Separations chiral

CE is playing a major role in the separation of chiral compounds, a field that is gaining increasing attention in pharmaceutical sciences as well as in forensic toxicology (Lurie, 1994 Novotny et al., 1994 Ward, 1994). The chirally active selectors used in CE include optically active complexes such as Cu(II)-l-histidine, Cu(II)-aspartame, cyclodextrins, modified CDs, bile salts, crown ethers, and proteins (bovine serum albumin, aracid glycoprotein, etc.). 

Different mechanisms have been devised to achieve chiral resolution by electromigration, but often we have to deal with mixed-mode separations rather than pure processes. In any case, chiral resolution results from stereospecific interactions of a chiral selector, with the enantiomers of the compound giving rise to a difference in migration velocity between the two entities. Chirally selective ligands, such as Cu(II)-L-histidine and Cu(II)-aspartame, have been used for derivatized amino acid mixtures. 

Packed capillary columns with chirally selective stationary phases (e.g., flr acid glycoprotein), as well as wall-immobilized, CD-based stationary phases, have been successfully used in CE chromatographic separations. Also, macro-cylic crown ethers, forming sterically selective complexes with the guest molecule, have been used for the resolution of optically active amines. 

MEKC with chirally selective micelles has proved very powerful for enantiomer separation. Chiral surfactants (e.g., bile acid salts) forming chirally active micelles have widely been used. Mixed micellar solutions containing SDS and chiral surfactants or derivatized CDs are becoming more popular. Mixedmode, chiral, and nonchiral, interactions increase the resolving power of these systems. 

This chapter discussed the concepts and principles of high performance capillary electrophoresis. A brief history of CE was followed by an analysis of the components of an CE system. Two types of separation were presented, electrophoretic migration and electroosmosis. Several types of CE were dis- 

Enantiomers need an isotropic medium to show different properties. In separation methods, there are three ways to make enantiomers and chiral selectors interact (1) a chiral derivatization agent can be used to react with the enantiomeric pair turning it into a diastereoisomeric pair that can be separated by classical means (2) a chiral selector can be added to the mobile phase so that labile diastereoisomers can be formed with the enantiomeric pair during the separation process. Again a classical column will be able to separate the formed diastereoisomers (3) a chiral selector can be attached to the stationary phase. Labile diastereoisomers can be formed with the chiral stationary phase (CSP) producing different progression of the two enantiomers within the chiral column. 

The importance of this mode of separation lies in the shear scale of the market for optically pure molecules. The sales of single enantiomer chiral drugs is currently U 180 billion per annum and —65% of active pharmaceutical ingredients (APIs) currently in development have at least one chiral centre. Apart 

Chiral purity is generally introduced in one of three ways  

In order to fully explain chiral chromatography it is necessary to have an understanding of chirality itself. It is worth describing how chiral separations work in order to help to dispel some of the myths surrounding the complexity of this mode of separation. A few brief definitions and formulae required to understand the summary are listed below. 

A chiral object cannot be superimposed on its mirror image whilst an achiral object can be superimposed on its mirror image. The two forms of a chiral molecule are known as enantiomers. A single chiral isomer is said to be enantiomerically or optically pure. Whereas an equimolar mixture of enantiomers is said to be racemic. 

The central point of chirality in a molecule is known as the stereo-centre and diastereoisomers occur when there is more than one stereo-centre in a molecule. Stereo-centres are given the absolute configurations of Rectus (R) and Sinister (S). Diastereoisomers that differ in absolute configuration at the stereo-centres are called epimers. Diastereoisomers differ in conformation so it is actually possible to purify these mixtures by normal phase and reversed phase HPLC. However, better separation factors are often obtained using chiral stationary phases. 

When a racemic mixture is introduced into a chromatographic system containing cyclodextrin, the two isomers will have a different affinity for the chiral phase, affording what is called the enantioselectivity of the cyclodextrin phase. Finally, one other approach to the separation of enantiomers that has been applied in forensic work is enantioselective derivation. Rather than using an optically active stationary phase, this technique achieves separation by derivatizing the chiral compounds with chiral agents resulting in different selectivities for chiral molecules. 

