Improved enantioseparation via the twin-column based recycling high performance liquid chromatography

• Twin-column R-HPLC was used for improving enantioseparation for the first time.
• Experimental comparison of two R-HPLC methods was reported for the first time.
• Performance of two R-HPLC methods was evaluated on the enantioseperation.
• Intractable four stereoisomers of isoxazolidines were separated by baseline.

A long-neglected recycling high performance liquid chromatography (R-HPLC) strategy was adopted to improve the enantioseparation efficiency in this work. Taking intractable isoxazolidine mixtures as the example, two different R-HPLC methods including single-column and twin-column based strategy were applied to improve the enantioseparation efficiency. Under the optimized conditions, these two methods present distinct separation results: the conceptually simple and commonly used single-column based R-HPLC led to severe peak broadening and degraded separation efficiency; on the contrary, the enantioseparation was significantly improved by adopting the relatively complicated twin-column strategy. This observed difference might be mainly attributed to less peak spreading and sample mixing of the latter, as a result of avoiding redirecting the effluent back to LC pump.