Method Development for Chiral Separation

Zhao, Y., Woo, G, Thomas, S., Semin, D., Sandra, P. Rapid method development for chiral separation in drug discovery using sample pooling and supercritical fluid chromatography-mass spectrometry. J. Chromatogr. A 2003, 1003, 157-166. 

Method development for chiral separation is a multidisciplinary task. It requires knowledge of stereochemistry, organic chemistry, and separation techniques. Separation of enantiomers is not linked to a certain technique (i.e., GC, HPLC, etc.) but rather to an understanding of the specific interactions between the enantiomeric analytes and a certain chiral stationary phase. Knowing these types of relationships will enable one to easily understand the formation of transient diastereomeric complexes between enantiomers and a chiral stationary phase during a chromatographic separation as well as their stereochemical relationship within the complex. Once such dependencies are established, development of a method for the separation of enantiomers becomes an easy process. Based on such a relationship, chiral stationary phases can be divided in five categories [161] ... 

Matthijs, N., Perrin, C., Maftouh, M., Massart, D.L., Vander Heyden, Y. Knowledge-based system for method development of chiral separations with capillary electrophoresis using highly sulphated cyclodextrins. J. Pharm. Biomed. An. 2002, 27, 515-529. 

Methods development for chiral analyses has been one of the most challenging separation problems for the analytical chemist in the pharmaceutical industry. Racemic drug substances have a variety of chemical structures and several chiral selectors are available for the analyst to choose in order to obtain the enan-tioselectivity needed for chiral resolution. To alleviate this problem, a fast capillary electrophoresis procedure for the enantiomeric separation of acidic and basic compounds using native and modified cyclodextrins has been described (200). The technique is called cyclodextrin array chiral analysis. A generalized optimi-... 

Matthijs N, Perrin C, Maftouh M (2004) Definition and system implementation of strategies for method development of chiral separations in normal- or reversed-phase liquid chromatography using polysaccharide-based stationary phases. J Chromatogr A104L119-133... 

CE is generally more suited to analytical separations than to preparative-scale separations. However, given the success of CE methods for chiral separations, it seems reasonable to explore the utility of preparative electrophoretic methods to chiral separations. Thus, the purpose of this work is to highlight some of the developments in the application of preparative electrophoresis to chiral separations. Both batch and continuous processes will be examined. 

Comparisons of LC and SFC have also been performed on naphthylethylcar-bamoylated-(3-cyclodextrin CSPs. These multimodal CSPs can be used in conjunction with normal phase, reversed phase, and polar organic eluents. Discrete sets of chiral compounds tend to be resolved in each of the three mobile phase modes in LC. As demonstrated by Williams et al., separations obtained in each of the different mobile phase modes in LC could be replicated with a simple CO,-methanol eluent in SFC [54]. Separation of tropicamide enantiomers on a Cyclobond I SN CSP with a modified CO, eluent is illustrated in Fig. 12-4. An aqueous-organic mobile phase was required for enantioresolution of the same compound on the Cyclobond I SN CSP in LC. In this case, SFC offered a means of simplifying method development for the derivatized cyclodextrin CSPs. Higher resolution was also achieved in SFC. 

The development of chiral separation methods for dipeptides is of relevance for purity controls, for checking racemization processes in peptide syntheses, and for the investigation of peptide and protein hydrolysates. Since their introduction as chiral... 

Roos, N., Ganzler, K., Szeman, J., Fanali, S. Systematic approach to cost- and time-effective method development with a starter kit for chiral separations by capillary electrophoresis. J. Chromatogr. A 1997, 782, 257-269. 

From a practical point of view, and related to method development for impurity testing, it can be recommended that a method be developed in such a way that the impurity elutes before the main component. It will be easier to obtain a baseline separation and one that reduces quantification problems that might occur when the impurity elutes in the tail of the main peak. Moreover, the peak obtained for the main compound might be rather broad and tailing in chiral chromatography (as will be shown further in this chapter) which favors even more the development of methods with the impurity eluting first. 

