Effects of Porous Structure of Separation Column on Chromatograms Obtained by Phase-Separation Mode in HPLC

A novel HPLC system utilizing a phase-separation multiphase flow as the eluent has been developed, referred to as phase-separation mode. This research explores the influence of the porous structure in an octadecyl-modified silica (ODS) column (with a pore diameter of 12 nm) on chromatographic outcomes under the phase-separation mode in HPLC. The chromatograms obtained from the porous ODS column were compared to those generated with a non-porous ODS column. In preliminary experiments, twenty-four mixed solutions, comprising combinations of water/acetonitrile/ethyl acetate and water/acetonitrile, were introduced as eluents at a column temperature of 20 °C. A model mixture of 2,6-naphthalenedisulfonic acid (2,6-NDS) and 1-naphthol (1-NA) was injected into the system, with separation achieved in most solutions except for some highly organic solvent-rich solutions where 2,6-NDS eluted faster than 1-NA, indicating reverse-phase mode operation. Subsequently, the separation of the model mixture was assessed at 0 °C, and four specific ternary mixtures were analyzed in detail at both 20 °C and 0 °C. These ternary mixtures, defined by their volume ratios, exhibited a two-phase separation, establishing a phase-separation multiphase flow. Consequently, the solution flow was homogeneous at 20 °C and heterogeneous at 0 °C. For instance, solutions with water/acetonitrile/ethyl acetate ratios of 20:60:20 (organic solvent-rich) and 70:23:7 (water-rich) were introduced as eluents at both 20 °C and 0 °C. At 0 °C in the organic solvent-rich eluent, 1-NA eluted faster than 2,6-NDS, characteristic of the phase-separation mode. In contrast, the water-rich eluent resulted in faster elution of 2,6-NDS at both temperatures. The porous ODS column displayed improved separation efficiency at 0 °C compared to the non-porous column, which can be attributed to the porous effect under phase-separation conditions.