Bile acid derivatives isomer separation

Separation of Isomers and Enantiomers by Bile Acid Derivatives... 

Sometimes the methyl esters are better separated than the free bile acids. This is especially true when neutral solvents are used. In acidic solvents the difference is small, particularly with trisubstituted bile acids. Thus, the same types of acidic solvents can be used for free and methylated bile acids. When bile acid methyl esters are acetylated or converted into trimethylsilyl ethers their mobility is considerably increased. The separation of configurational isomers is then often impaired whereas the separation of positional isomers may be improved (4). This is often the case when neutral derivatives are analyzed in neutral solvent systems (12). The influence of the nature and position of nuclear substituents on the mobility of a bile acid can be estimated... 

Improved separations of bile acid methyl esters can be observed with increasing amounts of phenyl substituents in the stationary phase (5% in SE-52, 20% in PhSi-20, and 50% in OV-17). Positional and configurational isomers are better resolved than on SE-30 or OV-1 columns. For instance the valuable separation of the trimethylsilyl ethers of 3,6,7-substituted methyl cholanoates from the corresponding cholic acid derivative on OV-17 should be noted. The pronounced effect of a 7/3-hydroxy substituent on retention times on columns of SE-52 and PhSi-20 is noteworthy. The large separation factors between the diacetate derivatives of chenodeoxy- and ursodeo.xycholic acids may be most useful in work with bile acids of biological origin. 

Spectra of O-methyloximes of methyl 3,6-diketo-5a- and 3,6-diketo-5/3-cholanoates have been reported by Allen et al. (30). The O-methyloximes, prepared as described by Fales and Luukkainen (49), were found to be particularly useful derivatives since the 5a- and 5 -isomers could be separated by gas chromatography, which was not the case with the free ketones. Considerable differences in relative intensities of the peaks were noted between the spectra of the two isomers. The 5y3 compound gave a base peak at mje 138 (probably ring A) whereas the molecular ion was base peak in the spectrum of the 5a-epimer. These results show the value of O-methyloximes in mass spectrometry of ketonic bile acids. It should be recognized that partial derivative formation may occur, and that formation of syn- and o //-isomers may result in a complex mixture of products from a pure compound. 

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