Resolution Separating Enantiomers

The process of separating the enantiomers of a racemic mixture is called a resolution. To accomplish this task, the environment must be made chiral so that the enantiomers have different properties. These different properties can then be employed in the separation process. 

Another resolution method that is sometimes employed involves the selective reaction of one enantiomer of a racemic mixture with one enantiomer of a chiral reagent, 

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Sharpless epoxidations can also be used to separate enantiomers of chiral allylic alcohols by kinetic resolution (V.S. Martin, 1981 K.B. Sharpless, 1983 B). In this procedure the epoxidation of the allylic alcohol is stopped at 50% conversion, and the desired alcohol is either enriched in the epoxide fraction or in the non-reacted allylic alcohol fraction. Examples are given in section 4.8.3. 

Three general methods exist for the resolution of enantiomers by Hquid chromatography (qv) (47,48). Conversion of the enantiomers to diastereomers and subsequent column chromatography on an achiral stationary phase with an achiral eluant represents a classical method of resolution (49). Diastereomeric derivatization is problematic in that conversion back to the desired enantiomers can result in partial racemization. For example, (lR,23, 5R)-menthol (R)-mandelate (31) is readily separated from its diastereomer but ester hydrolysis under numerous reaction conditions produces (R)-(-)-mandehc acid (32) which is contaminated with (3)-(+)-mandehc acid (33). 

This procedure is restricted mainly to aminodicarboxyhc acids or diaminocarboxyhc acids. In the case of neutral amino acids, the amino group or carboxyl group must be protected, eg, by A/-acylation, esterification, or amidation. This protection of the racemic amino acid and deprotection of the separated enantiomers add stages to the overall process. Furthermore, this procedure requires a stoichiometric quantity of the resolving agent, which is then difficult to recover efficiendy. Practical examples of resolution by this method have been pubUshed (50,51). 

Synthetic chiral adsorbents are usually prepared by tethering a chiral molecule to a silica surface. The attachment to the silica is through alkylsiloxy bonds. A study which demonstrates the technique reports the resolution of a number of aromatic compoimds on a 1- to 8-g scale. The adsorbent is a silica that has been derivatized with a chiral reagent. Specifically, hydroxyl groups on the silica surface are covalently boimd to a derivative of f -phenylglycine. A medium-pressure chromatography apparatus is used. The racemic mixture is passed through the column, and, when resolution is successful, the separated enantiomers are isolated as completely resolved fiactions. Scheme 2.5 shows some other examples of chiral stationary phases. 

Crystallization methods are widely used for the separation, or resolution, of enantiomer pairs. Enantiomer mixtures may essentially crystallize in two different ways. In around 8 per cent of cases, each enantiomer crystallizes separately, giving rise to a mechanical mixture of crystals of the two forms, known as a conglomerate. Conglomerates may usually be separated by physical methods... 

HPLC separations are one of the most important fields in the preparative resolution of enantiomers. The instrumentation improvements and the increasing choice of commercially available chiral stationary phases (CSPs) are some of the main reasons for the present significance of chromatographic resolutions at large-scale by HPLC. Proof of this interest can be seen in several reviews, and many chapters have in the past few years dealt with preparative applications of HPLC in the resolution of chiral compounds [19-23]. However, liquid chromatography has the attribute of being a batch technique and therefore is not totally convenient for production-scale, where continuous techniques are preferred by far. 

With regard to the resolution of enantiomers, some applications can be found with modified silica gel supports. Thus, a Pirkle-type CSP was used for the separation of 200 mg of a racemic benzodiazepinone [75]. Also tris-(3,5-dimethylphenyl)carba-mate of cellulose coated on silica [91, 92] was applied successfully to the resolution of the enantiomers of 2-phenoxypropionic acid and to oxprenolol, alprenolol, propranolol among other basic drugs. However, the low efficiency of this technique and the relative high price of the CSPs limits its use to the resolution of milligram range of sample. 

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). 

Racemic mixtures of sulfoxides have often been separated completely or partially into the enantiomers. Various resolution techniques have been used, but the most important method has been via diastereomeric salt formation. Recently, resolution via complex formation between sulfoxides and homochiral compounds has been demonstrated and will likely prove of increasing importance as a method of separating enantiomers. Preparative liquid chromatography on chiral columns may also prove increasingly important it already is very useful on an analytical scale for the determination of enantiomeric purity. 

Kinetic Resolution. Since enantiomers react with chiral compounds at different rates, it is sometimes possible to effect a partial separation by... 

Most methods for the resolution of enantiomers contained in a reaction mixture consist in the conversion of the compounds into stable or transient diastereoisomers and separation of the latter on the basis of their different physico-chemical properties. 

