The quantitative separation of estrogens by paper partition chromatography.

The varying success of known chromat’ographic procedures for the separation and identification of microquantities of estrogens (1, 2) has led to the development of paper partit!ion chromatographic techniques which are reported here. These t’echniques permit rapid separation, identification, and quantitative estimation of free est.rogens in biological materials. The solvent systems of o-dichlorobenzene-formamide, methylene chloride-formamide, and cyclohexene-formamide proved most satisfactory. Among others, chloroform, pentachloroethane, benzene, and cyclohexane did not give the best chromatographic resolution or the most appropriate time intervals for development. Although some color tests for the identification of free estrogens have been described (3-5), in this study known calorimetric procedures (6, 7), among other spot tests, have been successfully adapted for the detection of microquantities of these compounds on paper chromatograms. Spectrophotometric techniques for the identification and measurement of steroids (8-10) were also investigated. The application of the new Oechniques is demonstrated by the identification of some of the estrogens in the urine of normal and of pregnant females, as well as of estrogenic metabolites found in the bile of a completely ovariectomized, hysterectomized dog after the injection of estradiol-17p dibenzoate intramuscularly.