The study reports the development of a high-performance liquid chromatography (HPLC) system in which a phase-separation multiphase flow as the eluent and a silica-particle based packed column as the separation column were combined to form the phase separation mode. Twenty-four types of mixed solutions of water/acetonitrile/ethyl acetate and water/acetonitrile were applied as eluents to the system at 20 °C. 2,6-Naphthalenedisulfonic acid (NDS) and 1-naphthol (NA) were injected as model analytes into the system. They showed separation tendency in organic solvent-rich eluents in normal-phase mode and NA was detected earlier than NDS. Subsequently, seven types of the ternary mixed solutions were examined as eluents in the HPLC system at 20 °C and 0 °C. These mixed solutions worked as a two-phase separation mixed solution, providing a phase-separation multiphase flow at 0 °C in the separation column. In the organic solvent-rich eluent, the mixture of analytes was separated at both 20 °C (normal-phase mode) and 0 °C (phase-separation mode), with NA being detected earlier than NDS. The separation at 0 °C was more efficient than at 20 °C. In the water-rich eluent, the mixture of NDS and NA was not separated at 20 °C but was separated at 0 °C (phase-separation mode), with NDS being detected earlier than NA. We also discussed the separation mechanism of phase-separation mode in HPLC together with the computer simulation for the multiphase flow in the cylindrical tube having sub-μm inner diameter.
Keywords: HPLC; Phase-separation mode; Phase-separation multiphase flow; Silica-particle packed column.
© 2023. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.