The resolution of enantiomers by liquid chromatography using chiral stationary phases is based on the formation of reversible diastereomeric complexes of different stability between the sample and stationary phase. Since the formation of the complexes is strongly dependent on the structure of the sample, there are no universal chiral stationary phases. The specific advantages of TLC for enantiomeric separations result from its low cost, convenience and speed (10,97,98). The main limitation, particularly with respect to column liquid chromatography, is the small number of phases currently available. 

A chiral GC column is able to separate enantiomers of epoxy pheromones in the Type II class, but the applications are very limited as follows a custom-made column packed with a p-cyclodextrin derivative as a liquid phase for the stereochemical identification of natural 3,4- and 6,7-epoxydienes [73, 74] and a commercialized column of an a-cyclodextrin type (Chiraldex A-PH) for the 3,4-epoxydiene [71] (See Table 3). The resolution abilities of chiral HPLC columns have been examined in detail, as shown in Table 7 and Fig. 14 [75,76, 179]. The Chiralpak AD column operated under a normal-phase condition separates well two enantiomers of 9,10-epoxydienes, 6,7-epoxymonoenes and 9,10-epoxymonoenes. Another normal-phase column, the Chiralpak AS column, is suitable for the resolution of the 3,4-epoxydienes. The Chiralcel OJ-R column operated under a reversed-phase condition sufficiently accomplishes enantiomeric separation of the 6,7-epoxydienes and 6,7-epoxymonoenes. 

In addition to Rh-catalysed hydroformylation, this special phase behaviour has been successfully applied to other continuous catalytic reactions - such as Ni-catalysed, enantioselective hydrovinylation [66] and the lipase-catalysed kinetic resolution and enantiomer separation of chiral alcohols [67]. 

Several CD derivatives (charged and uncharged) are available which should allow the separation of most chiral molecules with at least one of them. However, due to the complexity of chiral recognition mechanisms, the determination of the best selector based on the analyte structure is challenging. Eurthermore, separations using CDs are influenced by numerous factors, so that no general rule can be applied for the successful resolution of enantiomers. ... 

An extremely important aspect in pharmaceutical research is the determination of drug optical purity. The most frequently applied technique for chiral separations in CZE remains the so-called dynamic mode where resolution of enantiomers is carried out by adding a chiral selector directly into the BGE for in situ formation of diastereomeric derivatives. Various additives, such as cyclodextrins (CD), chiral crown ethers, proteins, antibiotics, bile salts, chiral micelles, and ergot alkaloids, are reported as chiral selectors in the literature, but CDs are by far the selectors most widely used in chiral CE. 

Sanger-van de Griend et al. (29) determined the binding constants of several local anaesthetics with DM-/3-CD. These data showed that the achiral separation of analogues is a result of their mobility difference, whereas the resolution of enantiomers results from the difference in their binding constants with CDs. 

Compound (6) contains 3 centers of dissymmetry, and its resolution into separated enantiomers could effected using ( )-mandelic acid [7]. The least soluble diastereomer was found to be the (-)-mandelate salt (m.p. 164.5-164.8°C). Formation of the hydrochloride salts of both enantiomers gave (-i-)-isoxsuprine HCl (m.p. 196-196°C) and (-)-isoxsuprine HCl (m.p. 195-196°C). The two asymmetric centers at C-1 and C-2 were correlated with those of the erythro (p-OH-C6H4-CH(OH)-CH(CH3)-NH-) residue. 

For the separation of crystalline diastereomeric p,n-pairs, the same principles as for enantiomers are followed. Similar to the chromatographic resolution of enantiomers, the scale may vary significantly, but, in general, the separations of diastereomeric pairs are more reliable. For example, acids and lactones which may be separated via their phenylglycinol amide derivatives by MPLC on silica gel. Some examples with their derivatives and chromatographic method used for separation arc listed in Table 10. 

Because the physical properties of enantiomers are identical, they seldom can be separated by simple physical methods, such as fractional crystallization or distillation. It is only under the influence of another chiral substance that enantiomers behave differently, and almost all methods of resolution of enantiomers are based upon this fact. We include here a discussion of the primary methods of resolution. 

The resolution of an acyclic chiral amine into its separate enantiomers has not been achieved yet, and it appears that the enantiomers are very rapidly inter-converted by an inversion process involving a planar transition state ... 

Column selection is not yet a precise science. There are no uniform theories for the separation mechanisms that operate for the resolution of enantiomers using the wide array of chiral stationary phases that are currently available. It is therefore strongly recommended that the literature is searched to determine whether the analyte under investigation has been separated and which chiral stationary phase was used. Many chiral food volatiles have now been separated and a comprehensive database has been compiled that is continually updated and contains most reported separations (Koppenhoefer et al., 1993 Roussel and Piras, 1993). There are many other sources in the literature (Anonymous, 1993a,b Konig, 1993 Maas et al., 1994 Schreier et al., 1995 Juchelka et al., 1998 Miranda et al., 1998). 

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