Development of a HPLC system using a phase-separation multiphase flow as an eluent: an influence of column pressure on phase separation and chromatogram at room temperature.

Abstract

We have developed a HPLC system where phase-separation multiphase flow works in the separation column as an eluent. We call the novel separation mechanism a phase-separation mode. The ternary mixed solution of water/acetonitrile/ethyl acetate, which is one of the two-phase-separation mixed solutions, caused the phase-separation multiphase flow via phase change from homogeneous to heterogeneous with the temperature effect (from 20 to 0°C). In this study, we tried to perform phase-separation multiphase flow with the pressure effect instead of the temperature one. The fused-silica capillary tube (50cm length and 50µm inner diameter) was allied to the downstream of the column to apply the pressure of 5.5MPa to the system. Model analytes of 2,6-naphthalenedisulfonic acid (2,6-NDS) and 1-naphthol (1-NA) were examined. For example, solutions (the volume% of water/acetonitrile/ethyl acetate; 20:55:25, organic-rich) and (60:30:10, water-rich) were used as eluent. The model analytes were not separated with both solutions at the pressure of 1.5MPa and 20°C. But with the organic-rich solution, 1-NA and 2,6-NDS were separated in this order and with the water-rich solution, they were separated in the reverse order at the pressure of 5.5MPa and 20°C. The phase-separation mode could be performed at the high pressure even at the room temperature.