Indirect separation of enantiomers

If enantiomers are derivatized with a chiral, optically pure reagent a pair of diastereomers is formed. Diastereomers are molecules with more than one centre of asymmetry, which therefore differ in their physical properties. From the scheme in Fig. 21.5 it is clear that they are not mirror images. Diasetereomers can be separated with a non-chiral chromatographic system, but in any case the derivatization reagent must be chosen very carefully. 

For the reaction all hints for precolumn derivatization, as given in Section 19.10, need to be considered. Moreover, it is of the utmost importance that the reagent is of highest optical purity. Otherwise four isomers will be formed (two pairs of enantiomers), but the enatiomers cannot be separated in a non-chiral system and the result will be erroneous (see Table 21.2). 

Another important point to consider is the prevention of racemization during derivatization. It is also a problem that the reagent needs to be present in excess during the reaction if this excess cannot be removed prior to injection an interfering peak may occur in the chromatogram. 

It is favourable for the functional group to be derivatized to be situated close to the chiral centre of the molecule. Too large a distance from the centre of asymmetry can lead to the impossibility to resolve the diastereomers. If possible one should try to form amides, carbamates or ureas. All these classes of 

TABLE 21.2 Purity of reagent and obtained purity of sampie 

It is favourable for the functional group to be derivatized to be situated close to the chiral centre of the molecule. Too large a distance from the centre of asymmetry can lead to the impossibility to resolve the diastereomers. If possible one should try to form amides, carbamates or ureas. All these classes of compounds have a relatively rigid structure (in comparison with, for example, esters) which seems to facilitate the separation. If a choice is possible, one of the reagent s isomers should be taken that allows the minor compound of the pair of enantiomers to be eluted first then the small peak will not be lost in the tailing of the leading large one. 

Diastereomers may have differing detector properties. For quantitative analysis it is necessary to determine the calibration curve. 

Turiel and A. Martin-Esteban, Anal. Bioanal. Chem., 378, 1876 (2004) P. Spegel, L.L Andersson and S. Nilsson, in Giibitz and Schmid (first footnote of this chapter), p. 217 B. SeUergren, in Subramanian (first footnote of this chapter), p, 399. 

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I. Review on indirect separation of enantiomers as diastereomeric derivatives using UV, fluorescence and electrochemical detection, Biomed. Chromatogr., 6 163 (1992). 

W. Lindner, Indirect separation of enantiomers by hquid chromatography, in M. Zief and L. J. Crane (eds.), Chromatographic Chiral Separation, Marcel Dekker, New York, 1988, pp. 91-130. 

Lindner, W. Indirect separation of enantiomers by liquid chromatography. Chromatographic Science Series, 1988, 40, 91-130. 

Enantiomers can be separated by traditional chromatographic methods, provided they have been previously derivatized with a chiral compound to produce diaste-reomers. This method of indirect separation of enantiomers is explained in Section 22.5. 

Sublimation method for indirect separation of enantiomers is very similar, in principle to the above mentioned distillation-based procedure. In the next example the diastereoisomeric molecular complexes could be separated using the significant difference between their thermal stability. 

Chapter 8 provides a discussion of the indirect separation of enantiomer pairs, which, as described above, can be obtained with the help of a nonchiral chromatographic system. The author addresses such crucial issues as the principle of derivatization, structural demands imposed on derivatizing agents, reasons for the choice of a given agent, and, finally, present the compounds most frequently used for derivatization. The chapter ends with an overview of the separations of diastereoisomers performed with the aid of TLC. 

Separation of enantiomers can be performed via two different kinds of approaches, direct and indirect ones. In the indirect approach the enantiomers are derivatized prior to their separation, while in the direct approach they are placed in a chiral environment and are not subjected to a chemical reaction. 

There are several important requirements that must be met for the successful use of the indirect chromatographic separation of enantiomers. 

