A: Solvent mixture—Mix 96 volumes of chloroform and 4 volumes of acetone.

Chromatographic plates— Use suitable thin-layer chromatographic plates (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel. Pre-develop the plates by placing in a chromatographic chamber saturated with ether. Remove the plates from the chamber, allow the ether to evaporate, and immerse the plates in a 2.5% solution of boric acid in alcohol. After about 1 minute, withdraw the plates, allow them to dry at ambient temperature, and activate them at 110 for 30 minutes. Keep the plates in a desiccator.

Procedure— Apply 10 µL of a 6% solution of Glyceryl Behenate in chloroform and 10 µL of a 6% solution of USP Glyceryl Behenate RS in chloroform on a Chromatographic plate. Develop the chromatogram in Solvent mixture until the solvent front has moved about 12 cm. Remove the plate from the developing chamber, and allow the solvent to evaporate. Spray the chromatogram with a 0.02% solution of dichlorofluorescein in alcohol. Examine the spots under short-wavelength UV light: the RF values of the spots obtained from the test solution correspond to those obtained from the Standard solution.

B: Dissolve about 22 mg of Glyceryl Behenate in 1 mL of toluene in a screw-cap vial having a polytef-lined septum. Add about 0.4 mL of 0.2 N methanolic (m-trifluoromethylphenyl) trimethylammonium hydroxide, attach the cap, and mix. Allow the vial to stand at room temperature for not less than 30 minutes. Introduce a suitable volume into a gas chromatograph equipped with a flame-ionization detector and a 4-mm × 1.8-m column packed with 10% liquid phase G7 on support S1A, maintained at a temperature of about 225: the retention time of the main peak in the resulting chromatogram corresponds to that of the main peak in a similar preparation of USP Glyceryl Behenate RS concomitantly chromatographed. The ratio of the response of the main peak to the sum of all the responses is not less than 0.83.

Periodic acid solution— Dissolve 5.4 g of periodic acid in 100 mL of water, add 1900 mL of glacial acetic acid, and mix. Store in a light-resistant, glass-stoppered bottle or in a clear glass-stoppered bottle protected from light.

Chloroform— Use chloroform that meets the following test. To each of three 500-mL flasks add 50.0 mL of Periodic acid solution. Add 50 mL of chloroform and 10 mL of water to two of the flasks, and add 50 mL of water to the third flask. To each flask add 20 mL of potassium iodide TS, mix gently, and proceed as directed under Procedure, beginning with “allow to stand for not less than 1 minute.” The difference between the volumes of 0.1 N sodium thiosulfate required in the titrations with and without the chloroform is not greater than 0.5 mL.

Procedure— Melt the Glyceryl Behenate at a temperature not higher than 80, and mix. Transfer about 1 g, accurately weighed, to a 100-mL beaker, and dissolve in 25 mL of Chloroform. Transfer the solution, with the aid of an additional 25 mL of Chloroform, to a separator, wash the beaker with 25 mL of water, and add the washing to the separator. Place the stopper in the separator tightly, shake vigorously for 30 to 60 seconds, and allow the layers to separate. Add 1 mL to 2 mL of glacial acetic acid to break any emulsion formed. Collect the aqueous layer in a glass-stoppered, 500-mL conical flask, and extract the nonaqueous layer again, using two 25-mL portions of water. Retain the combined aqueous extracts for the test for Free glycerin. Transfer the nonaqueous layer to a glass-stoppered, 500-mL conical flask, and add 50.0 mL of Periodic acid solution to this flask and to a blank flask containing 50 mL of chloroform and 10 mL of water, swirling the flasks during the addition. Allow to stand for not less than 30 minutes but not longer than 90 minutes. To each flask add 20 mL of potassium iodide TS, and allow to stand for not less than 1 minute but not longer than 5 minutes before titrating. Add 100 mL of water, and titrate with 0.1 N sodium thiosulfate VS, using a magnetic stirrer to keep the solution mixed, to the disappearance of the brown iodine color. Then add 2 mL of starch TS, and continue the titration to the disappearance of the blue color. [note—If the Glyceryl Behenate titration is less than 0.8 of the blank titration, discard, and repeat using a smaller weight of Glyceryl Behenate.] Calculate the percentage of 1-monoglycerides, as glyceryl monobehenate, by the formula: in which 20.73 is one-twentieth of the molecular weight of glyceryl monobehenate; N is the normality of the sodium thiosulfate; B and S are the volumes, in mL, of 0.1 N sodium thiosulfate consumed by the blank and the Glyceryl Behenate, respectively; and W is the weight, in g, of Glyceryl Behenate taken; between 12.0% and 18.0% is found.

Periodic acid solution and Chloroform—Prepare as directed in the test for Content of 1-monoglycerides.

Procedure— To the combined aqueous extracts obtained as directed in the test for 1-Content of monoglycerides add 50.0 mL of Periodic acid solution. Prepare a blank by adding 50.0 mL of Periodic acid solution to a glass-stoppered conical flask containing 75 mL of water. Proceed as directed for Procedure in the test for Content of 1-monoglycerides, beginning with “Allow to stand for not less than 30 minutes.” Calculate the percentage of free glycerin by the formula: in which 2.30 is one-fortieth of the molecular weight of glycerin; N is the normality of the sodium thiosulfate; b and s are the volumes, in mL, of 0.1 N sodium thiosulfate consumed by the blank and the Glyceryl Behenate, respectively; and W is the weight, in g, of Glyceryl Behenate taken. Not more than 1.0% is found.