Recent developments in the high-throughput separation of biologically active chiral compounds via high performance liquid chromatography

Bioactive compounds, including active pharmaceutical ingredients (APIs), are often chiral molecules where stereoisomers have different biological and therapeutic activity. Nevertheless, the preparation of these molecules can lead to racemic or scalemic mixtures (it is not trivial to produce just the optically pure compound). The evaluation of the enantiomeric purity of bioactive compounds, and therefore quality, is indeed of fundamental importance for regulatory scopes. Chiral high performance liquid chromatography (HPLC) is the gold standard technique to separate and to purify enantiomers. This comes from the wide availability of commercial chiral stationary phases (CSPs) and operational modes, which makes the technique extremely versatile. In recent years, the most relevant trend in the field of chiral analytical HPLC has been the development of CSPs suitable for fast or even ultrafast separations, thus favoring the high throughput screening of biologically active chiral compounds. This process has somehow lagged behind compared to achiral HPLC, due to a series of practical and fundamental issues. The experience has shown how in chiral chromatography even very basic concepts, such as the supposed kinetic superiority of core-shell (pellicular) particles over fully porous ones to improve the chromatographic efficiency, cannot be taken for granted. In this review, the most relevant fundamental and practical features that must be taken into consideration to design successful high-throughput, fast enantioseparations will be discussed. Afterwards, the main classes of CSPs and the most relevant, recent (last five-year) high-throughput applications in the field of the separation of chiral bioactive compounds (for pharmaceutical, forensic, food, and omics ap-plications) will be considered.