Indirect Enantiomer Separation

It becomes obvious from the clustered data points that the binding constant for the 7 -enantiomer is too small to be accurately determined by this method. Hence, indirect affinity CE (resolution method) was utilized to determine the binding constant for the 7 -enantiomer. Indirect affinity CE makes use of the knowledge of the constant for one enantiomer (here, A s) and in addition of experimental separation data as obtained with the racemate of DNB-Leu in presence of the tBuCQN selector as BGE additive. By use of Equation 1.10, an enantioselectivity factor may be defined as the ratio of the binding constants of S- and 7 -enantiomers yielding the following equation ... 

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. 

There are several characteristics of diastereomeric chiral separations (also known as indirect enantiomeric separations) that are worth mentioning. Achiral phases that are cheaper, more rugged, and widely commercially available are used. The elution order can be controlled by choice of the chirality of the derivatizing agent. This feature is useful for the analysis of trace levels of enantiomers. The separation can be designed such that the minor enantiomer is eluted first, allowing for more accurate quantitation. 

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. 

Using the indirect approach to achieve a chiral separation diminishes the need for a CS in the running buffer. Prior to analysis with CE, the enantiomers are derivatized with an optically pure agent to form diastereomers. An achiral environment, in which a pseudo-stationary phase is present, is sufficient to separate these diastereomers because they possess different physicochemical properties. The indirect separation is an efficient and versatile approach mainly because of the availability of numerous chiral derivatization reagents. These derivatizing reagents can contain chromo-phore, fluorophore, or electrochemical groups, which improves the detection of hardly detectable compounds. 

The drawbacks of the indirect approach are expensive derivatization reagents, the need for rapid and ending reactions, and the inevident recovery of the pure enantiomer after analysis. These are the main reasons why indirect separations are not applied firequentiy for chiral separations. 

When the direct separation approach is applied, the CS is added to the BGE solution. The CS and the enantiomers will form diastereomer complexes, which differ in stability constants. One enantiomer will thus interact more strongly with the selector compared with the other, resulting in a separation. Direct chiral separations are applied more frequently than indirect separations because of the above-mentioned drawbacks. Many selectors can be used in direct separations. Therefore, a discussion on the most important selectors took place. For each selector type, a few... 

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. 

The indirect separation method of a racemate into its enantiomers can be achieved by its derivatization with a chiral derivatizing agent before chromatography (Figure 8.2), resulting in a diastereomeric complex/salt (Figure 8.11) [11,56],... 

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). 

Peter, A. et al., A comparison of the direct and indirect LC methods for separating enantiomers of unusual glycine and alanine amino acid analogues, Chromatographia, 56, S79, 2002. 

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. 

The indirect approach has been widely applied since many functional groups can be derivatized with various chiral reagents and the covalent diastereoisomers can be separated with inexpensive non-chiral systems. Other advantages of the indirect approach are that method development is rather straightforward and that the detection sensitivity of the enantiomers can be improved by the selection of an appropriate CDR having a strong chromophor or fluorophor. 

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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