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

Chromatographic plates— Use suitable thin-layer chromatographic plates (see Chromatography á621ñ)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 1minute,withdraw the plates,allow them to dry at ambient temperature,and activate them at 110for 30minutes.Keep the plates in a desiccator.

Procedure— Apply 10µLof a 6%solution of Glyceryl Behenate in chloroform and 10µLof a 6%solution of USP Glyceryl Behenate RSin chloroform on a Chromatographic plate.Develop the chromatogram in Solvent mixtureuntil the solvent front has moved about 12cm.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 UVlight:the RFvalues of the spots obtained from the test solution correspond to those obtained from the Standard solution.

B: Dissolve about 22mg of Glyceryl Behenate in 1mLof toluene in a screw-cap vial having a polytef-lined septum.Add about 0.4mLof 0.2Nmethanolic (m-trifluoromethylphenyl)trimethylammonium hydroxide,attach the cap,and mix.Allow the vial to stand at room temperature for not less than 30minutes.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 G7on 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 RSconcomitantly 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 5.4g of periodic acid in 100mLof water,add 1900mLof 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-mLflasks add 50.0mLof Periodic acid solution.Add 50mLof chloroform and 10mLof water to two of the flasks,and add 50mLof water to the third flask.To each flask add 20mLof potassium iodide TS,mix gently,and proceed as directed under Procedure,beginning with “allow to stand for not less than 1minute.”The difference between the volumes of 0.1Nsodium thiosulfate required in the titrations with and without the chloroform is not greater than 0.5mL.

Procedure— Melt the Glyceryl Behenate at a temperature not higher than 80,and mix.Transfer about 1g,accurately weighed,to a 100-mLbeaker,and dissolve in 25mLof Chloroform.Transfer the solution,with the aid of an additional 25mLof Chloroform,to a separator,wash the beaker with 25mLof water,and add the washing to the separator.Place the stopper in the separator tightly,shake vigorously for 30to 60seconds,and allow the layers to separate.Add 1mLto 2mLof glacial acetic acid to break any emulsion formed.Collect the aqueous layer in a glass-stoppered,500-mLconical flask,and extract the nonaqueous layer again,using two 25-mLportions of water.Retain the combined aqueous extracts for the test for Free glycerin.Transfer the nonaqueous layer to a glass-stoppered,500-mLconical flask,and add 50.0mLof Periodic acid solutionto this flask and to a blank flask containing 50mLof chloroform and 10mLof water,swirling the flasks during the addition.Allow to stand for not less than 30minutes but not longer than 90minutes.To each flask add 20mLof potassium iodide TS,and allow to stand for not less than 1minute but not longer than 5minutes before titrating.Add 100mLof water,and titrate with 0.1Nsodium thiosulfate VS,using a magnetic stirrer to keep the solution mixed,to the disappearance of the brown iodine color.Then add 2mLof starch TS,and continue the titration to the disappearance of the blue color.[NOTE—If the Glyceryl Behenate titration is less than 0.8of 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 20.73is one-twentieth of the molecular weight of glyceryl monobehenate;Nis the normality of the sodium thiosulfate;Band Sare the volumes,in mL,of 0.1Nsodium thiosulfate consumed by the blank and the Glyceryl Behenate,respectively;and Wis 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 1-Content of monoglycerides.

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