Colorimetric Determination of alpha-Estradiol in Estrogenic Mixtures Using Partition Chromatography

Abstract

The use of iron-Kober reagent for the determination of α-estradiol in estrogen preparations has been limited by the interfering reactions of some other phenolic estrogens. A simple partition chromatographic procedure is now described for separating the estradiols and dihydroequilin as a group from complex estrogenic mixtures. The partition system comprises 0.4 N NaOH fixed on Celite as the immobile phase with benzene as the mobile phase. By dilution of iron-Kober reagent with 1 N HCl, a new reagent for the determination of α-dihydroequilin in the presence of the estradiols has been prepared. Results of analysis of mixtures of α- and β-estradiols with α-dihydroequilin containing up to 40 μg. of estrogens in various proportions show good precision and accuracy for each of the estrogens by the combined iron-Kober procedures. By using previously described methods for the extraction and separation of the ketosteroids in combination with the new methods, an analytical procedure for the colorimetric determination of α-and β-estradiols in complex mixtures of estrogens is presented. The results obtained by six laboratories in the analysis of two oil solutions of such complex mixtures show that the procedure is satisfactory.