This chapter has presented a whirlwind tour of the instruments employed in forensic chemistry. These instruments are usually not the newest, nor do they necessarily reflect the cutting edge of analytical instrumentation—and there is good reason for this. First, analytical techniques must prove their worth and exhibit a good cost-benefit ratio as applied to forensic casework, a process that takes years of testing and research. Second, instruments and methods must be rigorously tested and validated to meet legal requirements. Third, cost and intercomparability of those results are essential. 

It appears (Barron 2007) that Kelvin was the first to coin the word chirahty to represent the concept of right- or left-handedness in science I call any geometrical figure or group of points chiral, and say that it has chirality if its image in a plane mirror, ideally reahzed, cannot 

One approach to chiral separations of racemic mixtures is to devise a chemical reaction with an optically active reagent to produce diastereomeric derivatives that have different physical properties depending on the original enantiomers these can then be separated by conventional (achiral) chromatographic or SPE approaches, such as a recent demonstration (Xia 2006) of the utility of porous graphitic carbon for this purpose. However, this approach is not always possible and, even when it is, complex derivatization procedures are not desirable in trace analytical procedures and are avoided if possible. 

Development of chiral stationary phases for HPLC separations of enantiomeric mixtures is a highly active field. A useful review (Dappen 1986) covers earlier work, and more recent overviews (Beesley 2004 Bojarski 2005) of such developments are supplemented by a review of chiral LC/MS quantitation in the pharmaceutical industry (Chen 2005). Chiral stationary phases have specific requirements concerning mobile phases and this raises nontrivial questions concerning the ionization technique (Chapter 5) that is best suited to interfacing with a mass spectrometer in any particular case. 

Pirkle-type columns are performed in normal phase mode, and this in turn determines the type of LC/MS interface that is most appropriate (Chen 2005). 

CSPs based on proteins are not as widely used as the polysaccharide types although they can provide enantiomeric separations of a wide range of analytes as a result of their multiple binding interactions and/or multiple 

Chiral separation of drng molecules and of their precursors, in the case of synthesis of enantiomerically pure drugs, is one of the important application areas of HPLC in pharmaceutical analysis. Besides HPLC, capillary electrophoresis (CE) is another technique of choice for chiral separations. Chapter 18 provides an overview of the different modes (e.g., direct and indirect ones) of obtaining a chiral separation in HPLC and CE. The direct approaches, i.e., those where the compound of interest is not derivatized prior to separation, are discussed in more detail since they are cnrrently the most frequently used techniques. These approaches require the use of the so-called chiral selectors to enable enantioselective recognition and enantiomeric separation. Many different molecnles have been nsed as chiral selectors, both in HPLC and CE. They can be classified into three different groups, based on their 

Hyphenated analytical techniques such as LC-MS, which combines liquid chromatography and mass spectrometry, are well-developed laboratory tools that are widely used in the pharmaceutical industry. Eor some compounds, mass spectrometry alone is insufficient for complete structural elucidation of unknown compounds nuclear magnetic resonance spectroscopy (NMR) can help elucidate the structure of these compounds (see Chapter 20). Traditionally, NMR experiments are performed on more or less pure samples, in which the signals of a single component dominate. Therefore, the structural analysis of individual components of complex mixtures is normally time-consuming and less cost-effective. The 

Chromatographic techniques represent one of the most significant sources of analytical data found in today s pharmaceutical laboratories. All of the chromatographic techniques produce data that must be acquired, interpreted, quantified, compared, reported, and finally archived (see Chapter 21). Whether the analysis is qualitative or quantitative in nature, the data must somehow be interpreted and reported so that meaningful decisions can be made. It may be as simple as a qualitative decision that indicates whether a reaction has reached completion successfully, or it may be a series of quantitative analyses that help determine if a batch or lot of product meets its specifications and may now be released. This chapter also examines the evolution and perhaps the revolution that has taken place within the chromatography data system (CDS) marketplace. 

The resolution of optical isomers and subsequent fractionation of the biologically active isomer from its enantiomer, which may be inactive, inhibit biological activity or indeed in some cases even be toxic, is essential for a pharmaceutical grade preparation. A number of polymeric liquid chromatography adsorbents have been used for 

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Chiral liquid crystals Chiral recognition Chiral separation Chiral separations Chiral shift reagents... 

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