From the method development and robustness point of view, the temperature is a parameter that controls equilibria such as pK and enantiomer—chiral selector complexation, or induces structural changes in, e.g., proteins.For chiral separations, generally a lower temperature results in better enantioseparation, but even the opposite has been observed. Sometimes a raise in temperature does not so much affect the enantiomeric separation, but increases the resolution between an enantiomer and a matrix component. ... 

Another method for creating a chiral environment is lo add an optically pure chiral selector to a bulk liquid phase. Chiral additives have several advanlages over chiral stationary phases and continue lo be the predominant mode for chiral separations by tic and capillary electrophoresis (cc). First of all, the chiral selector added to a bulk liquid phase can be readily changed. The use of chiral additives allows chiral separations lo be done using less expensive, conventional stationary phases. A wider variety of chiral selectors are available [ be used as chiral additives than are available as chiral stationary phases, thus, providing the analyst with considerable flexibility. Finally, the use of chiral additives may provide valuable insight into (he chromatographic conditions and/or likelihood ol success with a potential chiral stationary-phase chiral selector. This is particularly important for the development of new chiral stationary phases because of the difficulty and cosl involved. 

The first chiral separation using pSFC was published by Caude and co-workers in 1985 [3]. pSFC resembles HPLC. Selectivity in a chromatographic system stems from different interactions of the components of a mixture with the mobile phase and the stationary phase. Characteristics and choice of the stationary phase are described in the method development section. In pSFC, the composition of the mobile phase, especially for chiral separations, is almost always more important than its density for controlling retention and selectivity. Chiral separations are often carried out at T < T-using liquid-modified carbon dioxide. However, a high linear velocity and a low pressure drop typically associated with supercritical fluids are retained with near-critical liquids. Adjusting pressure and temperature can control the density of the subcritical/supercritical mobile phase. Binary or ternary mobile phases are commonly used. Modifiers, such as alcohols, and additives, such as adds and bases, extend the polarity range available to the practitioner. 

The use of CDs for chiral separations has, to date, been the most common approach when using CE or MEKC, so it would be difficult to discuss and detail every aspect relating to their chemistry, effects on separation, and application in this held. The emphasis will, thus, be placed on a short description of the principle and mechanism of chiral separation, typical method development procedures, and an outline of the influential experimental parameters using CE and MEKC. References to recent published review and research literature will enable the reader to explore this vast area further. It is also beyond the scope of this short introductory review to actually outline the actual CE or MEKC separation principles in detail, but an in-depth discussion can be found in this encyclopedia and references to recent textbooks and can be readily found elsewhere. It must, of course, be pointed out that CDs are not the only useful chiral selectors that can be employed using electrophoretic techniques. The use of chiral surfactants (bile salts), crown ethers, metal-chelation agents, carbohydrates, proteins, and glycopeptides have all been used effectively [2]. 

In this section, chiral separation by MEKC with chiral micelles is mainly treated. The development of novel chiral surfactants adaptable to pseudo-stationary phases in MEKC for enantiomer separation is continuously progressing. It seems somewhat difficult for a researcher to find an appropriate mode of CE when one wants to achieve a specific enantioseparation. However, nowadays, various method development kits for chiral separation have been commercially available and some literature on the topic is also available, so that helpful information may be obtained without difficulty. 

More than half of small druglike molecules are chiral. The Food and Drug Administration (FDA) requires testing of pure enantiomers. Such testing is most useful early on in drug development. SFC is dramatically superior to HPLC for chiral separations. SFC offers dramatically faster method development and should be the technique of choice for any molecules soluble in organic solvents (i.e., most druglike molecules). Further, unlike capillary electrophoresis, SFC is fully scalable. A method developed at the analytical scale should work equally well at the semiprep level. 

HPLC for chiral separations. On the analytical scale, the higher-speed chromatography and faster reequilibration makes method development dramatically faster. 

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