High Performance Liquid Chromatography at −196 °C

Ultralow temperature high-performance liquid chromatography (HPLC) was developed using a liquefied gas as the mobile phase. HPLC separation of low molecular weight alkanes at −196 °C with liquid nitrogen mobile phase was successfully achieved, whereas their GC separation at −196 °C using helium gas mobile phase failed to elute the analytes due to strong adsorption. Prior to the further study of HPLC at −196 °C, the effect of column temperature on the chromatographic behavior was investigated, and it was found that the retention of analytes drastically increased when the column temperature was over the boiling point of the mobile phase. As the study of retention control in HPLC at −196 °C, the mobile phases of nitrogen and methane mixtures were investigated. The addition of methane to the nitrogen mobile phase suppressed the retention of the analytes (tetra-deuterated methane, ethane, and propane), that is, the retention on HPLC at ultralow temperature could be controlled by the mobile phase composition, akin to the typical retention in HPLC. The selectivity toward the n- and iso-alkane in HPLC at −196 °C was altered compared with that in GC separation at room temperature. A significant enhancement of retention of alkanes compared with alkanes were observed in HPLC at −196 °C.