Development of an HPLC device with water/acetonitrile with NaCl mixed solution that induces a phase-separation multiphase flow as the eluent in the separation column.

We developed a novel HPLC device where the phase-separation multiphase flow worked as the eluent in the separation column by using a water/acetonitrile/ethyl acetate triple mixed solution as a dual-phase-separation solution. Dual-phase-separation solutions form a phase-separation multiphase flow in a microscopic space. The new separation mechanism in the HPLC is called phase-separation mode. In this study, we used water and acetonitrile with NaCl mixed solution as a dual-phase-separation solution instead of the triple mixed solution. Octadecylsilyl (ODS)-modified particle- and porous silica particle-packed separation columns were united with the HPLC device for phase-separation mode caused by phase-separation multiphase flow. NA (1-naphthol) and NDS (2,6-naphthalenedisulfonic acid) were analyzed by the device as model sample. Using the water and acetonitrile with NaCl mixed solution at the solvent volume ratio of 5:5, NA and NDS were not separated on either column at 25 °C. On the other hand, they were separated with the order NDS and NA on the ODS column and separated with the order NA and NDS on the silica column in phase-separation mode at 0 °C. We discuss the separation mechanism of phase-separation mode using the water and acetonitrile with NaCl mixed solution at 0 °C.

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

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.