Separation of Racemic Compounds

Pasteur could separate the optical isomers due to the crystalline forms of sodium ammonium tarttate [22], Natural tartaric acid rotates the plane of polarized light. However, tartaric acid from laboratory synthesis does not show any rotation of the plane of polarized light. 

Pasteur observed that the crystals of sodium ammonium tartfate are a mixture of two asymmetric forms that are mirror images of one another. He could separate the two forms of crystals manually and he arrived at two forms of sodium ammonium tartrate. One form rotated the plane of polarized light clockwise and the other form rotated the plane of polarized light anticlockwise. The separation in this way is possible for tartaric acid only below 27°C, because above this temperature the isomers of tartaric acid do not form separate crystals. 

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Additional enantiomerically pure 4,5-dihydroisoxazoles are prepared by separation of racemic compounds via chiral sulfoxides5-8, or by microbial reduction of 5-acetyl-4,5-di-hydroisoxazoles 34. 

Separation of racemic compounds into enantiomers by resolutions that involve ... 

In schematic terms the separation of racemic compound R,S into enantiomers by reaction with a single enantiomer of R is represented in Scheme 3.1. RR and SR are diastereomers, and can be covalent compounds or salts. R is chosen so that the solubilities of RR and SR in a particular solvent are different. The racemic mixture, now in combination with R, i.e. RR and SR, is now recrystallized and one diastereoisomer, say RR, will crystallize out whereas the other, SR, will stay in solution. Sometimes when the first crop of crystals is merely enriched in RR, a number of further recrystallizations will be necessary. Salts are the preferred diastereoisomers (RR and SR ), because they are both easily formed and readily re-converted to the starting enantiomers, following separation. 

There is no doubt that enantiomeric separation of racemic compounds will become increasingly important. The enantiomeric separation method should, however, be simple, green and economical. In this chapter, some examples of separations by inclusion complexation with chiral or achiral host compounds are described. In future, chiral host compounds for enantiomeric separation should be improved. More green and cheap chiral sources should be developed for more efficient and safe enantioseparations. For example, naturally occurring chiral sources such as sugars, amino acids, terpenes, alkaloids and cellulose could become useful and important... 

One area of chirotechnology which is undergoing rapid development is chiral HPLC, whereby the use of chiral stationary phases (CSPs) permits the direct separation of racemic compounds into constituent enantiomers. Despite the capital outlay required, for example, for columns costing upwards of 3000, the use of preparative chiral HPLC in drug discovery has a number of benefits. After development of an appropriate method based on a previously defined analytical separation has been carried out, rapid and quantitative separation of racemates can be achieved, with evaporation of solvent from column fractions affording pure enantiomers directly. Although preparative chiral HPLC is less amenable to scale-up than other resolution techniques, it may be ideal for preliminary screening of both enantiomers in circumstances where manipulation of small quantities of material, for example, by crystallization, is impractical and prone to contamination problems. 

At the separation of racemic compounds using another chiral material (namely resolution) the behaviour of enantiomeric mixture forming the racemate determines the efficiency of... 

Park JH, Whang YC, Jung YJ, Okamoto Y, Yamamoto C, Carr PW, et al. Separation of racemic compounds on amylose and cellulose dimethylphenylcarbamate-coated zirconia in HPLC. J Sep Sd 2003 26 1331-6. 

Polenske D, Lorenz H, Seidel-Morgenstern A. Potential of different techniques of preferential crystallization for enantio-separation of racemic compound forming systems. Chirality 2009 21 728 737. 

The type of CSPs used have to fulfil the same requirements (resistance, loadabil-ity) as do classical chiral HPLC separations at preparative level [99], although different particle size silica supports are sometimes needed [10]. Again, to date the polysaccharide-derived CSPs have been the most studied in SMB systems, and a large number of racemic compounds have been successfully resolved in this way [95-98, 100-108]. Nevertheless, some applications can also be found with CSPs derived from polyacrylamides [11], Pirkle-type chiral selectors [10] and cyclodextrin derivatives [109]. A system to evaporate the collected fractions and to recover and recycle solvent is sometimes coupled to the SMB. In this context the application of the technique to gas can be advantageous in some cases because this part of the process can be omitted [109]. 

E. Erancotte, Chromatography as a separation tool for the preparative resolution of racemic compounds in Chiral separations, applications and technology, S. Ahuja (Ed.), American Chemical Society, Washington (1997) Chapter 10. 

Statistically, of the compounds enantioresolved by macrocyclic glycopeptide CSPs, new polar organic mode accounts for more than 40 %, balanced by reversed-phase mode, while typical normal-phase operation resulted in approximately 5 % of separations. Some categories of racemic compounds that are resolved on the glycopeptide CSPs at different operating modes are listed in Table 2-4. 

Since the proline residue in peptides facilitates the cyclization, 3 sublibraries each containing 324 compounds were prepared with proline in each randomized position. Resolutions of 1.05 and 2.06 were observed for the CE separation of racemic DNP-glutamic acid using peptides with proline located on the first and second random position, while the peptide mixture with proline preceding the (i-alamine residue did not exhibit any enantioselectivity. Since the c(Arg-Lys-0-Pro-0-(i-Ala) library afforded the best separation, the next deconvolution was aimed at defining the best amino acid at position 3. A rigorous deconvolution process would have required the preparation of 18 libraries with each amino acid residue at this position. 

Erancotte E. (1996) Chromatography as a Separation Tool for the Preparative Resolution of Racemic Compounds, in Chiral Separations. Applications and Technology, Ahuja S. (ed.), American Chemical Society, p. 271-308. 

