Enantiomers, separation approaches

An interesting and practical example of the use of thermodynamic analysis is to explain and predict certain features that arise in the application of chromatography to chiral separations. The separation of enantiomers is achieved by making one or both phases chirally active so that different enantiomers will interact slightly differently with the one or both phases. In practice, it is usual to make the stationary phase comprise one specific isomer so that it offers specific selectivity to one enantiomer of the chiral solute pair. The basis of the selectivity is thought to be spatial, in that one enantiomer can approach the stationary phase closer than the other. If there is no chiral selectivity in the stationary phase, both enantiomers (being chemically identical) will coelute and will provide identical log(Vr ) against 1/T curve. If, however, one... 

B. Sellergren, Enantiomer separation using tailor-made phases prepared by molecular imprinting in A practical approach to chiral separations by liquid chromatography, G. Subramanian, VCH, Weinheim (1994) Chapter 4. 

Conglomerate crystallization in the above case indicates that the inclusion approach may be further extended into the realm of the salt-type associates. Such an attempt is especially interesting due to the obvious role in enantiomer separation which relies heavily on the solubility difference of the enantiomeric salts under certain circumstances 137). 

A copolymerization approach of 0-9-[2-(methacryloyloxy)ethylcarbamoyl] cinchonine and cinchonidine with methacryl-modified aminopropylsilica particles was utilized by Lee et al. [71] for the immobilization of the cinchona alkaloid-derived selectors onto silica gel. The CSPs synthesized by this copolymerization procedure exhibited merely a moderate enantiomer separation capability and only toward a few racemates (probably because they were based on less stereodifferentiating cinchonine and cinchonidine). Moreover, the chromatographic efficiencies of these polymer-type CSPs were also disappointing. 

Three approaches can be employed to separate peptide stereoisomers and amino acid enantiomers separations on chiral columns, separations on achiral stationary phases with mobile phases containing chiral selectors, and precolumn derivatization with chiral agents [111]. Cyclodextrins are most often used for the preparation of chiral columns and as chiral selectors in mobile phases. Macrocyclic antibiotics have also been used as chiral selectors [126]. Very recently, Ilsz et al. [127] reviewed HPLC separation of small peptides and amino acids on macrocyclic antibiotic-based chiral stationary phases. 

Another direct approach to chiral polymeric stationary phases is the modification of commercially available polysiloxanes which contain reactive side groups. Thus, the diamide phase was linked to a modified XE-60 polysiloxane phase (Table 2). In one case (XE-60-L-Val-(/ or 5)-a-pea)124 another center of stereogenicity (R or S configuration) has been introduced in the amide group. An XE-60-L-Val-(S)-x-pea column was used for the enantiomer separation of racemic. V-rert-butoxycarbonyl amino acids after their methylation with diazomethane (serine and threonine as the O-trimethylsilyl derivatives) (Figure 12)124. 

Phinney KW, Sub- and supercritical fluid chromatography for enantiomer separations, in Chiral Separation Techniques A Practical Approach, (Suhramanian G, Ed.), p. 299, VCH Verlag, Weinheim, Germany (2001). 

The preparative-scale enantiomeric separations of enflurane, isoflurane and desflurane (cf. Figure 16) by gas chromatography on modified cyclo-dextrins have been achieved. The separation was aided by unprecedentedly large separation factors a of the enantiomers. Two approaches were followed. 

Another insect pheromone synthesis illustrates one of the drawbacks of chiral pool approaches. The ambrosia beetle aggregation pheromone is called sulcatol and is a simple secondary alcohol. This pheromone poses a rather unusual synthetic problem the beetles produce it as a 65 35 mixture of enantiomers so, in order to mimic the pheromone s effect, the chemist has to synthesize both enantiomers separately and mix them together in the right proportion. 

Thus far, the most successful approach to M IP-based CEC utilises capillary columns filled with a monolithic, super-porous imprinted polymer [39-41]. The morphology of a certain MIP monolith is depicted in Fig. 16.3. Using this system enantiomer separations with baseline resolution have been carried out in less than 2 minutes. The M IP-filled capillaries are obtained by an in situ photo-initiated polymerisation process (Fig. 16.4]. The capillary is filled with a pre-polymerisation mixture of imprint molecule, functional and cross-linking monomers (MAA and TRIM, respectively), radical initiator (2,2 -azobisisobutyronitrile) and solvent (toulene). Both ends of the capillary are sealed and the polymerisation is performed... 

Depending on the nature of the substituents on nitrogen, this reaction may give a complex product mixture, but in some cases such derivatization could be the basis of a useful indirect enantiomer separation. When one or two of the three N-substituents are methyl, for example, demethylation may be favored, and the reaction may be usable as a precolumn derivatiza-tion for chromatographic resolution. This approach was used in the analysis of encainide, a tertiary-amine antiarrhythmic drug (73). 

The three general approaches to enantiomer separation entail a chiral stationary phase, a chiral mobile phase, or a chiral reagent. Tandem columns, with reversed and chiral stationary phases, were used to separate 18 D-L pairs of PTC-amino acids in 150 min. OPA-amino acid enantiomers have been separated on both ion-exchange and reversed-phase columns using a sodium acetate buffer with a L-proline-cupric acetate additive. Chiral reagents, such as Marphey s reagent and OPA/IBLC (A-isobutiril-L cysteine), were successfully used for racemization analysis within 80 min. 

Another stimulating factor was the breakthrough in the development in process-scale chromatography around this time. During 1993 the first scaled-down versions of SMB units had been presented for use in pharmaceutical product recovery, focusing on enantiomer separation. This triggered discussion on how to develop generic approaches for quick method development and implementation, and led to the... 

It also needs to be emphasized that it was the development of robust and broadly applicable CSPs that has laid the foundations for economic chromatographic enantiomer separation on a preparative scale. Although indirect [57-62] and CMPA-based direct [63-65] chromatographic methodologies have seen some use in preparative enantiomer separation, the considerable efforts associated with chemical manipulation and/or recovery of the products render these approaches economically unattractive [66]. Preparative enantiomer separations employing CSPs are not subject to these limitations. With CSPs enantiomers can be processed directly (i.e. without prior derivatization) with readily volatile achiral mobile phases (devoid of SOs), simplifying product recovery to a trivial solvent evaporation step. 

For the separation of chiral molecules into their respective enantiomers, several approaches are possible by HPLC. These include precolumn derivatization to form diastereomers, followed by the use of normal-phase or reversed-phase HPLC, or addition of the derivatization reagent to the chromatographic mobile phase to form dynamic diastereomers during the separation process. Alternatively, specialty columns prepared with cyclodextrins or specific chiral moieties as stationary phases may be used. 

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