Paper Chromatography of Steroids

In Paper I of this series (1) a method was described for the paper chromatographic analysis of certain ketonic steroids as hydrazones formed with Girard’s Reagent T. When that technique was applied to the adrenal cortical hormones, however, certain deficiencies appeared. (a) The RI values of the individual steroid-Reagent T hydrazones were not sufficiently different to permit identification. (b) Each compound frequently gave rise to two spots on the chromatogram corresponding to the monoand dihydrazones, respectively. No improvement resulted, furthermore, when various other carbonyl reagents were substituted for Girard’s Reagent T. These included carboxymethoxime, nicotinic acid hydrazide, N-methyl/3-carbohydrazide-pyridinium iodide, y-aspartyl hydrazide, and 2-quinolylhydrazine. Since the corticoids, as a group, possess somewhat higher water solubilities than most other ketosteroids, the chromatographic behavior of the free compounds in aqueous solvents was investigated. Although separation of these steroids was obtained with systems such as benzene-water, undesirable “tailing” of the spots appeared. Efforts to eliminate this defect included the substitution of various non-polar solvents for benzene, the use of several grades and types of filter paper, and the preliminary treatment of papers with cellulose acetate,’ latex (2), and chlorosilanes.2 These attempts were ineffective. Replacement of the water by certain polar organic solvents overcame the “tailing” and, furthermore, gave greater chromatographic resolution.