Enantiomers chiral separation

CE has been established as a very efficient technique for the separation of drug enantiomers. Chiral separations in CE are also based on the formation of diastereomeric complexes between the enantiomers and a chiral selector. The main advantages of the technique are its high efficiency, short analysis times, versatility due to the great variety of chiral selectors that can be added to the BGE, short equilibration times required when changing the chiral selector and low consumption of selector. °... 

The FITC labeling method was also applied to chiral separations of amino acids on a microchip to determine the enantiomeric ratios of amino acids found on a meteorite [27], Since biotic amino acids are normally single enantiomers, chiral separations of amino acids are not truly clinical in nature, but illustrate the potential for chiral separations of small molecules of clinical interest. Ma-thies and co-workers used this technique to search for evidence of life in extraterrestrial environments. Enantiomeric forms of Val, Ala, Glu, and Asp could be discriminated by addition of a-, (3-, or y-cyclodextrin (CD) to the run buffer. Improved resolution with faster separations was found with respect to conventional CE. This method has been modified, by addition of SDS to the buffer, to perform cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) [28]. Increasing the SDS concentration decreased the magnitude of elec-troosmotic flow (EOF), increasing the effective migration distance, and therefore the resolution on the microchips. 

Chiral separations are particularly important for biologically active chiral compounds (e.g., pesticides) where the active ingredient is only one enantiomer. Chiral separations have been used to track biological degradation rates and changes in source characteristics. GC-based applications include chiral chordane and a-HCH in the polar bear food chain and levetiracetam and its enantiomer in dog plasma and urine whilst LC applications include amino acids in water and chiral aroma compounds in alcoholic drinks. 

Chiral separations are concerned with separating molecules that can exist as nonsupetimposable mirror images. Examples of these types of molecules, called enantiomers or optical isomers are illustrated in Figure 1. Although chirahty is often associated with compounds containing a tetrahedral carbon with four different substituents, other atoms, such as phosphoms or sulfur, may also be chiral. In addition, molecules containing a center of asymmetry, such as hexahehcene, tetrasubstituted adamantanes, and substituted aHenes or molecules with hindered rotation, such as some 2,2 disubstituted binaphthyls, may also be chiral. Compounds exhibiting a center of asymmetry are called atropisomers. An extensive review of stereochemistry may be found under Pharmaceuticals, Chiral. 

Traditionally, chiral separations have been considered among the most difficult of all separations. Conventional separation techniques, such as distillation, Hquid—Hquid extraction, or even some forms of chromatography, are usually based on differences in analyte solubiUties or vapor pressures. However, in an achiral environment, enantiomers or optical isomers have identical physical and chemical properties. The general approach, then, is to create a "chiral environment" to achieve the desired chiral separation and requires chiral analyte—chiral selector interactions with more specificity than is obtainable with conventional techniques. 

Most chiral chromatographic separations are accompHshed using chromatographic stationary phases that incorporate a chiral selector. The chiral separation mechanisms are generally thought to involve the formation of transient diastereomeric complexes between the enantiomers and the stationary phase chiral ligand. Differences in the stabiHties of these complexes account for the differences in the retention observed for the two enantiomers. Often, the use of a... 

Chiral separations present special problems for vaUdation. Typically, in the absence of spectroscopic confirmation (eg, mass spectral or infrared data), conventional separations are vaUdated by analysing "pure" samples under identical chromatographic conditions. Often, two or more chromatographic stationary phases, which are known to interact with the analyte through different retention mechanisms, are used. If the pure sample and the unknown have identical retention times under each set of conditions, the identity of the unknown is assumed to be the same as the pure sample. However, often the chiral separation that is obtained with one type of column may not be achievable with any other type of chiral stationary phase. In addition, "pure" enantiomers are generally not available. 

As in tic, another method to vaUdate a chiral separation is to collect the individual peaks and subject them to some type of optical spectroscopy, such as, circular dichroism or optical rotary dispersion. Enantiomers have mirror image spectra (eg, the negative maxima for one enantiomer corresponds to the positive maxima for the other enantiomer). One problem with this approach is that the analytes are diluted in the mobile phase. Thus, the sample must be injected several times. The individual peaks must be collected and subsequently concentrated to obtain adequate concentrations for spectral analysis. 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

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... 

From the pioneering studies of Ito et al. [117], CCC has been mainly used for the separation and purification of natural products, where it has found a large number of applications [114, 116, 118, 119]. Moreover, the potential of this technique for preparative purposes can be also applied to chiral separations. The resolution of enantiomers can be simply envisaged by addition of a chiral selector to the stationary liquid phase. The mixture of enantiomers would come into contact with this liquid CSP, and enantiodiscrimination might be achieved. However, as yet few examples have been described in the literature. 

Although some applications for preparative-scale separations have already been reported [132] and the first commercial systems are being developed [137, 138], examples in the field of the resolution of enantiomers are still rare. The first preparative chiral separation published was performed with a CSP derived from (S -N-(3,5-dinitrobenzoyl)tyrosine covalently bonded to y-mercaptopropyl silica gel [21]. A productivity of 510 mg/h with an enantiomeric excess higher than 95% was achieved for 6 (Fig. 1-3). 

S. G. Allenmark, Separation of enantiomers by protein-based chiral phases in A practical approach to chiral separations by liquid chromatogra.phy, G. Subramanian, VCH, Weinheim (1994) Chapter 7. 

Enantiomeric separations have become increasingly important, especially in the pharmaceutical and agricultural industries as optical isomers often possess different biological properties. The analysis and preparation of a pure enantiomer usually involves its resolution from the antipode. Among all the chiral separation techniques, HPLC has proven to be the most convenient, reproducible and widely applicable method. Most of the HPLC methods employ a chiral selector as the chiral stationary phase (CSP). 

In general, a liquid membrane for chiral separation contains an enantiospecific carrier which selectively forms a complex with one of the enantiomers of a racemic mixture at the feed side, and transports it across the membrane, where it is released into the receptor phase (Fig. 5-1). 

The second system studied was the separation of the chiral epoxide enantiomers (la,2,7,7a-tetrahydro-3-methoxynaphth-(2,3b)-oxirane Sandoz Pharma) used as an intermediate in the enantioselective synthesis of optically active drugs. The SMB has been used to carry out this chiral separation [27, 34, 35]. The separation can be performed using microcrystalline cellulose triacetate as stationary phase with an average particle diameter greater than 45 )tm. The eluent used was pure methanol. A... 

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