The tools for analytical resolution of enantiomers have evolved in recent years with advances in chromatographic techniques. As in any analytical method, the separation of enantiomeric pairs should achieve a rapid and complete resolution of the two chromatographic peaks and must also be reproducible and robust. Ideally it should also separate sample impurities, reach a low LOD/LOQ (limit of detection/limit of quantification) and show an appropriate elution order. Moreover, the ideal chromatographic conditions should ensure the stability of the sample during the analysis in addition to the compatibility of the sample medium with the mobile phase and the column.
Such a list of conditions for the resolution of challenging samples requires decisions concerning both the chiral stationary phases and the chromatographic modes employed. Our goal to find the best chromatographic method must be tempered with the use of a minimum number of chiral stationary phases (CSPs), thus, an efficient screening strategy is essential.
The starting point is to decide which technology is to be used with a specific type of selector. Both liquid chromatography (LC) and supercritical fluid chromatography (SFC) are powerful tools that have been used successfully. The decision, therefore, is typically based upon equipment availability and suitability for the scale and type of molecule. Although SFC lacked sensitivity in its early development, recent advances have resulted in the efficient use of SFC with mass spectrometry (MS) detectors.
In LC method development, one may use organic solvent mixtures or water-compatible mobile phases. Normal-phase applications have historically been more widely used for the separation of enantiomers. However, the reversed-phase separations, together with polar organic modes, should be seriously considered when samples are in aqueous media to exploit the LC–MS compatibility of these mobile phase systems.
The present article aims to cover the practical approaches applied in our laboratories to screen analytical samples. Based upon extensive experimental work, we will focus on the results achieved with a range of 3- and 5-μm silica-based phases, containing amylose and cellulose derivatives as chiral selectors in a coated or immobilized fashion. Strategies for efficient HPLC method development with these CSPs in normal phase, polar mode and reversed phase conditions, as well as SFC, will be addressed. The different modes will be reviewed with the preferred primary screening, as well as the potential alternatives for higher peak efficiency (switch to smaller particles), fast analysis and unique selectivities with new selectors.