Preparation of modified rosin bonded silica high performance liquid chromatographic stationary phase and separation of <italic>Panax notoginseng</italic> saponins

The sanqi is the dried root of Panax notoginseng (Burk.) F. H. Chen. The main components responsible for the drug actions of sanqi are notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd, which account for about 80% of the saponin content in sanqi. It is widely used in the treatment of anemia, coronary heart disease, hypertension, stroke sequelae, and other diseases. However, sanqi has many chemical components with complex and similar structures, which are difficult to separate. In this study, alkylated silica gel bonded with hydrogenated rosin hydroxyethyl acrylate (HRHA) was prepared via mercapto-ene click chemistry. A new type of modified rosin-bonded silica stationary phase (SiO₂@HRHA) for high performance liquid chromatography was prepared for the separation of five saponins (R1, Rg1, Re, Rb1, and Rd). It was characterized by thermogravimetric analysis, Fourier-transform infrared spectroscopy, specific surface area and microporous physical adsorption and elemental analysis. The results showed that SiO₂@HRHA had a regular spherical shape with porous surfaces, along with a specific surface area of 308.55 m²/g and an average pore diameter of 6.78 nm. Performance evaluation of the column revealed that the SiO₂@HRHA column showed typical reversed-phase chromatographic behavior with better flowability and reproducibility. Results of the Tanaka test showed that SiO₂@HRHA column had good stereoselectivity and hydrogen bond capacity. Compared to other stationary phases, e. g. silica modified with acrylopimaric acid (16-hydroxyethyl-34-hydroxyethyl acrylate) ester (AAE) and dihydroterpineol (DTP), which were prepared in our laboratory at the same time, the SiO₂@HRHA column demonstrated better resolution (R_s) for the separation of the five saponins under optimal chromatographic conditions. The R_s values for R1, Rg1, Re, Rb1, and Rd were 3.33, 3.54, 20.17 and 9.72, respectively on the SiO₂@HRHA column. R_s between Rg1 and Re was also better than that obtained on a C18 column. Panax notoginseng saponins were separated on the SiO₂@HRHA column using acetonitrile and water as the mobile phases at the flow rate of 1.0 mL/min at 25 ℃. The optimal UV detection wavelength was 203 nm. It was found that the five saponins could be separated better using the SiO₂@HRHA column than the SiO₂@AAE and SiO₂@DTP columns. Because the ternary phenanthrene skeleton of the rosin group in SiO₂@HRHA had structural similarity and good stereoselectivity to the polycyclic compounds (Panax notoginseng saponins). In addition, according to the hydrophobicity evaluation, the SiO₂@HRHA column showed the best hydrophobicity among the three columns, which may be conducive to the separation of the five saponins. Thus, this study can provide a new avenue for the separation and purification of Panax notoginseng saponins from actual samples.