Achiral HPLC

Chromatographic Method. Progress in the development of chromatographic techniques (55), especially, in high performance Hquid chromatography, or hplc, is remarkable (56). Today, chiral separations are mainly carried out by three hplc methods chiral hplc columns, achiral hplc columns together with chiral mobile phases, and derivatization with optical reagents and separation on achiral columns. All three methods are usehil but none provides universal appHcation. 

Online CD detectors are now commercially available for use with HPLC that are inherently more sensitive than corresponding OR detectors and not affected by solvent changes to the same extent and are thus more gradient compatible [121]. Provided Ae and the concentration of an analyte are known with good precision/accuracy, the measurement of CD will allow the determination of enantiomeric purity. In addition, with CD-based detection systems, both chiroptical and ordinary absorbance can be determined simultaneously allowing the measurement of the g-factor (or dissymmetry factor), which is defined as the ratio of the CD to the absorbance (AA/A) [122]. The g-factor is concentration independent and its measurement allows a more reliable determination of enantiomeric purity (without using a CSP) with reference to standards of known enantiomeric composition irrespective of their concentration [123]. A small number of recent literature examples have suggested the potential use of achiral HPLC with online CD detection for the determination of extreme enantiomeric ratios [121, 124-126] however, chiral separation techniques currently provide a more reliable measurement of enantiomeric purity. 

The sample should be as pure as possible, but standards have been used where the major constituent is approximately 85% of the mixture, as long as the remainder is made up of known compounds. Suggestions to use mixtures as reference standards have been made to reduce the number of assays run and the number of samples handled. This is clearly the case for a racemic mixture in an achiral HPLC assay, where both enantiomers have the same retention and detector response. It can also be beneficial to use known mixtures of diastereomers as a standard, provided there is sound reasoning that they give equivalent response to the method of detection. 

Enantiomeric resolution can be obtained by direct and indirect methods. The derivatization of a mixture of enantiomers with an optical active agent (149,150) into diastereomers is the indirect mode. The diastereomers are separated using achiral HPLC or GC. The direct mode depends on the formation of labile diastereomers (formed by hydrogen bonding, dipole-dipole, jt-jt and/or hydrophobic interactions) between the enantiomers and chiral environment, a chiral stationary phase, with which they interact. HPLC, using chiral stationary phases and derivatization reactions (151-156), has dominated this field, but the increased selectivity and versatility of CE, as well as the ease of incorporating various chiral selectors, make it a viable alternative. The number of chiral selector... 

The diastereomeric compounds RS and SS obtained by this way may be resolved using achiral HPLC columns, which are less expensive. In addition, the chiral derivatizing reagents available in both configurations allow us to revert the migration order of enantiomers. These are advantages of indirect enantioseparation techniques. 

A. P. Bereford, Advantages of achiral HPLC as a preparative step to chiral analysis in biological samples and its use in toxicokinetic studies, Xenobiotica, 22(No7)( 1992)789. 

FIG. 4 Self-disproportionation of Michael adduct enantiomers during the purification on achiral HPLC column. 

Besides the usual reasons for mistakes that are familiar from achiral HPLC [24], there are other sources of errors in enantioselective H P LC, which, in many cases, can be excluded by taking simple precautions. In view of the price of HPLC columns, some basic care can result in significant cost savings. 

Williams, R. C., Edwards, J. R, Joshi, A. S. and Aubry, A.-F. Chiral analysis of drug substance in clinical plasma extracts using achiral HPLC with circular dichroism detection. /. Pharm. Biomed. Anal 25 501, 2001. 

Derivatization with Optically Active Reagents and Separation on Achiral Columns. This method has been reviewed (65) a great number of homochiral derivatizing agents (HD A) are described together with many appHcations. An important group is the chloroformate HD As. The reaction of chloroformate HD As with racemic, amino-containing compounds yields carbamates, which are easily separated on conventional hplc columns, eg (66),... 

D acquarica, I., Gasparrini, F., Giannoli, B., Badaloni, E., Galletti, B., Giorgi, F., Tinti, M.O., Vigevani, A. (2004). Enantio- and chemo-selective HPLC separations hy chiral-achiral tandem-columns approach the combination of CHIROBIOTIC TAG and SCX for the analysis of propionyl carnitine and related impurities. J. Chromatogr. A 1061, 167-173. 

He, J., Shibukawa, A., Nakagawa, T., Wada, H., Fujima, H., Imai, E., Go-oh, Y. (1993). Direct injection analysis of atenolol enantiomers in plasma using an achiral/chiral coupled column HPLC system. Chem. Pharm. Bull. 41, 544—548. 

Guo, Z., Wang, H., and Zhang, Y., Chiral separation of ketoprofen on an achiral C8 column by HPLC using norvancomycin as chiral mobile phase additives, J. Pharm. Biomed. Anal, 41, 310, 2006. 

A second example of the use of ionic chiral auxiliaries for asymmetric synthesis is found in the work of Chong et al. on the cis.trans photoisomerization of certain cyclopropane derivatives [33]. Based on the report by Zimmerman and Flechtner [34] that achiral tmns,trans-2,3-diphenyl-l-benzoylcyclopropane (35a, Scheme 7) undergoes very efficient (0=0.94) photoisomerization in solution to afford the racemic cis,trans isomer 36a, the correspondingp-carboxylic acid 35b was synthesized and treated with a variety of optically pure amines to give salts of general structure 35c (CA=chiral auxiliary). Irradiation of crystals of these salts followed by diazomethane workup yielded methyl ester 36d, which was analyzed by chiral HPLC for enantiomeric excess. The results are summarized in Table 3. 

Gagne and coworkers utilized this combination to discover enantioselec-tive receptors for (-)-adenosine [12]. A racemic dipeptide hydrazone [( )-pro-aib] generated a stereochemically diverse DCL of n-mer. The dimers were composed of two chiral (DD/LL) and one achiral isomer (DL), the four trimers (DDD, LLL, DDL, and LLD), the tetramers of four chiral and two achiral isomers, etc. Two techniques were used to measure the enan-tio-imbalance that was caused by the enantioselective binding of the chiral analyte to the enantiomeric receptors (Fig. 5.11). Since the unperturbed library is optically inactive, the optical enrichment of each library component could be measured by a combined HPLC optical rotation detection scheme (laser polarimeter, LP). LP detection differentiated unselective binding (amplification but not optical enrichment) from enantioselective recognition of the analyte (amplification and optical enrichment). In this manner the LL dimer (SS) of the dipeptide was amplified and identified as the enantioselective match for (-)-adenosine. 

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