Separation of Steroid Hormones on Chromatographic Columns 
Of the 100 pl of chloroform extract, as much as possible was added 
directly to a reamed-out portion of the column, and to a porous stopper which 
rested on the top of the colum. Three ml of the developing solvent (ethyl 
acetate:cyclohexane, 1:1) were added to the filling heads, and the chromato- 
grams developed for 5 min by centrifugation at 2500 G. The ethyl acetate: 
cyclohexane system was selected from among twelve other solvent systems 
because it more readily separated steroids of the androstane series from 
those of the estrane series, and each of these from cholesterol (Randerath, 
1963). The silica columns were extruded on trays and dried 10 min at 65°C. 
Visualization of Steroid Hormones on Chromatographic Columns 
Staining with iodine 
Bands of steroid hormones were stained by exposing the chromatograms to 
iodine crystals in a closed container. The Rm values (distance of migration 
of steroid hormone bands from top of column) of unknown bands were compared 
to those of authentic steroid hormones. Further confirmation of the identity 
of individual steroid hormones was by sulfuric acid charring, viewing colors 
of bands in daylight, or fluorescence under ultraviolet light, or by elution 
and scanning of absorption peaks. 
Charring with sulfuric acid 
When charred, dried chromatograms were transferred to spatulas, inserted 
into the charring chamber, six drops of concentrated sulfuric acid added to 
the heating elements, and 90 volts applied until a satisfactory color had 
developed. The colors of individual bands were viewed in daylight. The 
charred chromatograms were also exposed to ultraviolet light, which caused 
certain steroid hormone bands to fluoresce. 
Scanning of Absorption Peaks 
When individual steroid hormones were to be scanned, cut bands with Rm 
values corresponding to steroid hormone standards were eluted with 2 ml of 
absolute chloroform, taken to complete dryness under nitrogen, and redissolved 
in 100 ul of chloroform. Fifty ul of this solution were added to 500 ul of 
concentrated sulfuric acid in a microcuvette. Sulfuric acid chromogen spectra 
over the range of 190 - 600 nm (Zaffaroni, 1953) were compared in eluted 
samples, using a Beckman Acta II, recording spectrophotometer. 
Check of Recovery Using Radioactive Steroids 
Percent recovery of l4c_testosterone and !'cC-8-estradiol was estimated by 
radiochemical techniques. Known amounts of radioactive steroids were added to 
5 ml plasma from male or female Japanese quail. Aliquots (0.2 ml) of each 
were removed, oxidized on a biological material oxidizer,“ collected in 
2 Beckman Instruments, Inc., Silver Spring, Md. 
2 
