Indicator solution— Transfer about 0.675g of bromocresol purple,accurately weighed,to a 1-Lvolumetric flask.Dissolve in 25mLof 0.10Nsodium hydroxide,dilute with water to volume,and mix.

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

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

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

Calibration— Determine the absorbances of the Calibration solutionsin a 1-cm cell at the wavelength of maximum absorption of the basic form of bromocresol purple at about 589nm,with a suitable spectrophotometer,using water as the blank.The absorbance difference,AS,between the 0.0µg per mLsolution and the other solutions adheres to Beer's law over the range stated under Calibration solutions.Plot ASversus 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 10mLof the Test solutionthrough a polytef syringe filter that has been presoaked in isopropyl alcohol.Determine the absorbance of the filtered Test solutionin a 1-cm cell at about 589nm 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 100is the total volume,in mL,of the Test solution;CUis the concentration of free acid,calculated as acetic acid,in µg per mL,based on the absorbance difference between the Control solutionand the Test solutionread directly from the calibration plot;and WUis the weight,in g,of Cellaburate taken to prepare the Test solution.[NOTE—If the CUvalue is greater than 2.8µg 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 4.6mg per mL,and store it in a tightly closed container.

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

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

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

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

Chromatographic system (see Chromatography á621ñ)— 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 8mLper 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 0.45for acetic acid,0.85for butyric acid,and 1.00for 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 1µLof the Standard preparationinto 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 10mLof the Internal standard solutionby the formula: in which 10/50is the volume ratio of the Internal standard solutionin the Test preparationto that in the Standard preparation;qRAis the weight,in g,of acetic acid in the Standard preparation;and RSAis the average peak response ratio of acetic acid to isovaleric acid.Similarly,calculate the average unit weight response,FSB,of butyric acid per 10mLof the Internal standard solutionby the formula: in which 10/50is the volume ratio of the Internal standard solutionin the Test preparationto that in the Standard preparation;qRBis the weight,in g,of butyric acid in the Standard preparation;and RSBis the average peak response ratio of butyric acid to isovaleric acid.

Procedure— Inject about 1µLof the upper clear solution from the Test preparationinto 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 43/60is the ratio of the formula weights of acetyl and acetic acid;RUAis the peak area response ratio of acetic acid to isovaleric acid in the Test preparation;FSAis as defined under Calibration;and WUis 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 71/88is the ratio of the formula weights of butyryl and butyric acid;RUBis the peak area response ratio of butyric acid to isovaleric acid in the Test preparation;FSBis as defined under Calibration;and WUis the weight,in g,of Cellaburate taken to prepare the Test preparation.NF23