Spectrodensitometry Application to Analytical Identification of Estradiol, Hydrocortisone, Testosterone and Cholesterol on Diol Plates

Abstract

The selected steroid compounds such as: estradiol (E), hydrocortisone (H), testosterone (T), and cholesterol (CH) were separated by using thin-layer chromatography on glass plates precoated with Diol F254s (E. Merck, #1.05636) and using a chloroform as mobile phase. The densitometric detection of these compounds with and without the use of sulphuric acid solutions as visualizing reagents was compared. A robust and sensitive detection procedure for selected steroid compounds using the sulphuric acid as a visualizing reagent was described. Spot intensities on the plates were quantified after dipping in sulphuric acid solutions and heating at temperatures from 80°C to 140°C for times ranging from 5 to 30 min. The best detection conditions for high signal intensity [AU] were determined. Particularly robust and sensitive detection of investigated compounds separated was observed by dipping the plate for 15 s in the solution of sulphuric acid in methanol in the volume composition 1:19, and for temperature equal to 120°C and for heating for 10 min. The spectrodensitograms of hydrocortisone, estradiol, testosterone, and cholesterol on DiolF254 plates and by the use of sulphuric acid as visualizing reagents are different than the spectrodensitograms obtained on the plate without the use of a visualizing reagent. The obtained spectrodensitograms of investigated compounds after the detection with the use of sulphuric acid as visualizing reagents differ in the number and intensity of additional absorption bands. This fact has analytical and pharmaceutical significance in the identification of hydrocortisone, estradiol, testosterone, and cholesterol. Comparison and characterization of chromatographic spots of examined compounds on the basis of resolution (RS), separation factor (α), constant of the pair separation (), and ΔRF values were discussed. It was stated, that the resolution Rs values are similar at the wavelengths 250, 275, 300, 325, 375, and 400 nm for the particular separated compounds analyzed after the use of sulphuric acid solution as visualizing reagent.