Study on the separation of triterpene saponins from Panax notoginseng by off-line two-dimensional supercritical fluid chromatography–ultrahigh-performance liquid chromatography

Abstract

The off-line two-dimensional supercritical fluid chromatography (SFC)–ultrahigh-performance liquid chromatography (UHPLC) was selected to separate the triterpene saponins from Panax notoginseng. The separation by SFC was performed on an Atlantis® HILIC silica column. Methanol was selected as a modifier, and the most time-saving gradient was developed. The decrease of the column temperature and the increase of the back pressure could shorten the retention time but had no effect on the separation selectivity. Then, the back pressure, column temperature, and flow rate were set as 131 bar, 45 °C, and 4.0 mL min ⁻¹, respectively. The retention behavior of the saponins from P. notoginseng was different between SFC and reversed-phase liquid chromatography (RPLC), which facilitated to construct an off-line SFC/RPLC–mass spectrometry (MS) system. In first dimension, a total of eight fractions were collected under SFC and further analyzed by RPLC–MS in second dimension. The result indicated that the retention behavior of triterpene saponins was mainly controlled by the hydrogen bonding interactions which were affected by the number and types of sugars, as well as the aglycone in the structure of triterpene saponins. Thus, the presence of “clustering effect” under SFC was observed, namely, one SFC peak always contained several saponins with same number of sugars and similar structure of aglycone. The clustering effect of triterpene saponins promised SFC to be used as first dimension to complete the preliminary crude separation in the two-dimensional liquid chromatography.