VARIABLES WHICH AFFECT COLOR DEVELOPMENT IN A MODIFICATION OF ZIMMERMANN'S REACTION AND A SPECTROPHOTOMETRIC ANALYSIS OF COLORS DEVELOPED WITH KETOSTEROIDS12

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Abstract
The variable factors which affect the course of color development in Zimmermann''s m-dinitrobenzene reaction for androgen assay have been studied in detail. These include the temp, and duration of the reaction, alkalinity of the soln., relative amts. of the reactants, cone, of ale. and of m-dinitrobenzene, and the intensity of light to which the reaction mixture is exposed. A modification of the Zimmermann reaction for use in the Oesting colorimeter was devised on the basis of these data. The colored solns., which were prepared from crystalline and urinary androgens by this modification, and the optical system of Oesting''s colorimeter were then subjected to spectrophotometric studies in order to eliminate the subjective factor inherent in visual colorimetry. These observations show that the colors which are developed in solns. of androgens by the present modification of the Zimmermann procedure give good chromaticity and brightness matches with the discs of the Oesting colorimeter when a specific attempt is made to secure such a match by adjusting the cone, of hormone in soln. The numerous factors which affect the rate and intensity of color development in the m-dinitrobenzene reaction and the instability of the unknown colored compound which is formed are undesirable features of this method of assay. Although this procedure has proved useful in practice, it would be highly desirable from a chemical viewpoint to replace it with another reaction which is free of this criticism.