Development of Cryogenic HPLC for Wide Range Low Temperatures Using Liquefied Propane as Mobile Phase and Its Application to Hydrogen/Deuterium Compounds Separation

Abstract

In this study, a novel cryogenic high-performance liquid chromatography (HPLC) system, where the separation temperature can be controlled from -196 to -50 °C, was developed using liquefied propane as the mobile phase. Pyrene retention in a bare silica-gel column increased as the column temperature decreased, ranging from -130 to -50 °C. The linear van’t Hoff plot for pyrene (r² = 0.9926) suggested that a variation in the retention mechanism for pyrene did not occur in this temperature range. The separation of pyrene and pyrene-d₁₀ was enhanced by the decrease in the separation temperature in the bare silica-gel column, that is, the H/D selectivity increased with a decrease in temperature. The H/D pyrene was not separated in an ODS column even though the cryogenic HPLC at -90 °C. This suggested that hydrogen bonding between H/D pyrene and hydroxy groups in the mobile phase chemicals is essential for H/D separation in reversed-phase HPLC.