Sulfuric acid, both concentrated and variously diluted with ethanol and water, has been widely employed in steroid analysis. Three types of reactions with neutral steroids are: (a) development of characteristic absorption spectra at room temperature after addition to a dried sample of steroid (l-3), ( 5) development of a characteristic major absorption peak in the visible range after heating with the reagent and diluting (4), and (c) development of fluorescence (5-8).
The blue color given by dehydroepiandrosterone (DHA) after heating with the Allen reagent and diluting with ethanol is well known (4). Little attention has, however, been paid to the absorption spectrum developed by this and other steroids in the reagent itself, without heating or diluting. Our attention was directed to this phenomenon by the observation of a deep blue color from A5-pregnene-3P,17cY,20a-triol
(5-PT) at room temperature. Other A5-3P-OH steroids were then found to give distinctive spectra with this "cold" Allen reagent. Meanwhile Oertel and Eik-Nes (3) showed that several A5-3/?-OH steroids gave an intense yellow color in a more dilute ethanolic sulfuric acid reagent.
The present communication compares absorption spectra of a number of A5-3,&OH steroids in the Allen and Oertel reagents and in concentrated sulfuric acid. A group of other steroids has been studied with the Allen reagent alone. It is shown that spectra in the diluted reagents are useful (a) for identification purposes, (5) for quantitative determination of several steroids for which no simple method existed, and (c) as spot tests on paper chromatograms, or for testing column eluates.
MATERIALS AND METHODS
Reagents and Equipment
Concentrated Sulfuric Acid. Baker and Adamson Co., reagent grade. 402 8PECTRA OF SOME HTDROXY STEROIDS 403 Ethanol. U. S. Industrial Chemical Co. "U.&I. Absolute pure ethyl alcohol" U.S.P.-N.F.
reagent quality. Negligible absorption in the visible and ultraviolet wavelengths was found when 5 ml of this ethanol was evaporated to dryness and the residue treated with sulfuric acid.
EthanoE, 90% and 95%. Prepared from absolute ethanol by adding distilled water.
Allen Reagent (4). Eighty milliliters of cont. sulfuric acid was added to 20 ml of 90% ethanol, with cooling.
Oertel Reagent (3). Sixty-seven milliliters of cont. sulfuric acid was added to 33 ml of 95% ethanol, with cooling.
Spectrophotometer. Cary model 14 recording speetrophotometer. Applied Physics Corp., Pasadena, Cal.
Spectrophotometer Cuvettes. One milliliter capacity, 10 mm light path.