Since Louis Pasteur discovered chirality in 1984, it has become an important part of life. The systematic investigation of the biological activity of individual stereoisomers has become the rule for all new chiral drugs and chiral considerations are now integral parts of drug research and development and of the regulatory process. In this context, there has been a considerable development of enantioselective synthetic methodologies, which have now reached a high degree of diversity and complexity. Simultaneously, this trend has created an intensive demand for stereoselective separation techniques and analytical assays for precise determination of the enantiomeric purity of chiral compounds. The development of chiral stationary phases (CSPs) or chiral selectors for gas chromatography (GC), liquid chromatography (LC) and capillary electrophoresis (CE) rapidly opened a new dimension in the area of separation technologies.

Need for chiral separation
The separation of enantiomers is of great importance because many chiral drug enantiomers exhibit wildly differing pharmacokinetic, pharmcodynamic and toxicological profiles. Some examples of activity differences are given in Table 1.

While enantioselective chromatography has become the method of choice for analytical determinations of enantiomeric purity, the prominence of the technique on a preparative scale is also gaining increasing recognition as a powerful alternative for the supply of pure enantiomers of bioactive compounds. In particular, the concomitant introduction of both, efficient chiral stationary phases, and efficient separation techniques, such as simulated moving-bed (SMB) chromatography, offers new possibilities in the field of chromatographic separations which were not feasible some years ago.

Approaches for chiral separation
The successful application of chiral chromatography as a valuable tool to the separation of chiral twins on a preparative and even production scale has attracted the attention of most pharmaceutical industries. Basically, two major options exist for preparing single enantiomers of chiral compounds. The “chiral approach”, consists in designing an enantioselective synthesis of the desired enantiomer. If both enantiomers are needed, it is usually necessary to develop two independent syntheses. The chiral approach includes enantioselective synthesis using substances from the chiral pool and or chiral auxiliaries, enzymes or stereoselective catalytic processes. In contrast to the chiral approach, the “racemic approach” implies the preparation of the racemate, which is subsequently resolved into the corresponding enantiomers.

This preparation is usually achieved by a reaction sequence which generally presents a much lower degree of difficulty than that for the corresponding optically active forms. In the racemic approach, separation methods comprise the widely used technique of crystallization of diastereoisomers, membrane systems and chromatographic methods.

Chiral chromatographic separation
Among the chromatographic methods, the most used approach is undeniably liquid chromatography (LC) separation on chiral stationary phases (CSPs). Over the last two decades a large number of chiral stationary phases have been described in the literature and some of them are commercially available viz. Pirkle CSPs, Polysaccharide CSPs, Cavity CSPs and Macrocyclic glycopeptide CSPS. A literature search has identified HPLC as the most popular enantiomer separation technology, followed by CE-, GC- and, finally, CEC-related techniques (Fig. 1.). In addition, the interest in HPLC-based enantiomer separation technologies appears to be growing, while the number of publications devoted to GC-, CE- and CEC-based assays is stagnant or even in decline. A striking advantage of HPLC-based over GC- and CE/CEC-based protocols is their ready scalability.

Generally, chromatographic enantiomer separation protocols developed for analytical applications can conveniently be transformed into preparative procedures, providing quick access to enantiomerically pure drug candidates. Usually, an analytical HPLC separation will be developed first and can be used as a starting point for a preparative method. Several considerations particular to prep LC separation should be kept in mind. Some of the salient features are mentioned here.

1. Normal-Phase chromatography with organic solvents as the mobile phase is often preferred. The removal of volatile organic solvents from the product will be easier than for reversed-phase HPLC with aqueous mobile phase.
2. Solubility of the sample is very important in prep LC because it desirable to inject large weight of the sample dissolved in a relatively small volume of mobile phase.
3. In prep LC it is advantageous to use larger particles (> 7 um or larger), higher flow rate   (>> 10 ml/min) and large ? Value.
4. Touching band separation is sufficient for prep separation.

Particularly in the early development stages, where time constraints prevail over cost considerations, chromatographic enantiomer separation is appreciated as the most efficient way to produce enantiomerically pure drug candidates in gram- to kg-amounts. Recent advances in chromatographic process engineering have adapted simulated moving bed (SMB) technology to enantiomer separation.

This continuously operating chromatographic technology allows enantiomer separation at the multi-ton scale, at costs that can compete with conventional manufacturing options.

The number of commercial CSPs has dramatically increased over the last 10 years, even though only several CSPs are available for large scale applications. Among them, the polysaccharide derivatives, manufactured by Daicel (Japan), stand out in the field because of their high selectivity and high loadability shown towards a large number of different molecules. Their applications go from high-performance analytical separations to the largest scale of preparative chromatography worldwide. The excellent separation properties of polysaccharide derivatives are due to their structural complexity. A number of chiral distinction models that have been put forward to account for the enantiomeric resolution by HPLC. These are often based on the three-point interaction model advanced by Dalgelish in 1952.

The chemical structure of these CSPs consists of a natural or synthetic polysaccharide, cellulose or amylose, within which the hydroxyl groups are derivatised with acid chlorides or isocyanates to form esters and carbamates, respectively. Figure 2 and 3 shows the chemical structure of the cellulose and amylose based chiral stationary phases. CSPs were applied as a preparative tool for many years before their potential as a powerful technique for the analysis of chiral compounds was recognized.

However, the real potential of enantioselective chromatography for the preparative separation of enantiomers was definitely established in 1973 by Hesse and Hagel who introduced fully acetylated cellulose (triacetylcellulose) as a new efficient chiral CSP. They successfully achieved the preparative separation of the enantiomers of various chiral compounds.  

For many years, triacetylcellulose was practically the only chiral stationary phase available for preparative separations and it has been used for the chromatographic resolution of a broad variety of chiral molecules. Most widely employed commercially available preparative chiral stationary phases are given in Table 2.

With the exponential explosion of chiral technology a large number of analytical chiral columns are available and are now routinely used for the determination of the enantiomeric composition of mixtures of enantiomers from enantioselective syntheses, from biological investigations, or from pharmacokinetic or toxicology studies. Some of these phases have also become extremely useful for enantioselective preparative separations of chiral molecules.

There are currently a limited number of reported applications of chromatographic chiral separations at the process scale. This is probably may be due o the confidential nature of these activities. Table 3 gives a brief list of preparative chiral resolutions.   

Conclusion
Chiral chromatography whether on the analytical/laboratory scale it is now the method of choice as it is rapid, easily and generally applicable, and it further provides both the enantiomers. On the pilot and process scale, the chromatographic approach allows a continuous supply of enantiopure substances in quantities required to perform the desired investigations in drug discovery and development process. Even on the production scale, and especially since the introduction of the simulated moving bed technology, chromatography must now be considered as one of the possible approaches for obtaining single enantiomers. However, as cost is a major factor at production scale, it will be determining for deciding which approach should be applied, and the choice will remain a case by case decision. Nevertheless, the implementation of the SMB technique at a scale of several tons per year already demonstrates the real potential of this approach.

Without any reservation chromatography can be considered as a powerful alternative for the preparation of enantiopure compounds.