Photometric Determination of Estrogens

The Kober color test for the identification of natural estrogens (1) is a 2-phase reaction. The first effect of heating estrogens with the phenolsulfonic acid reagent is the production of a yellowish brown-colored solution; when this solution is chilled, carefully diluted with water, and then reheated, its color gives way to a characteristic pink. The Z-phase nature of the reaction renders its application to quantitative assay work somewhat tedious, since both the initial color production and the conversion to the final pink are subject to a number of variables, some of which are difficult to control. Moreover, although the final pink color obtained in the reaction appears to be common to most of the natural estrogens, insufficient attention has been paid to the observation of Cohen and Marrian (2) and of Venning, Evelyn, Harkness, and Browne (3) that both the speed and extent of its formation vary appreciably among different members of this group of substances; thus, assays of mixtures of estrogens by Kober’s original method cannot give accurate results unless the identities and proportions of the various ingredients are known beforehand. Because of these difficulties a study of the reaction was undertaken, as a result of which it was found possible to simplify the procedure of color production, and thereby to investigate more extensively than has heretofore been feasible the precise chromo-