Chiral HPLC columns

Giovanni Boocaletti is gratefully acknowledged for the large number of experiments that paved the way to enantioselective Lewis-acid catalysis in water. Furthermore, we kindly thank the Syncom company for the use of the chiral HPLC column. 

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. 

Chiral Hplc Columns. There are about 40 commercially available chiral columns which are suitable for analytical and preparative purposes (57). In spite of the large number of commercially available chiral stationary phases, it is difficult and time-consuming to obtain good chiral separation. In order to try a specific resolution meaninghilly, a battery of chiral hplc columns is necessary and this is quite expensive. 

Resolution of Optical Isomers), Gakkai Shuppan Senta, Tokyo, Japan, 1989, Chapt. 11, pp. 132—143 Chiral HPLC columns are available from Regis, Spelco, and AST in the United States Merck, Nagel, and Serva in Germany LKB in Sweden and Daisem, Sumitomo, and Toso in Japan. 

The structure of a natural product is shown without any specification of stereochem-istiy. It is a pure substance which gives no indication of being a mixture of stereoisomers and has zero optical rotation. It is not a racemic mixture because it does not yield separate peaks on a chiral HPLC column. When the material is completely hydrolyzed, it gives a racemic sample of the product shown. Deduce the complete stereochemical structure of the natural product fiom this information. 

A chiral GC column is able to separate enantiomers of epoxy pheromones in the Type II class, but the applications are very limited as follows a custom-made column packed with a p-cyclodextrin derivative as a liquid phase for the stereochemical identification of natural 3,4- and 6,7-epoxydienes [73, 74] and a commercialized column of an a-cyclodextrin type (Chiraldex A-PH) for the 3,4-epoxydiene [71] (See Table 3). The resolution abilities of chiral HPLC columns have been examined in detail, as shown in Table 7 and Fig. 14 [75,76, 179]. The Chiralpak AD column operated under a normal-phase condition separates well two enantiomers of 9,10-epoxydienes, 6,7-epoxymonoenes and 9,10-epoxymonoenes. Another normal-phase column, the Chiralpak AS column, is suitable for the resolution of the 3,4-epoxydienes. The Chiralcel OJ-R column operated under a reversed-phase condition sufficiently accomplishes enantiomeric separation of the 6,7-epoxydienes and 6,7-epoxymonoenes. 

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... 

Fig. 14A-C Chromatography of the racemic monoepoxy derivatives (I—III) of Z3,Z6,Z9-18 on chiral HPLC columns A Chiralpak AD B Chiralpak AS C Chiralcel OJ-R. The solvent system for the former two normal-phase columns is 0.1% 2-propanol in n-hexane (0.45 ml/min), and that of the third column is 15% water in MeOH (0.45 ml/min). Homo-conjugated dienes, epo3,Z6,Z9-18 H (I) and Z3,Z6,epo9-18 H (III), were detected by UV (215 nm), and Z3,epo6,Z9-18 H (II) was detected by RID. The earlier eluting isomers have a 3S,4R, 6S,7R, or 9R,10S configuration...

Table 7 Enantiomeric separation of monoepoxides derived from Z3,Z6,Z9-trienes and Z6,Z9-dienes with aC17-C23 straight chain on chiral HPLC columns [75,76,179]... 

Much has been written about the usefulness of MIPs for chiral HPLC or chiral CEC separations. As long as one has an expert in chiral separation and can afford the expensive chiral HPLC columns which are commercially available, there seems to be little incentive to use MIPs for this purpose [25]. Chiral separations are today often required in the pharmaceutical industry, where the expertise and the financial resources are available to solve most problems. In the future, however, there may be more interest in chiral separation by other scientists, e.g., biochemists, and this could contribute to a wider use of MIPs in analytical chiral separations. 

Pirkle-lJ Chiral HPLC Column, Regis Technologies, Morton Grove, IL (1995). 

The synthetic ( )-calanolide A was resolved into its enantiomers, (+)-calanolide A (1) and ( )-calanolide A, by using a semipreparative chiral HPLC column packed with amylose carbamate eluting with hexane/ethnol (95 5). The ultraviolet detection was set at a wavelength of 254 nm. (+)-calanolide A and its enantiomer (—)-calanohde A were collected, and their chemical structures were identified based on their optical rotations and spectroscopic data, as compared with the corresponding natural and synthesis compounds. 

Application Guide for Chiral Column Selection Crownpak-Chiralcel-Chiralpak. Chiral HPLC Column for Optical Resolution. 2nd Ed.. Daicel Chemical Industries. Ltd.. Tokyo. 

There has been some recent interest in the separation of enantiomers of 1,3-dioxolane systems by chromatography on chiral stationary phases and examples include separation of the dioxolan-4-ones (66) on a chiral HPLC column <89LA103> and of the dioxolane esters (67) by chiral GC <93MI... 

