Place a mixture of toluene and acetone (95:5) or a mixture of hexane and ether (65:35) in the chamber to a depth of approximately 1 cm. Close the chamber with the appropriate cover, and leave for at least 30 minutes. Strips of filter paper dipping into the eluent may be placed on the internal surfaces of the chamber. [note—The developing mixture should be replaced for every test to ensure reproducible elution conditions.] Apply 0.3 mL of Test solution A approximately 2 cm from the lower edge in a streak which is as thin and as uniform as possible. In line with the streak place 2 to 3 µL of Reference solution A at one end of the plate. Develop the chromatograms in an equilibrated chamber with a Developing solvent system until the solvent front reaches approximately 1 cm from the upper edge of the plate. Remove the plate from the developing chamber, and evaporate the solvent under a current of hot air [note—Avoid excessive heat.] or by leaving the plate for a short while under a hood. Spray the plate with a 0.2% alcoholic solution of 2,7-dichlorofluorescein, and examine in UV light at 254 nm. [note—The plates pretreated with UV indicator are also commercially available and used equivalently.] In each of the plates, mark the limits of the sterol band identified through being aligned with the stain obtained from Reference solution A along the edges of the fluorescence, and additionally include the area of the zones 2 to 3 mm above and below the visible zones corresponding to Reference solution A. Remove the silica gel in the marked area into a filter funnel with a G3 porous septum. Add 10 mL of hot chloroform, mix carefully with the metal spatula, filter under vacuum, and collect the filtrate in the conical flask attached to the filter funnel. Wash the residue in the funnel three times with ether, about 10 mL each time, and collect the filtrate in the same flask attached to the funnel. Evaporate the filtrate to a volume of 4 to 5 mL, transfer the residual solution to a previously weighed 10-mL test tube with a tapering bottom and a sealing stopper, and evaporate to dryness by mild heating in a gentle stream of nitrogen. Dissolve the residue in a few drops of acetone, and evaporate again to dryness. Place at 105 for approximately 10 minutes, and allow to cool in a desiccator, and weigh.

Treat Test solution B and Test solution C the same way as prescribed for Test solution A.

Table 1. Relative Retention Times of Sterols for Two Different Columns