Finding the Best Separation for Enantiomeric Mixtures

Sep 01, 2010

The separation of enantiomers by chromatography is a well-established technique in chemical and pharmaceutical research. There are currently a very large number of columns commercially available and, to meet time constraints, an efficient screening strategy for the selection of conditions with a high success rate is required. A study of screening procedures has been performed which demonstrated that a relatively small set of polysaccharide-derived columns can be used on a routine basis in different chromatographic modes to meet modern analytical needs.

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 resolution of racemic mixtures by chromatography has reached maturity in the last decade (1,2). Although most racemates and diastereomeric mixtures can be separated at analytical level, the challenge is to achieve complete resolution of the components while reaching high-speed, preserving efficiency and with limited screening efforts.

Figure 1: Decision tree in method development in the resolution of enantiomers by chromatography.
Resolution of enantiomers by chromatography is predominantly performed by using chiral stationary phases. A number of chiral selectors are available commercially at present, such as proteins, polysaccharides, antibiotics, brush-type molecules, ionic exchangers, crown ethers, cyclodextrins and multiple polymers (1,2). One of the most widely used groups of CSPs is the group consisting of silica-based polysaccharide-derived chiral supports. They can operate in different modes and, therefore, a preliminary decision tree to devise the screening strategy is substantial when a new sample is received (see Figure 1).

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.