The Separation of Optical Isomers

One problem with a special emphasis in the pharmaceutical industry involves the isolation of stereoisomers (optical isomers). An organic molecule with no asymmetric carbon atoms is denoted as achiral, but if it contains one or more asymmetric carbon atoms, it would be denoted as a stereoisomer. If a single asymmetric carbon exists, there will be two enantiomers, while with two asymmetric carbon atoms the molecule forms four diastereomers. A reader not familiar with the general concept of steriochemistry should refer to a introductory organic chemistry text. Biological systems are inherently based on enantiomeric or stereoisomeric biochemistry. Thus, there has been a trend toward selecting a single stereoisomer as a new chemical entity for pharmaceuticals (Collins et al. 1997 Stinson 1999). 

In practice, there are multiple methods for creating and isolating a stereoisomer, which may apply to the system of interest. It may be possible to synthesize the molecule with chiral chemistry or to use chiral-inducing enzymatic chemistry. It is often possible to separate stereoisomers by chromatography, and it is sometimes possible to crystallize stereoisomers. 

The history of the crystallization of stereoisomers is fascinating. Pasteur noted the visible difference in morphology between crystals of the isomers of ammonium sodium tartrate in 1848. This turned out to be a fortuitous circumstance as ammonium sodium tartrate happens to crystallize as conglomerate crystals, that is, a eutectic mixture containing crystals of the two pure isomers. Such behavior has since been found to be infrequent, although it has proved industrially important. Pasteur separated the crystals manually to achieve the first documented isomeric separation  

The second important observation on stereoisomer separation also involved ammonium sodium tartrate. Thirty-four years after Pasteur s observation, Jungfleisch (1882) observed that carefully introducing crystals of the individual isomers into different areas of a supersaturated solution of ammonium sodium tartrate resulted in the growth of isomerically pure crystals. These two observations form the basis for most industrial scale crystallizations for the purification of enantiomers or diastereoi-somers. However, it is more common for a solute to crystallize with the thermodynamically stable crystal form being a compound of the two isomers. This is typically denoted as a racemic compound. Secor (1963) made the first systematic review of optical isomer separation by crystallization, based upon phase behavior. Collet, Brienne, and Jacques (1980) applied systematic thermodynamics to the phase behavior, and developed straightforward methods for correlating the solubilities of isomers. 

Before proceeding, we first review the thermodynamics that apply to stereoisomers. The isomers have the same chemical 

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Chiral Chromatography. Chiral chromatography is used for the analysis of enantiomers, most useful for separations of pharmaceuticals and biochemical compounds (see Biopolymers, analytical techniques). There are several types of chiral stationary phases those that use attractive interactions, metal ligands, inclusion complexes, and protein complexes. The separation of optical isomers has important ramifications, especially in biochemistry and pharmaceutical chemistry, where one form of a compound may be bioactive and the other inactive, inhibitory, or toxic. 

Particular examples for the separation of optical isomers in the (pharmaceutical) industry include prazinquatel [51], 3-blockers [52], chiral epoxide [6],thia-diazin EMD5398 [18] and hetrazipine [7]. The Belgian company UCB Pharma uses a large-scale SMB from NOVASEP to perform optical isomer separation at a scale of several tons per year. Almost all of these separations are performed on cellulose-based stationary phases using organic eluents [4]. 

Other recent applications of suitable secondary chqmical equilibria in the mobile phase include the separation of optical isomers by using opti-... 

In MEKC, mainly anionic surface-active compounds, in particular SDS, are used. SDS and all other anionic surfactants have a net negative charge over a wide range of pH values, and therefore the micelles have a corresponding electrophoretic mobility toward the anode (opposite the direction of electro-osmotic flow). Anionic species do not interact with the negatively charged surface of the capillary, which is favorable in common CZE but especially in ACE. Therefore, SDS is the best-studied tenside in MEKC. Long-chain cationic ammonium species have also been employed for mainly anionic and neutral solutes (16). Bile salts as representatives of anionic surfactants have been used for the analysis of ionic and nonionic compounds and also for the separation of optical isomers (17-19). 

The growing interest in supramolecular chirality stems not only from the intrinsic relevance of such studies for the origin of chirality in life processes, but also from the potential technological applications, such as the separation of optical isomers for the pharmaceutical or food industries. 

B. A bonded stationary phase for the separation of optical isomers has the structure... 

Chemically bonded phases may also be tailored to a specific separation problem. A case in point is the synthesis of chiral stationary phases for the separation of optical isomers. Another application of polar bonded phases, which is beyond the scope of this book,... 

It seems tc, also be worth mentioning that the described procedure has been used for micro-preparative separations of mephenytoin and hexobarbital enantiomers (26) p -CD solutions were also successfully used for resolution of 1-[2-(3-hydroxyphenyl)-l-phenylethylJ-4-(3-me-thyl-2-buteny1) piperazine enantiomers in RP systems (.20). An especially interesting example of the application of -CD is the separation of optical isomers of D,L - norgestrel (27). 

The first step in method development is selecting an adequate HPLC mode for the particular sample. This choice depends on the character of the sample compounds, which can be either neutral (hydrophilic or lipophilic) or ionic, low-molecular (up to 2000 Da) or macromolecular (biopolymers or synthetic polymers). Many neutral compounds can be separated either by reversed-phase or by normal-phase chromatography, but a reversed-phase system without ionic additives to the aqueous-organic mobile phase is usually the best first choice. Strongly lipophilic samples often can be separated either by non-aqueous reversed-pha.se chromatography or by normal-phase chromatography. Positional isomers are usually better separated by normal-phase than by reversed-phase chromatography and the separation of optical isomers (enantiomers) requires either special chiral columns or addition of a chiral selector to the mobile phase. 

