THE SEPARATION AND IDENTIFICATION OF MIXTURES OF Czl AND Cl9 STEROIDS BY PAPER CHROMATOGRAPHY* BY LEONARD R. AXELROD WITH THE TECHNICAL ASSISTANCE OF MADELINE GOLDSTEIN

Paper chromatographic techniques have been reported for the separation and identification of steroid compounds related to progesterone and testosterone, in which impregnated filter paper (l-4) and steroid complexes (5, 6) are utilized. After preliminary investigations on the separation of progesterone and its metabolites (7), however, it became apparent that progesterone-like Czl compounds from biological media (i.e., blood and urine) could not be chromatographically separated by any of the above procedures without gross contamination with metabolit.es of the Cl9 series and vice versa. This is probably related to the fact that the solubilities and “polarities” of the two groups are similar. The need for procedures by which mixtures of closely related free steroids of the two series can be separated has led to the development of techniques reported here. The solvent systems of Decalin-formamide, cyclohexene-formamide, methylcyclohexane-propylene glycol, and methylcyclohexane-1,3-butanediol proved most satisfactory, giving the best chromatographic resolutions and the most convenient time intervals for development. Modifications of the Zimmermann (8), Lund (9), and van Zijp (10) tests have been adapted to the detection of micro quantities of the steroids on paper chromatograms. Triphenyltetrazolium chloride (11)) fuming sulfuric acid (15 per cent free SOS) (12), and antimony pentachloride (12) were also used in this investigation for identifying the steroids. The technique in which the absorption spectra of corticosteroid sulfuric acid chromogens (13) were utilized was adapted to the Czl and Cl9 steroids for further aid in identification.