Development of a HPLC system using a phase-separation multiphase flow as an eluent coupled to a silica-particle packed column.

Abstract

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.