The immohilization of the cage complexes on the surface through apical groups offers interesting application possibilities. This approach enables one to obtain ion-exchange resins especially selective for metal ions. The immobilized optically active cations allow one to obtain ion-exchange resins for the separation of optical isomers, such as racemic amino acids or optically active complexes. 

Szepesi et al. reported an ion-pair separation of eburnane alkaloids on a chemically bonded cyanopropyl stationary phase. As counter-ion, di-(2-ethyl hexyl)phosphoric acid or (+)-10-camphorsulfonic acid were used in a mobile phase consisting of hexane - chloroform -acetonitrile mixtures (Table 8.8, 8.9). Because of the poor solubility of the latter pairing ion, diethylamine (Table 8.9) was added to the mobile phase. Addition of diethylamine considerably reduced the k1 of the alkaloids, due to suppression of the ionization of the alkaloids. However, due to the strong acidic character of the pairing ion, ion-pairs were still formed under these conditions. The camphorsulfonic acid containing mobile phases were found to be very useful for the separation of optical isomers (Table 8.10, 8.11, Fig.8.8) 6. It was also found that the selectivity of the system could be altered by choosing different medium-polarity solvents (moderator solvents) as dioxane, chloroform or tetrahydrofuran. The polar component of the solvent system affected peak shape. Based on these observations, a method was developed to analyze the optical purity of vincamine and vinpocetine. For the ana-... 

For the ferric siderophore complexes, comparison of the CD spectra of the chromium complexes of ferrichrome and enterobactin with the CD spectra of their iron complexes [and the separation of optical isomers of even ferric(benzhydroxamate)3 complexes in nonaqueous solution 192)] have shown that the same rule applied to the CD spectra for chromium complexes can be used for iron siderophore complexes as well iron(III) complexes will have a predominant A configuration in solution if the CD band in... 

The successful application of enantioselective chromatography as a valuable approach to the separation of optical isomers on a preparative and even production scale has attracted the attention of most pharmaceutical companies. 

Of particular interest is the use of esters of a-oxyphosphonic acid for the analysis of trace amounts of carboxylic acids using a selective phosphorus detector [353]. TMS esters are also used in chromatographic analysis [254,354-366]. Interesting methods of analysing keto-acids have been described [367-372] and the separation of optical isomers has been studied [199,373,374]. 

Such differences can indeed be small for very similar compounds, such as certain isomers. However, exploitations of some very selective interactions of solute molecules with the stationary phase have now been documented the separation of optical isomers on optically active stationary phases, as will be discussed in more detail in one of the following sections, is definitely the best example of such a situation. Less... 

GC was the first instrumental technique that allowed the separation of optical isomers [89], This technique offers relatively high peak efficiency compared with HPLC, but the volatility and thermostability requirements of the analyte limit the application of GC for the separation of drug enantiomers. However, GC is used for the enantioseparation of several drugs and many drug intermediates, natural compounds, food and beverage components, essential oils and perfumes. 

The overwhelming majority of GC methods, employing CDs as stationary phase or as stationary phase additive, deal with the separation of optical isomers. The increasingly... 

Tubocurarine was used as a chiral selector for the separation of optical isomers of a series of organic carboxylates (amethopterin, ketoprofen, TV-protected amino acids) using CE in the pH range 5-7. In several cases, Rs values of about 2 were observed, but there were no clear correlations between structures of analytes and efficiency of chiral resolution. 

The dramatic influence of the pH of the medium was first encountered at the separation of optical isomers of a s-permethrinic add carried out with half an equivalent amount of (S)-2-benzylaminobutanol ((S)-BAB). The resolution started in the presence of a 25 mol % excess of sodium hydroxide. In such a medium the diastereoisomeric salt containing the almost pure (S)-CPA crystallized ee %%) with a yield of 27% counted to the amount of racemic acid. After removal of the precipitate by filtration, the excess alkali was neutralized with a counted amount of hydrochloric add. That time the resolving agent remained in the filtrate crystallized with (R)-CPA enantiomer as diastereoisomeric salt and in the second mother liquor an almost racemic CPA-Na salt was found (it can be recycled into the resolution). 

The separation of optical isomers presents an entirely different problem. By definition, dextro and levo isomers have identical dipole moments, vapor pressures, etc. Therefore, there can be no separation unless an asymmetric environment exists within the column. In gas-liquid chromatography this condition is fulfilled when an optically active liquid is employed as the... 

HPTLC separation material is available in the form of precoated layers supported by glass, plastic sheets or aluminum foil. Although precoated layers of aluminum oxide, cellulose, polyamide, ion exchange materials, reversed phase silica (alkyl bonded) have been commercially available for quite some time, the vast majority of TLC separations is carried out on normal phase silica gel, particularly in quantitative TLC analysis. Recently introduced plates modified with amino, cyano and diol functional groups bonded to the silica can be used as multimodal media. Depending on the developing solvent used, they extend normal phase or reversed phase chromatographic properties. These layers may affect the predominant role of normal phase silica gel in TLC to a certain extent. They are described in Chapter 4 of this book. Also chiral layers for the separation of optical isomers are available and cause increased interest (11). 

De Haan AB, Simandi B. Extraction technology for the separation of optical isomers. In Marcus Y, Shatma MM, Marin-sky JA, editors. Ion exchange and solvent extraction. New York Marcel Dekker 2001. p 255-294. 

The first paper on the employment of such technique for the separation of optical isomers by column chromatography was carried out by Wulff et al. [42] in 1977 while more recently (1994) Kriz et al. [43] used MIPs for resolution of racemates by planar chromatography. 

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