Chromatography chiral HPLC

Several methods to resolve racemic mixtures of a- amino acids have been worked out, including separation of diastereomeric salts by crystallization or amides by chromatography. Chiral HPLC on phases carrying d- and L-pro-line-copper complexes has been scaled up to 20 g quantities of amino acid race-mates. Resolution with immobilized amidases, which deacetylate only L-amino acid acetamides, and subsequent precipitation of the D-amino acid acetamides work on a 500-kg scale. All kinds of labels ( C, D) can thus be introduced... 

Thin-Layer Chromatography. Chiral stationary phases have been used less extensively in tic as in high performance Hquid chromatography (hplc). This may, in large part, be due to lack of avakabiHty. The cost of many chiral selectors, as well as the accessibiHty and success of chiral additives, may have inhibited widespread commerciali2ation. Usually, nondestmctive visuali2ation of the sample spots in tic is accompHshed using iodine vapor, uv or fluorescence. However, the presence of the chiral selector in the stationary phase can mask the analyte and interfere with detection (43). 

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. 

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...

All aldehydes used in the experiment were freshly distilled or washed with aqueous NaHC03 solution to minimize the amount of free acid. Chiral HPLC was performed using a chiral OJ-H column (0.46 cm x 25 cm, Daicel industries) with a water 717 auto sampler and a UV-vis detector (254 nm). The eluting solvent used was different ratios of hexane and 2-propanol. Chiral gas chromatography analysis was performed in a Shimadzu auto sampler with cyclodextrins columns as chiral stationary phase (fused-silica capillary column, 30 m X 0.25 mm x 0.25 gm thickness, /3-Dex-120 and /3-Dex-325 from Supelco, USA) using He as a carrier gas (detector temperature 230 °C and injection temperature 220 °C). 

For high-performance liquid chromatography (HPLC) analysis samples (0.5 mL) were clarified by centrifugation at 14000 Gav for 5 min and the supernatant was decanted, filtered through a 0.2 yim in-line syringe filter and analysed directly by chiral HPLC (see below). 

Diastereomers of nitro derivative of 45 (R = NO2) were separated on a silica gel column chromatography, and enantiomers of cis-3H,7H derivative of 45 (R = H) were separated by chiral HPLC (06USA2006/0004028). 

Specialized Stationary Phases for Liquid Chromatography Chiral Stationary Phases for Liquid Chromatography Detectors for Liquid Chromatography Ultraviolet Detection of Chromophoric Groups Derivatizing Reagents for HPLC... 

The enantiomeric purity of the sulfamidates 55a and 55b was established using Whelk 0-2 chiral high-performance liquid chromatography (HPLC) <2005TA1583>, and in each case chiral HPLC confirmed an enantiomeric excess of at least 97%. These two sulfamidate oils proved to be very stable when stored for up to a year at — 20 °C. 

The analysis of chiral compounds to determine their optical purity is still not a trivial task. The analysis method has to differentiate between the two antipodes and, thus, has to involve a chiral agent. However, the development of chiral chromatography, especially HPLC (high-performance liquid chromatography), has done a significant amount to relieve this problem. The purpose of this book is to discuss large-scale synthetic reactions, but the reader is reminded that the development of chiral analytic methods may not have been a trivial undertaking in many examples. 

In a typical reaction, a solution of the antibody in phosphate buffered saline (PBS) is added to a solution of the racemic substrate (ca. 50-100 mM) in either toluene or chlorobenzene. The mixture is shaken, while the substrate ee is monitored by chiral HPLC. When the desired ee is reached, the reaction mixture is cooled (—20°C), allowing easy separation of the organic layer from the frozen aqueous antibody solution. The aldol product is purified by column chromatography, and the antibody solution is thawed for reuse. 

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