The effect of column structure and chromatographic conditions (flow rate, inlet pressure, and temperature) on separation behavior in low-temperature HPLC using pure liquid CO2 mobile phase was investigated. Three types of packed columns (fully porous, small nonporous, and core-shell particles) and a monolithic column (fully porous) were used. The effect of temperature (-5 to -30°C), inlet pressure (5.3 or 6.3 MPa), and flow rate (1 or 2 mm/s) on chromatographic behavior was evaluated. The retention factors of the analytes (naphthalene, anthracene, and pyrene) were not affected by either inlet pressure or flow rate. Although the column temperature somewhat affected the separation efficiency, the typical relationship between temperature and separation efficiency was not observed. On the other hand, both inlet pressure and flow velocity affected the separation efficiency. In this study, the highest separation efficiency was achieved in the core-shell column with a flow rate of 1 mm/s and inlet pressure of 6.3 MPa. The best separation efficiency under this condition was 12 m in theoretical plate height (85 000 N/m) for pyrene at -30°C. In addition, the supplied liquid CO2 was found to solidify in the column at low temperatures and its behavior also depends on both the chromatographic conditions (particularly for flow rate) and the column structure. The typical relationship between solidification and chromatographic behavior was not observed clearly.
2017 The Society for Chromatographic Sciences