Preparative separation of ten flavonoids from Scutellaria baicalensis Georgi roots using two-dimensional countercurrent chromatography with an online-storage, dilution, and mixing interface

The process of counter-current chromatography (CCC) separation for natural products typically necessitates the use of multiple solvent systems to accommodate constituents with a wide range of polarities. However, the incompatibility between these different solvent systems often results in unsuccessful online 2D successive separations. In this study, a 2D CCC system was developed, featuring an interface for online-storage, dilution, and mixing. It facilitated the implementation of online 2D CCC using different solvent systems. The method was subsequently applied for the preparative isolation of flavonoids from Scutellaria baicalensis Georgi roots. For ¹D CCC, n-heptane-ethyl acetate–methanol-water (HepEMWat, 5:5:4:6, v/v) was utilized, while for ²D CCC, ethyl acetate-n-butanol-water (EBuWat, 0:5:5, v/v) was employed. The eluent with low resolution in ¹D CCC was stored online, diluted three times using the lower phase of EBuWat (0:5:5, v/v), and subsequently transferred into ²D CCC for further isolation utilizing the same EBuWat (0:5:5, v/v) system. As results, six lipophilic compounds were isolated in ¹D CCC in a normal mode, while two major hydrophilic constituents were isolated in a pH-peak-focusing mode in ²D CCC. Additionally, two additional compounds were purified through subsequent semi-preparative HPLC separation in order to resolve co-elution in ²D CCC. The developed 2D CCC system with a multifunctional interface demonstrated to be an exceptionally efficient and promising approach for the high-throughput purification of complex natural products.