TLC of Selected Bile Acids: Detection and Separation

Abstract

The selected bile acids such as: cholic acid (C), glycocholic acid (GC), glycolithocholic acid (GLC), deoxycholic acid (DC), chenodeoxycholic acid (CDC), glycodeoxycholic acid (GDC), lithocholic acid (LC) were separated by using thin layer chromatography on glass plates precoated with silica gel 60 with a concentrating zone. A robust and sensitive detection procedure for selected bile acids using the sulphuric acid as a visualizing reagent was described. Phosphomolybdic acid (10%) in ethanol was used as the comparative visualizing reagent. Spot intensities on the plates were quantified after dipping in sulphuric acid solutions and heating at temperatures from 60°C to 120°C for times ranging from 2 to 45 min. The best detection conditions for high signal intensity [AU] were determined. Particularly robust and sensitive detection of investigated bile acids separated was observed using the solution of sulphuric acid in methanol in the volume composition 1:19, and for temperature equal to 90°C and for heating for 20 min. Comparison and characterization of chromatographic spots of examined compounds on the basis of resolution (R _S ), separation factor (α), constant of the pair separation (R _F ^α), and ΔR _F values were discussed. The R _S parameter, serving to evaluate the substance separation, was determined by visual and densitometric methods. It was proven that the R _S parameter determined by the visual method for two adjacent substances is always larger than determined by the densitometric method. It was stated that the densitometric method is correct, objective, and assures standard conditions to the parameter R _S determination. All bile acids have been best separated only by developing with n‐hexane‐ethyl acetate –methanol‐acetic acid in volume composition 20∶20∶5∶2 as the mobile phase.

Keywords:

• Bile acids
• Adsorption TLC
• Spot visualization
• Sulphuric acid
• Separation
• Densitometric analysis
• Separation factors

Acknowledgment

This research was financed by the Ministry of Science and Higher Education by resources reserved for science in the years 2005‐2008 as research project No. 3 T09A 155 29.