Limit of free glycerin
Mix 10 mL of pyridine with 20 mL of propionic anhydride.
Internal standard solution
Dissolve a suitable quantity of tributyrin, accurately weighed, in chloroform, and dilute quantitatively with chloroform to obtain a solution having a concentration of about 0.2 mg per mL.
Transfer about 15 mg of glycerin and about 50 mg of tributyrin, both accurately weighed, to a glass-stoppered, 25-mL conical flask; add 3 mL of Propionating reagent;
and heat at 75
for 30 minutes. Volatilize the reagents with the aid of a stream of nitrogen at room temperature, add about 12 mL of chloroform, and mix. Dilute about 1 mL of this mixture with chloroform to about 20 mL, and mix.
Transfer about 50 mg of Glyceryl Monostearate, accurately weighed, to a glass-stoppered, 25-mL conical flask; add by pipet 5 mL of Internal standard solution;
and mix to dissolve. Immerse the flask in a water bath maintained at a temperature between 45
, and volatilize the chloroform with the aid of a stream of nitrogen. Add 3 mL of Propionating reagent,
and heat at 75
for 30 minutes. Volatilize the reagents with the aid of a stream of nitrogen at room temperature, add about 5 mL of chloroform, and mix.
Under typical conditions, the instrument is equipped with a flame-ionization detector, and contains a 4-mm × 2.4-m borosilicate glass column packed with 2% liquid phase G16 on 80- to 100-mesh support S1A. The carrier gas is helium, flowing at a rate of about 70 mL per minute. The column is maintained isothermally at a temperature between 190
, and the injection port and detector block temperatures are maintained at about 300
Chromatograph six to ten injections of the Standard solution as directed for Procedure: the resolution, R, between derivatized glycerin and tributyrin is not less than 4.0; and the relative standard deviation of the ratio of their peak areas is not more than 2.0%.
Inject a suitable portion of the Standard solution
into a suitable gas chromatograph, record the chromatogram, and measure the areas under the peaks, recording the values of the areas under the tripropionin and tributyrin peaks as AS
, respectively. Calculate the response factor, F
, by the formula:
(AD / AS)(WS / WD)
in which WS
are the weights, in mg, of glycerin and tributyrin, respectively, in the Standard solution.
Similarly inject a suitable portion of the Test solution
, record the chromatogram, and measure the areas under the peaks, recording the values of the areas under the tripropionin and tributyrin peaks as aU
, respectively. Calculate the percentage of glycerin by the formula:
100F(aU / aD)(wD / wU)
in which wD
is the weight, in mg, of tributyrin in 5 mL of Internal standard solution
; and wU
is the weight, in mg, of Glyceryl Monostearate in the Test solution:
the limit is 1.2%.
Change to read:
Assay for monoglycerides
Transfer about 40 mg of Glyceryl Monostearate, accurately weighed, to a 5-mL volumetric flask, dissolve in and dilute with tetrahydrofuran to volume, and mix.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a refractive index detector and a 7.5-mm × 60-cm column containing 5-µm 100-
packing L21. The column and detector temperatures are maintained at 40
Two or three 7.5-mm × 30-cm L21 columns may be used in place of the one 60-cm column, provided that system suitability requirements are met; and the column temperature may be lowered to ambient temperature, although working at 40
provides stable separation conditions and ensures better sample solubility.]
The flow rate is about 1 mL per minute. Chromatograph the Assay preparation,
and record the peak responses as directed for Procedure:
the relative retention times are about 1.0 for glycerin, 0.86 for monoglycerides, 0.81 for diglycerides, and 0.77 for triglycerides; and the relative standard deviation for replicate injections determined from the monoglycerides peak is not more than 2.0%.
Inject a volume (about 40 µL) of the Assay preparation
into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of monoglycerides in the portion of Glyceryl Monostearate taken by the formula:
100(ri / rs)
in which ri
is the peak response for the monoglycerides; and rs
is the sum of the responses for all the glyceride peaks.NF24