A new brush-type CSP, the Whelk-0 1, was used by Blum et al. for the analytical and preparative-scale separations of racemic pharmaceutical compounds, including verapamil and ketoprofen. A comparison of LC and SFC revealed the superiority of SFC in terms of efficiency and speed of method development [50]. The Whelk-0 1 selector and its homologues have also been incorporated into polysiloxanes. The resulting polymers were coated on silica and thermally immobilized. Higher efficiencies were observed when these CSPs were used with sub- and supercritical fluids as eluents, and a greater number of compounds were resolved in SFC compared to LC. Compounds such as flurbiprofen, warfarin, and benzoin were enantioresolved with a modified CO, eluent [37]. 

The selectivity of another cellulose-based CSP, Chiralcel OJ, has also been examined in SFC [60]. Separations of racemic drugs such as benoxaprofen, temazepam, and mephobarbital were obtained. Acetonitrile proved to be a better modifier than methanol for some of the compounds investigated. The four optical isomers of a calcium channel blocker were resolved by Siret et al. on the Chiralcel OJ CSP [30]. In LC, two CSPs were required to perform the same separation. 

Competing amines such as triethylamine and di-rc-butylamine have been added to the mobile phase in reversed-phase separations of basic compounds. Acetic acid can serve a similar purpose for acidic compounds. These modifiers, by competing with the analyte for residual active sites, cause retention time and peak tailing to be reduced. Other examples are the addition of silver ions to separate geometric isomers and the inclusion of metal ions with chelating agents to separate racemic mixtures. 

Bicchi C, Artuffo G, D Amato A, Manzin V, Galfl A, GaUi M, Cyclodextrin derivatives in the GC separation of racemic mixtures of volatile compounds, Part V Heptakis 2,6-dimethyl-3-pentyl- 3-cyclodextrins,Chromatogr 15 710-714, 1992. 

In fluorine-18 chemistry some enzymatic transformations of compounds already labelled with fluorine-18 have been reported the synthesis of 6-[ F] fluoro-L-DOPA from 4-[ F]catechol by jS-tyrosinase [241], the separation of racemic mixtures of p F]fluoroaromatic amino acids by L-amino acylase [242] and the preparation of the coenzyme uridine diphospho-2-deoxy-2-p F]fluoro-a-o-glucose from [ F]FDG-1-phosphate by UDP-glucose pyrophosphorylase [243]. In living nature compounds exhibiting a carbon-fluorine bond are very rare. 

Tablet Separation of racemic grafted NucleosU 300-5 compounds on 6-0-(norborn-2-ene-5-carboxyl)- 8-CD- ...

The synthesis of a-branched amines caught our attention, as these compounds exhibit parhcular biological activity. In several of our ongoing projects involving the synthesis of biologically achve compounds, we required the asymmetric synthesis of a-branched chiral amines. a-Branched amines can be prepared by various routes, all performed in an asymmetric fashion. Currently, enzymatic and chemical separation of racemic a-branched amines and also diastereoselective methods still play a major role on an industrial scale [25]. However, due to poor separation by the latter methods and for economic reasons, catalytic approaches will be favored. 

The chromatographic methods use gas or liquid separately as the mobile phase, hence the terms gas chromatography (GC) and liquid chromatography (LC). Gas chromatography could not be accepted as the method of choice for the chiral resolution of racemic compounds mainly because of its requirement for the conversion by derivatization of the racemic compound into a volatile species. Besides, the separated enantiomers cannot be collected for further pharmacological and other studies. Moreover, GC cannot be used at the preparative scale. 

The evolution of CDs as chiral selectors in the liquid chromatographic separation of enantiomers has been a subject of interest for the last two decades. The presence of the chiral hollow basket, or cavity, makes these molecules suitable for the chiral resolution of a wide range of racemic compounds. At present, the use of CDs as chiral selectors for enantiomeric resolution by liquid... 

Cyclodextrin-based CSPs are among the most popular materials used for the chiral resolution of racemic compounds. These CSPs have a wide range of applications because they can be used successfully in all three mobile phase modes normal, reversed, and polar organic. There are numerous examples of chiral separations on CDs and CSPs based on their derivatives. Some of the important chiral separations are discussed herein. 

The chiral resolution on CD-based CSPs depends on the formation of inclusion complexes in the cavities and, therefore, the structures and sizes of analytes are very important for the chiral resolution of racemates on these phases. Amino acids often are considered to be the best class of racemic compounds to use in structural studies. In 1987, Han and Armstrong [55] studied the chiral resolution of amino acids on -CD-based CSPs. It was observed that different retention, separation, and resolution factor values were obtained for different amino acids under identical chromatographic conditions, which indicated that the structures and sizes of amino acids govern their chiral resolution. The same observations may be found in the work of Fujimura et al. [70]. 

The Pirkle-type chiral stationary phases are quite stable and exhibit good chiral selectivities to a wide range of solute types. These CSPs are also popular for the separation of many drug enantiomers and for amino acid analysis. Primarily, direct chiral resolution of racemic compounds were achieved on these CSPs. However, in some cases, prederivatization of racemic compounds with achiral reagents is required. The applications of these phases are discussed considering re-acidic, re-basic, and re-acidic-basic types of CSP. These CSPs have also been found effective for the chiral resolution on a preparative scale. Generally, the normal phase mode was used for the chiral resolution on these phases. However, with the development of new and more stable phases, the reversed phase mode became popular. 

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