Stationary Phases for Chiral Separations

Separation of optically active isomers is one of the most important areas of HPLC apphcation in the pharmaceutical industry. Since most of biological systems are predominantly homochiral, different enantiomers of the same drug could have different effect and potency, and the development of enan-tioselective analytical (and preparative) separation methods is very important. Detailed description of chiral HPLC separation is given in Chapter 22 of this book here we only briefly review the specifics of distinctive types of chiral stationary phases (CSP). 

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Type V CSPs are protein phases. Because of the well established chemo- and stereospecificity of enzymes, a large number of experimentalists have adapted proteins in one form or another as stationary phases for chiral separations. The intermolecular forces responsible for analyte binding to these biopolymers are the same as for most other CSPs but the size and complexity of proteins makes them difficult to study computationally. One would think that with approximately 400 entries in the Brookhaven Protein Databank to select from, separation scientists would have used one of these proteins as a chiral selector and then use those atomic coordinates to carry out molecular modeling studies. Only one example has appeared in the literature where information from the PDB has been used to serve as a beginning point for molecular modeling of a protein CSP. In all other examples the CSP is viewed as having an unknown structure and Quantitative Structure-Enantioselective Retention Relationships (QSERRs) have been carried out. 

It is particularly difficult to predict the appropriate stationary phase for chiral separations (where the solutes are chemically identical mirror images of each other). The standard approach is to try a large number and see which of those work. 

The traditional operating mode of CEC with a conventional packed column is the use of commerdally available chromatographic resins, as used for HPLC or (iHPLC. Examples are RP Qg-modified silica particles of typical diameter 3-5 pm, ion-exchange resins and stationary phases for chiral separations. The latter indude... 

It is hence possible to access cyclodextrins selectively perfunctionahzed in good yields withoutlong purification processes. Potential appHcations of these derivatives include stationary phase for chiral separations, model for the design of artificial enzymes, and building blocks for supramolecular architectures [4, 21]. 

Shamsi SA, Warner IM (1997) Monomeric and polymeric chiral surfactemts as pseudo-stationary phases for chiral separations. Electrophoresis 18 853-872 Gribitz G (1990) Separation of drug enantiomers by HPLC using chiral stationtuy phases -a selective review. Chromatographia 30 555-564... 

Alkaloids, for example, quinine, are widely applied as catalytic reagents in chiral organic syntheses and also in the production of HPLC stationary phases for chiral separations. Cinchona alkaloids also induce the enantioselection of chiral fluoro-compounds analyzed by FNMR spectroscopy [32]. (-)-Brucine and (-)-berberine mixed with silica gel were used for preparation of thin-layer plates to resolve racemic amino acids. Other optically active pure enantiomers of natural compounds are applied for impregnation of TLC plates (-l-)-tartaric acid, L-aspartic acid [33]. (—)-Menthol is used for preparation of diastereomeric derivatives in indirect enantiomeric separation of, for example, 2- and 3-hydroxy acids [34]. 

Scheme 2.5. Chiral Stationary Phases for HPLC Separation of Enantiomers...

Lubda, D., Cabrera, K., Nakanishi, K., Lindner, W. (2003). Monolithic silica columns with chemically bonded b-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals. Anal. Bioanal. Chem. 377, 892-901. 

Chiral stationary phases for the separation of enantiomers (optically active isomers) are becoming increasingly important. Among the first types to be synthesized were chiral amino acids ionically or covalently bound to amino-propyl silica and named Pirkle phases after their originator. The ionic form is susceptable to hydrolysis and can be used only in normal phase HPLC whereas the more stable covalent type can be used in reverse phase separations but is less stereoselective. Polymeric phases based on chiral peptides such as bovine serum albumin or a -acid glycoproteins bonded to... 

Chiralsil-val, 6 96-97 Chiral smectic C liquid crystals, 15 106-107 Chiral stationary phases, 6 79-82 Chiral supramolecular clusters, 24 61 Chiral synthons, 11 5 Chiral titanium complexes, 25 98—99 Chirobiotic phases, for chiral separations, 6 90-91... 

Table 7. Commercially Available Chiral Stationary Phases for Enantiomer Separation by LC...

The ability to design chiral ILs in which the cation and anion is of fixed chirality represents additional tuning features of ILs. Two approaches have incorporated ILs as new stationary phases for chiral GC. One method involves the use of chiral ILs as stationary phases in WCOT GC [37]. In the second approach, chiral selectors (e.g., cyclodextrins) were dissolved in an achiral IL and the mixture coated onto the wall of the capillary colunm [38]. Both approaches can separate a variety of different analytes, but the observed enantioselectivities and efficiencies do not rival those observed with commercially available chiral stationary phases (CSPs). 

Stationary HPLC Phases for Chiral Separations, IRIS Technologies, Lawrence, KS... 

Kip, J., Van Haperen, P., and Kraak, J.C., R-N-(pentafluorobenzoyl) phenylglycine as a chiral stationary phase for the separation of enantiomers by high-performance liquid chromatography, J. Chromatogr., 356, 423, 1986. 

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,... 

Chiral stationary phases for the separation of chiral and achiral compounds... 

In this mode of separation, active compounds that form ion pairs, metal complexes, inclusion complexes, or affinity complexes are added to the mobile phase to induce enantioselectivity to an achiral column. The addition of an active compound into the mobile phase contributes to a specific secondary chemical equilibrium with the target analyte. This affects the overall distribution of the analyte between the stationary and the mobile phases, affecting its retention and separation at the same time. The chiral mobile phase approach utilizes achiral stationary phases for the separation. Table 1 lists several common chiral additives and applications. 

As for the use of monomeric and polymeric chiral surfactants as pseudo-stationary phases for enantiomer separations in MEKC, a review article has been available. 

Only cellulose, cellulose triacetate, and silanized sihca gel impregnated with the copper(II) salt of derivatized L-hydroxyproline have been used as chiral stationary phases for the separation of enantiomeric solutes. 

Figure 22.5 one possibility of enantiomer resolution consists of the reaction of the compounds of interest with enantiomerically pure, chiral reagents to obtain diastereomers. With a proper choice of reagent the diastereomers can be separated on a reversed phase (or another nonchiral stationary phase). For the separation presented here the quantitative reaction with chloroformate was obtained within 30 min. 

Numerous racemates have been separated into their enantiomers since the first success in 1848 by Pasteur. The traditional method is to derivatize a racemate into a mixture of two diastereoisomers by means of so-called resolving agents, and then to separate the diastereoisomers by recrystallization or chromatography. Recent development of chiral stationary phases for chromatographic separation of the enantiomers made it possible to separate them even without derivatization. Another method of choice is to use enzymes for enantiomer separation. Examples in this chapter will illustrate the use of these methods. After enantiomer separation, the absolute configuration and the enantiomeric purity of the resulting enantiomers must be determined. 

See, e.g., R. DSppen, H. R. Karfunkel, and F. J. J. Leusen,/. Comput. Chem., 11,181 (1990). Computational Chemistry Applied to the Design of Chiral Stationary Phases for Enantiomeric Separation. R. J. Boudeau and S. M. N. Efange, Invest. Radiol., 27, 653 (1992). Computer-Aided Radiopharmaceutical Design. 

Okamato et al. [11] also examined the relative performance of cellulose and amylose derivatives for the separation of a group of racemic drugs. At the same time, they demonstrated generally the versatility of the cellulose based chiral stationary phases for the separation of a range of physiologically active compounds. Examples of such separations are shown in figure 11.13 (A-F). 

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