Resolution of saturated and unsaturated 5β-cholanoic acids by gas–liquid and thin-layer chromatography

The thin-layer and gas–liquid chromatographic properties of the methyl ester acetates were determined for a series of 20 monounsaturated 5β-cholanoic acids representing the simple chemical and enzymic dehydration products of the common bile acids. The unsaturated acids were generally indistinguishable from their saturated analogues by thin-layer chromatography on plain silica gel, but resolution was achieved on silica gel impregnated with silver nitrate for compounds having sterically exposed double bonds. The gas–liquid chromatographic behaviour of the unsaturated bile acids on the OV-225, SE-30, and Poly-S-179 liquid phases was closely similar to that observed for the saturated bile acids. The 5β-cholenoic acids obeyed the general rules of chromatographic mobility based on the overall shape of the molecule and the number and configuration of functional groups, with a constant retention factor attributable to the oleflnic bond. The structural information provided by the chromatographic behaviour of the standard unsaturated bile acids allows a distinction to be made among most of the isomeric 5β-cholenoates. A complete identification of all isomeric olefins is possible when chromatographic and mass spectrometric data are combined.