In deacylation, as the enzyme cleaved the phenylacyl group, phenylacetic acid was formed, which lowered the pH of the reaction medium. Base was added to maintain the starting pH. (Note Use of ammonium hydroxide led to the formation of desilylated byproducts desilylation was eliminated when bicarbonates were used.) This approach was not required in the acylation reaction. At pH above 7.5 the (R)-and (S)-amines are practically insoluble in water. Organic solvents were used to extract the free amines from the aqueous reaction medium at pH 8.0. p-Fluoro-benzoyl, 1-naphthoyl, and phenylacetyl derivatives of the racemic amine were prepared and their behavior on the chiral HPLC column was studied. Based on ease of preparation and HPLC analysis, the 1-naphthoyl derivatives (Fig. 7) were preferred. Reversed phase HPLC analysis on a Vydac-C18 analytical column used a gradient of acetonitrile (0.1% triethylamine) in water (0.05% phosphoric acid) to quantify the total amide in the reaction mixture. Chiral HPLC analysis on (S,S) Whelk-O Chiral column used isopropanol hexane (30 70) as a solvent system to separate and quantify the (R)- and (S)-enantiomers. 

The absolute configuration of the monosaccharides can be determined by analyzing the sugars (obtained from hydrolysis experiments) on a chiral HPLC column (86, 87). The absolute stereochemistry of the monosaccharides may also be derived by chiral GC analysis (88, 89). Moreover, one can also compare the observed value of the molecular rotation with the value calculated on the basis of Klyne s rule (90). 

The use of a simply prepared high efficiency chiral HPLC column capable of separating the enantiomers of thousands of compounds is described, documentation being provided by more than 120 specific examples covering 19 classes of compounds. Chiral recognition models are presented to account for elution orders of the enantiomers. Practical applications of the chiral column, including preparative separations, are described. 

Numerous chiral HPLC columns have been used for determination of chiral purity of 1,1 -bi-2-naphthol.8b 9b The submitters used Diacel Chiralpak OP(+) column (4.6 mm x 250 mm) at room temperature for their chiral assay. Typical retention times of 1,1 -bi-2-naphthol are 14 min (R-enantiomer) and 20 min (S-enantiomer) using methanol as an eluting solvent at 0.5 mL/min. The submitters detection limit of minor... 

The structure of nonactin is shown below without any specification of stereochemistry. It is isolated as a pure substance from natural sources and gives no indication of being a mixture of stereoisomers. Although it is not optically active, it does not appear to be a racemic mixture, because it does not yield separate peaks on chiral HPLC columns. When completely hydrolyzed, it yields racemic nonactic acid. Deduce the stereochemical structure of nonactin from this information. 

Due to its availability, synthetic racemic ( )-ABA has been used in most metabolic studies. It is only in recent years that chiral HPLC columns have become available. With these, one can rapidly resolve the two enantiomers of ABA (or their methyl esters) [128-131], of PA and the l 4 -diols of ABA [132], and of 7 -OH-ABA [133]. These methods can also be used to determine the optical purity of ABA and its metabolites [134,135]. 

These gas-phase observations paved the way for the first synthetic route towards azafullerenes, as described in the next section. The cage-opened structures 22 and 26 are asymmetric. Ketolactam 22 was later resolved on a chiral HPLC column, and the circular dichroism of the enantiomers was determined [64]. 

More recently, 13,14-dimethyldibenzo[6j][4,7]phenanthroline (55) was synthesized by the copper-catalyzed condensation reactions between 1,4-diiodobenzene (52) and 2-aminoacetophenone (53) to give 54 followed by sulfuric acid-promoted cyclization reactions (Scheme 10) [56]. Alternatively, 55 was also prepared by a one-pot procedure involving the ZnCl2-catalyzed reactions between AA -diphenyl-p-phenylenediamine (56) and acetic acid (Scheme 11) [57, 58]. The enantiomers of 55 were separated by a chiral HPLC column and were found to undergo rapid racemization at room temperature. A slower rate of racemization was observed for the 13,14-diethyl derivative 57. The X-ray structure of 57 confirmed the twisted aromatic framework. 

Cyclodextrins (and their derivatives) have been used widely in analytical chemistry, in particular, in separation technology. They have been incorporated into chromatographic applications, such as thin layer chromatography, gas chromatography, capillary electrophoresis and high performance liquid chromatography (HPLC), for the separation of similar chemical substances and even enantiomers (cyclodextrins are chiral). Conventional chiral HPLC columns are costly. A normal column containing an immobilised chiral cyclodextrin, such that one enantiomer forms a more stable complex over the other, can drastically reduce the cost. 

Figure 8 Enzymatic resolution of ( ) 10 (BCH189) at 90 g scale using free cytidine deaminase. Ninety grams of racemic 10 was incubated at 32 C with ISOO units of cytidine deaminase in a 15 liter volume in a 20 liter fermentor. The pH was adjusted to 7.0 with concentrated ammonia solu tion and maintained constant by the addition of 20% (v/v) acetic acid. At intervals, samples were analyzed by chiral HPLC [column. Cyclobond I acetyl (250 xO.46 mm) mobile phase, rriethylam-monium acetate (0.2% v/v), pH 5.5 flow rate, 1 inl/inin detection wavelength, 270 run]. (B) Epivit, g/liter (9) ( t-) 10, g/liter ( ) ee (Epivii) (T) conversion (% deamination of racemic 10). (Reproduced from Ref. 57 with the permisionofBurterwcMTh-Heineniann.)...

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