions appeared at the bead locations, surrounded by non-fluorescent areas. This indicates that the ionic liquid-rich phase, which contains Eosin Y, preferentially flows near the surfaces of the silica beads. These results provide the first direct visual evidence of PSMF behavior within a particle-packed microchannel. The findings support the proposed flow dynamics of PSMF in HPLC columns and validate the separation mechanism of the phase-separation mode.</p> Graphical abstract <p></p>