Unlimited recycling counter-current chromatography for the preparative separation of natural products: Naphthaquinones as examples.

Recycling counter-current chromatography (CCC) has been developed and widely used in preparative separation. Due to increasingly broader peaks with longer elution times, recycling elution must be stopped before a peak overlap occurs, resulting in the insufficient separation of target compounds. In this study, the concept of in situ concentration was proposed, and the corresponding technique was designed to compress the effluents with the reserved separation effect (peak resolution). By combining this technique with multi-stage recycling elution, a novel unlimited recycling CCC (URCCC) strategy was developed to overcome the recycling time limitation to improve the resolution. The URCCC strategy was successfully applied in the preparative separation of naturally occurring naphthaquinones, where the in situ concentration was used two times with three-stage recycling CCC elution. Finally, isobutyrylshikonin (1), β, β-dimethylacrylshikonin (2) and isovalerylshikonin (3) were separated with high resolutions (R = 1.38 and R = 1.26). A high yield of pure naphthaquinones was achieved (89.6%), and the purity of each exceeded 98%. In conclusion, the URCCC strategy can improve the recycling elution times until the target compounds achieve sufficient separation, which may enable a broader range of application in structurally related compounds separation, especially in natural product separation.