Enantiomer separation by HPLC

Enantiomer separation by HPLC chromatography using a column containing a chiral stationary phase. 

Yoshio Okamoto was born in Osaka, Japan, in 1941. He received his bachelor (1964), master (1966), and doctorate (1969) degrees from Osaka University, Faculty of Science. He joined Osaka University, Faculty of Engineering Science, as an assistant in 1969, and spent two years (1970— 1972) at the University of Michigan as a postdoctoral fellow with Professor C. G. Overberger. In 1983, he was promoted to Associate Professor, and in 1990 moved to Nagoya University as a professor. His research interest includes stereocontrol in polymerization, asymmetric polymerization, optically active polymers, and enantiomer separation by HPLC. He received the Award of the Society of Polymer Science, Japan, in 1982, the Chemical Society of Japan Award for Technical Development in 1991, the Award of The Chemical Society of Japan (1999), and the Chirality Medal (2001), among others. 

In the HPLC of drugs and poisons, derivatisation reactions, although sometimes used to stabilise an analyte, are seldom needed to achieve satisfactory chromatography except when performed to permit the separation of enantiomers for example Srinivas and Igwemezie list a number of derivatisation reagents that have been used to facilitate enantiomer separations by HPLC-ECD. However, derivative formation is chiefly used to enhance the selectivity and sensitivity of detection. Ruorescent derivatives are the most popular, but derivatisation has also been used to enhance UV or EC detection characteristics. " ... 

The stereochemistry of each enantiomer separated by the chiral HPLC has been studied after methanolysis of the epoxy ring. Examining the H NMR data of esters of the produced methoxyalcohols with (S)- and (R)-a-methoxy-a-(tri-fluoromethyl) phenylacetic acid by a modified Mosher s method [181], it has been indicated that the earlier eluting parent epoxides are (3S,4R)-, (6S,7R)-, and (9R,10S)-isomers (Table 7) [75, 76, 179]. The above three chiral HPLC columns show different resolution abilities but a different elution order is not observed. The resolution profile by the reversed-phase OJ-R column has been generalized with molecular shapes of the epoxy compounds considering the... 

The stereochemistry of each enantiomer separated by the chiral HPLC has been studied after methanolysis of the epoxy ring. Examining the NMR data... 

Han SM, Armstrong DW, HPLC separation of enantiomers and other isomers with cyclodextrin bonded phases rule for chiral recognition, in Chiral Separations by HPLC (Krstulovic AM, Ed.), Ellis Horwood, Chichester, p. 208 (1989). 

M. Pawlowska, Enantiomer separations by normal HPLC systems with permethylated /3-cyclodextrin dynamically coated on silica solid supports, J. Liquid Chromatogr., 14 2213 (1991). 

Lynam and Nicolas have evaluated chiral separations by HPLC versus SFC [14] The enantiomers studied were pharmaceutical synthetic precursors. Repeated injections of trans-stilbene oxide and carbobenzyloxy phe-nylalaninol were made and the chromatographic parameters Rs, N, and a were calculated daily. SFC gave superior enentiomeric resolution of peaks and there was a faster solvent equilibration. The columns were quite stable in both SFC and HPLC systems. 

N. R. Srinivas and L. N. Lgwemezie, Chiral separation by HPLC.l. Review of indirect separation of enantiomers as diastero-meric derivatives using ultraviolet, fluorescence and electrochemical detection, Biomed. Chromatogr., 6 163 (1992). 

Five dihydropyridine enantiomers (including nimodipine) were separated by HPLC using cyclodextrin as the chiral selector [25]. Columns containing (5-cyclodextrin covalently bonded to 5 pm silica (25 cm x 4 mm i.d.), or of (R)- or (S)-naphthyl ethyl carbomyl (1-cyclodextrin covalently bonded to 5 pm silica (25 cm x 4.6 mm i.d.), were used. The mobile phases (flow rate of 0.8 mL/min) were mixtures of ethanol or acetonitrile, with 0.1% triethylamine, adjusted to pH 5 with acetic acid. Detection was at 239 nm. 

Figure 4. Separation by HPLC of the D,L amino acid enantiomers using a chiral reagent. The D,L order Is obtained when L-Prollne Is added to the carrier In the presence of cupric sulfate. The enantiomer resolution Is reversed using D-Prollne. No optical resolution Is obtained when the carrier chlralty Is removed, l.e. with the use of the racemic D,L eluant (17).

The third approach to Isomer separation by HPLC Is based on the use of chiral stationary phases. The phase may be Inherently optically active (e.g., powdered d-quartz or starch) or may be a non-optlcally active material coated with or reacted with an optically active moiety. As early as 1938, enantiomers of a camphor compound were separated on a lactose column (54), and twenty years later, alumina coated with ortartarlc acid was used to separate Isomers of mandellc acid and phenylglyclne-methyl ester... 

Four novel isomeric cyclic thiosulfinates, the zeylanoxides, have been isolated from the tropical weed Sphenoclea zeylanica. The structures and absolute stereochemistry of these compounds, which are plant growth inhibitors, have been determined by synthesis from the enantiomers of glucose. Use of L-glucose as outlined in Scheme 35 gave a mixture, separable by HPLC, of zeylanoxide A (187, a-sulfoxide), epi-zeylanoxide A (187, p-sulfoxide), zeylanoxide B (188, a-sulfoxide), and epi-zeylanoxide B (188, p-sulfoxide), with optical rotations in good agreement with those of the natural products. ... 

In the area of cyclodextrin ethers the -compound has been converted into a set of five tris-Tbdms ethers, all substituted at their various 6-positions, which were separated by hplc and characterized by n.m.r. spectroscopy. Related work applied to y-cyclodextrin gave the various 6,6 -disubstituted ethers. 5-Bromo-l-pentene was used to produce the 2-0-mono-4-pentenyl ether of P-cyclodextrin which was then permethylated and the product was chemically bonded to silica gel to form an efficient hplc stationary phrase for the separation of enantiomers. Peroctyl a-cyclodextrin has been studied as a chiral receptor for the ephedrinium ion. Various octyl ethers of a-, P- and y-cyclodextrin ranging in their substitution from the diethers to completely alkylated products were characterized by electrospray mass spectrometry and n.m.r. methods applied to methylated derivatives. The 2,6-didodecyl derivative of p-cyclodextrin has been used as a potentiometric sensor. In the field of aromatic ethers, naphthyl carboxylate substituents have been bonded at the 6-positions and the products were able to transfer excitation energy to complexed merocyanine held in the cavities of those molecules. These phototransfer processes were extremely efficient.P-Substituted cyclodextrin derivatives with p-allyloxybenzoyl or various benzyl substituents at 0-2 or 0-3 were incorporated by hydrosilylation to give hydromethylpolysiloxane polymers used as chiral phases for chromatographic resolution of enantiomers. Cyclodextrins with complex benzyl-like eth are illustrated in 22 and 23. The latter were prepared as artificial redox enzymes. 

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