The chromatographic separation of enantiomers as diastereomers was first developed using GLC (1). Subsequently, many separations using GLC were reported, but modern LC has dominated the field of enantiospedfic drug analysis in recent years. Nevertheless, the arrival of high-resolution capUlary GLC has revived interest in the use of indirect enantiomer separation via this type of chromatography, and today GLC remains important in the analytical separation of enantiomers after derivatization with CDAs. The availability of sensitive detection methods, for example, mass spectrometry, electron capture, etc., enhances the applicability of GLC in indirect enantiospedfic drug analysis. 

The advent of modern column LC in the 1970s rapidly led to the use of this chromatographic technique in the separation of enantiomers as dia-stereomeiic derivatives. Today, most of the reported new developments in indirect enantiomer resolutions use LC, and LC is particularly important in the resolution of chiral pharmaceuticals. 

The large number and variety of applications of the chiral derivatization approach attest to the success, viability, and importance of the technique. It is expected that despite the predictable advances to be realized in the near future in the development of direct chromatographic— mainly chiral-stationary-phase-based—separations of enantiomers, the indirect approach will continue to be widely used to solve stereochemical problems in the pharmaceutical, pharmacological, and toxicological arenas. 

As mentioned above, the different solubility of diastereomers offers a challenge for their separation by diastereomeric crystallization. The indirect HPLC separation of enantiomers relies on their different interaction with a (achiral) stationary phase. The interaction of the enantiomeric pair (R, S) with one (let us assume it to have S configuration) enantiomer of a chiral derivatizing reagent may be expressed as follows ... 

Quinine, quinidine and cinchonidine, which are amino alcohols with high chiral capability, have been used as selectors for the separation of enantiomers of acids containing a hydrogen bonding function [24]. These chiral selectors have high UV absorbance, providing indirect detection possibilities for solutes without inherent UV absorbance (Figure 6). 

This is the indirect approach for the chromatographic separation of enantiomers. The reaction of the two forms of an enantiomer with an optically pure chiral reagent gives a mixture of diastereoisomers that are not mirror images of each other and therefore can be separated on a nonchiral, common LC phase. This is shown in Figure 4 where amino acids have been derivatized with o-phthaldialdehyde and N-isobutyryl-... 

GC separation of enantiomers can be performed either direct (use of a chiral stationary phase, CSP) or indirect (off-column conversion into diastereomeric derivatives and separation by non-chiral stationary phases). The direct method is preferred as being simpler and minimizing losses during sample preparation. The key, of course, is to find a chiral stationary phase with both selectivity and temperature stability. 

The most popular thin layer chromatography (TLC) techniques for separation of enantiomers are described here 1) use of non-chiral phases for indirect resolution of optical isomers after derivatization to obtain the corresponding diastereoisomers and 2) direct resolution of enantiomers using chiral stationary phases or chiral mobile phases. Advantages and limits of all reported techniques are discussed. 

Since all the physical properties of two given enantiomers are the same in the absence of a chiral, or optically active, medium, their chromatographic resolution needs a different approach from the relatively simple separation of geometrical isomers, stereoisomers or positional isomers. Two methods are used. The older technique of indirect resolution, requires conversion of the enantiomers to diastereoisomers using a suitable chiral reagent, followed by separation of the diastereoisomers on a non-chiral GC or LC stationary phase. This technique has now been largely superseded by direct resolution, using either a chiral mobile phase (in LC) or a chiral stationary phase. A variety of types of chiral stationary phase have been developed for use in GC, LC and SFC(21 23). 

In order to generally categorize the reaction schemes mentioned previously and the following ones in the course of indirect enantioseparation techniques, it has to be emphasized again, that the reciprocity principle should always be applicable. This means that if a chiral acid as the CDA can be used successfully to resolve the enantiomers of a chiral amine, then this optically pure amine as the CDA will equally well separate the enantiomers of the acid by the indirect method. The OPA reaction (see Figure 4) is therefore equally well suited for analyzing the optical purity of thiols, amines or amino acids. 

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