Procedure
Apply separately 20 µL of the
Test solution, 5, 10, 15, and 20 µL of the
Standard solution, and 20 µL of the
Identification test solution to a suitable thin-layer chromatographic plate (see
Chromatography
621![](images/chars/rang.gif)
) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, position the plate in a chromatographic chamber, and develop the chromatograms in a solvent system consisting of a mixture of toluene and ethyl acetate (4:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Spray the plate with a 1 in 100 solution of diphenylamine in methanol, and irradiate the plate with short- and long-wavelength UV light for about 15 minutes, and examine the chromatogram: any spot obtained from the
Test solution, other than the principal spot, is not more intense than the spot in the chromatogram from the 20-µL application of the
Standard solution. Compare the intensities of any secondary spots observed in the chromatogram of the
Test solution with those of the principal spots in the chromatograms of the
Standard solution (corresponding to 0.5%, 1.0%, 1.5%, and 2.0%, respectively): the sum of the intensities of the secondary spots obtained in the
Test solution is not more than 3%.
[NOTENitrates of glycerin typically have
RF values of about 0.21, 0.37, and 0.61 for mono-, di-, and tri-substituted glycerins, respectively.
]