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_1,2 = 1.38 and R_2,3 = 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.

已经开发了循环逆流色谱法（CCC），并将其广泛用于制备分离中。由于越来越宽的峰具有更长的洗脱时间，因此必须在出现峰重叠之前停止循环洗脱，导致目标化合物的分离不充分。在这项研究中，提出了原位浓缩的概念，并设计了相应的技术来压缩废水，并保留分离效果（峰值分辨率）。通过将此技术与多阶段回收洗脱相结合，开发了一种新颖的无限回收CCC（URCCC）策略，以克服回收时间限制，从而提高分离度。URCCC策略已成功应用于天然萘酚醌的制备分离中，其中原位浓缩两次使用三步循环CCC洗脱。最后，以高分辨率分离出异丁酰基紫草素（1），β，β-二甲基丙烯酰胺紫草素（2）和异戊酰基紫草素（3）（R _1,2  = 1.38和R _2,3  = 1.26）。获得了高产率的纯萘酚醌（89.6％），每种的纯度均超过98％。总而言之，URCCC策略可以改善回收洗脱时间，直到目标化合物实现足够的分离为止，这可以在结构相关的化合物分离中，尤其是在天然产物分离中应用更广泛。