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

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 (50 cm length and 50 µm inner diameter) was allied to the downstream of the column to apply the pressure of 5.5 MPa 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 1.5 MPa 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 5.5 MPa and 20 °C. The phase-separation mode could be performed at the high pressure even at the room temperature. Graphical Abstract