Innovative Off‐line Two‐dimensional Supercritical Fluid Chromatography, Separation, and Purification of Structurally Similar Polyphenols From <i>Gentiana szechenyii</i> Using Propyl Urea and Phenyl/Tetrazole Stationary Phases

Although traditional Tibetan medicine is a valuable source for the discovery of novel lead compounds, the isolation of structural analogs from natural sources remains a challenge in the field of separation science. The Tibetan medicine Gentiana szechenyii (GC) is widely to treat various types of inflammation and upper respiratory tract infections. Despite its strong medicinal value, a complex purification process has limited investigations into its chemical composition. Therefore, in this study, a novel off-line two-dimensional supercritical fluid chromatographic (2D-SFC) method was developed and successfully applied to the efficient separation and purification of complex compounds and structural analogs from GS. Initially, the crude GS sample was pretreated using a binary medium-pressure liquid chromatography system to obtain the target fraction (Fr16, 1.0 g). A novel propyl urea and phenyl/tetrazole stationary phases off-line 2D-SFC system was then developed and applied for the separation and purification of Fr16. Consequently, five polyphenolic compounds were successfully isolated with purities of >95%. The obtained results demonstrate the potential of off-line 2D-SFC for maximizing the discovery of novel structural analogs and isomers, thereby providing a theoretical basis for the further exploitation of natural products.