Indicator solution— Transfer about 0.675 g of bromocresol purple, accurately weighed, to a 1-L volumetric flask. Dissolve in 25 mL of 0.10 N sodium hydroxide, dilute with water to volume, and mix.

Calibration solutions— Pipet 1, 2, 3, and 4 mL of 0.001 N acetic acid VS into four 100-mL volumetric flasks, respectively. Pipet 4 mL of the Indicator solution into each flask and into an empty 100-mL volumetric flask, and dilute each flask with water to volume to obtain solutions containing 0.0, 0.60, 1.20, 1.80, and 2.40 µg of acetic acid per mL.

Control solution— Place 96 mL of water in a suitable bottle, add a stirring bar, cap the bottle, and stir for 75 minutes at room temperature. Pipet 4 mL of Indicator solution into the bottle, and mix.

Test solution— Transfer about 1 to 2 g of Cellaburate, accurately weighed, to a bottle, and add 96 mL of water. Add a stirring bar, cap the bottle, and stir for 75 minutes at room temperature. Pipet 4 mL of Indicator solution into the bottle, stir to mix, and allow the solid to settle for 2 minutes.

Calibration— Determine the absorbances of the Calibration solutions in a 1-cm cell at the wavelength of maximum absorption of the basic form of bromocresol purple at about 589 nm, with a suitable spectrophotometer, using water as the blank. The absorbance difference, AS, between the 0.0 µg per mL solution and the other solutions adheres to Beer's law over the range stated under Calibration solutions. Plot AS versus CS (the concentration of the acetic acid in µg per mL) on linear graph paper, and draw the straight line best fitting the points, including the origin.

Procedure— Pass 10 mL of the Test solution through a polytef syringe filter that has been presoaked in isopropyl alcohol. Determine the absorbance of the filtered Test solution in a 1-cm cell at about 589 nm on the same spectrophotometer, using water as the blank. In the same manner, determine the absorbance of the Control solution. Calculate the percentage of free acid, as acetic acid, in the portion of Cellaburate taken by the formula: in which 100 is the total volume, in mL, of the Test solution; CU is the concentration of free acid, calculated as acetic acid, in µg per mL, based on the absorbance difference between the Control solution and the Test solution read directly from the calibration plot; and WU is the weight, in g, of Cellaburate taken to prepare the Test solution. [note—If the CU value is greater than 2.8 µg per mL, reduce the test sample size by half in the Test solution, and repeat the determination.] Not more than 0.1% is found.

Internal standard solution— Prepare a solution of isovaleric acid in pyridine containing about 4.6 mg per mL, and store it in a tightly closed container.

Saponification solution— Place 250 mL of n-propyl alcohol in a 500-mL volumetric flask, add 65.5 g of potassium hydroxide, and mix to dissolve. Dilute with n-propyl alcohol to volume, and mix.

Acid solution— Place 250 mL of n-propyl alcohol in a 500-mL volumetric flask, add 166 mL of hydrochloric acid, and mix. Dilute with n-propyl alcohol to volume, and mix.

Standard preparation— Transfer about 0.20 g of glacial acetic acid and 0.31 g of butyric acid, each accurately weighed, to a 50-mL volumetric flask. Dilute with Internal standard solution to volume, and mix.

Test preparation— Transfer about 0.15 g of Cellaburate, previously dried at 105 for 1 hour and accurately weighed, into a 25-mm × 160-mm test tube. Pipet 10 mL of Internal standard solution into the test tube, and dissolve by stirring and heating at 110 for 30 minutes. While stirring, add 5 mL of Saponification solution slowly into the tube. Heat at 110 for 10 minutes. Cool, and add 5 mL of the Acid solution. Mix on a vortex mixer, and allow the precipitate to settle.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.53-mm × 30-m fused silica column bonded with a 1-µm layer of phase G35, and a split injection system with a split ratio of about 35:1. Helium is used as the carrier gas, flowing at a rate of about 8 mL per minute. The injection port, column, and detector block temperatures are maintained at 250, 125, and 250, respectively. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.45 for acetic acid, 0.85 for butyric acid, and 1.00 for isovaleric acid; the tailing factor for the butyric acid peak is not more than 1.5; and the relative standard deviation for replicate injections is not more than 3.0%.

Calibration— Inject about 1 µL of the Standard preparation into the chromatograph, and record the chromatogram as directed for Procedure. Repeat two more times. Calculate the average unit weight response, FSA, of acetic acid per 10 mL of the Internal standard solution by the formula: in which 10/50 is the volume ratio of the Internal standard solution in the Test preparation to that in the Standard preparation; qRA is the weight, in g, of acetic acid in the Standard preparation; and RSA is the average peak response ratio of acetic acid to isovaleric acid. Similarly, calculate the average unit weight response, FSB, of butyric acid per 10 mL of the Internal standard solution by the formula: in which 10/50 is the volume ratio of the Internal standard solution in the Test preparation to that in the Standard preparation; qRB is the weight, in g, of butyric acid in the Standard preparation; and RSB is the average peak response ratio of butyric acid to isovaleric acid.

Procedure— Inject about 1 µL of the upper clear solution from the Test preparation into the chromatograph, record the chromatogram, and measure the peak area responses. Calculate the percentage of acetyl in the portion of Cellaburate taken by the formula: in which 43/60 is the ratio of the formula weights of acetyl and acetic acid; RUA is the peak area response ratio of acetic acid to isovaleric acid in the Test preparation; FSA is as defined under Calibration; and WU is the weight, in g, of Cellaburate taken to prepare the Test preparation. Similarly, calculate the percentage of butyryl in the portion of Cellaburate taken by the formula: in which 71/88 is the ratio of the formula weights of butyryl and butyric acid; RUB is the peak area response ratio of butyric acid to isovaleric acid in the Test preparation; FSB is as defined under Calibration; and WU is the weight, in g, of Cellaburate taken to prepare the Test preparation.