Analytical separation, amine enantiomers

The brush-type of CSP was introduced by Pirkle who was one of the pioneers of modern enantioselective liquid chromatography [55]. The most frequently used 7i-acceptor phases are derived from the amino acids phenylglycine (DNBPG) (Fig. 6.8) or leucine (DNBLeu) covalently or ionically bonded to 3-aminopropyl silica gel [56, 57]. These CSPs are commercially available for analytical or preparative separation of enantiomers. Further CSPs based on amino acid or amine chiral selectors such as valine, phenylalanine, tyrosine [58] and l,2-tr s-diaminocyclohexane (DACH-DNB phase) [59] and 1,2-traus-diphenylethylene diamine (ULMO phase) [60] were also developed (Fig. 6.8). These CSPs have been applied for the preparative separation of the enantiomers of a few racemic compounds, but the number of reported preparative applications has remained very limited over the last 10 years. 

Anan eva, lA, EN Shapovalova, SA Lopatin, OA Shpigun, VP Varlamov, VA Davankov and DW Armstrong (2002). Use of unmodified and aminated /3-cyclodextrins for the separation of enantiomers of amino acid derivatives by high-performance liquid chromatography. Journal of Analytical Chemistry, 57(4), 331-337. 

The analytical capability of these matrices has been demonstrated for chiral amines [12, 13]. The procedure is illustrated in Fig. 8-4 for the separation of NapEtNH " CIO . Concentrated methanol/dichloromethane solutions of the racemic mixture were placed on a column containing the chiral macrocycle host. The enantiomers of the ammonium salts were resolved chromatographically with mixtures of methanol and dichloromethane as the mobile phase. The amounts of R and S salts in each fraction were determined by polarimetry. Because the chiral supported macrocycle interacts more strongly with S salts, the R salt passes through the column first and the S salt last, as seen in Fig. 8-4. 

For assaying the enantiomeric purity of dilevalol in the DBTA salt, the Schering Research Analytical Department worked out an efficient glc procedure, utilizing methylboronic acid. Although the method did not separate RR and SS enantiomers or RS and SR enantiomers, it served to quickly indicate the efficiency of the resolution process since no racemization of the R-amine moiety was ever found. A typical glc trace was as follows ... 

With chiral stationary phases, chromatographic separation of enantiomeric ferrocene derivatives is possible. An apparatus for the resolution of ferrocenyl alcohols and other compounds on triacetylcellulose has been described [60]. Analytical enantiomer separation of ferrocenyl alcohols, ethers, sulfides, and amines for the determination of enantiomeric excesses is best achieved on cyclodextrin bonded phases [61]. 

Recently, organic sulfonic acids have been suggested as normal-phase and SFC additives to improve peak shapes for basic analytes [125]. These strongly acidic additives proved particularly beneficial for the separation of a broad variety of amines on a CHIRALPAK AD CSP under normal-phase conditions. The addition of ethanesulfonic and methanesulfonic acid allowed successful separation of a selection of amines which had failed to resolve wifh less acidic additives.The enantiomer separation of a basic drug compound employing ethanesulfonic acids and trifluoroacetic acid is shown in Fig. 7.8. 

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. 

Microcrystalline cellulose triacetate, cyclodextrin- and crown ether-derived CSPs, as well as some chiral synthetic polymers, achieve enantiomer separation primarily by forming host-guest complexes with the analyte in these cases, donor-acceptor interactions are secondary. Solutes resolved on cyclodextrins and other hydrophobic cavity CSPs often have aromatic or polar substituents at a stereocenter, but these CSPs may also separate compounds that have chiral axes. Chiral crown ether CSPs resolve protonated primary amines. 

While native CF6 showed only limited enantioselectivity to a few compounds, derivatized CF6s appeared to be versatile chiral selectors and can be tuned for the best HPLC separation of different types of compounds. Aliphatic-derivatized cyclofructans operating in the polar organic mode provide best enantiomeric separations for primary amines. On the other hand, extensively aromatic-derivatized CF6 were able to separate a variety of different classes of enantiomers. Cyclofructans are the newest types of chiral selectors, and their further development and their applications in both analytical and preparative enantiomeric separations are not yet fully explored and are expected to grow significantly in the future. 

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