A: Under a hood with a good draft place a few drops of Isoflurophate in a small platinum crucible, quickly add 2 mL of sulfuric acid, and immediately cover the crucible with a small, clear watch glass. Allow to stand for 10 minutes, then heat on a steam bath for 5 to 10 minutes: the side of the watch glass exposed to the mixture is visibly etched.

B: Slowly heat under the hood the crucible and contents from Identification test A until copious white fumes are evolved. Cool, place the crucible in a beaker, and add a sufficient quantity of water nearly to cover the crucible. After a few minutes remove the crucible from the beaker with the aid of a glass rod, add 3 mL of nitric acid, and boil for a few minutes. Cool, cautiously add 6 N ammonium hydroxide with stirring until a slight odor of ammonia persists, then add 1 mL of nitric acid. Filter the liquid if not clear, warm to about 40, and add about 10 mL of ammonium molybdate TS: a yellow precipitate, which is soluble in 6 N ammonium hydroxide, is formed.

Mixed indicator— Mix 3 volumes of a 1 in 1000 solution of bromocresol green in alcohol and 1 volume of a 1 in 500 solution of methyl red in alcohol.

0.1 N Sodium hydroxide in dehydrated alcohol— Dissolve about 0.4 g of sodium hydroxide in 100 mL of dehydrated alcohol, and when solution is complete, standardize the alcoholic solution as follows. Pipet 25.0 mL of 0.1 N hydrochloric acid VS into a suitable container. Dilute with 50 mL of water, add 2 drops of Mixed indicator, and titrate with the alcoholic sodium hydroxide solution to the first appearance of a green color. Calculate the normality. [note—Store in tightly stoppered bottles.]

Test preparation— Prepare as directed in the Assay, except to draw 1.0 mL instead of 0.5 mL of Isoflurophate into the bulb.

Procedure— Carefully place the bulb containing the Test preparation in a 250-mL flask containing 20 mL of dehydrated alcohol. Break the bulb as directed in the Procedure under Ionic fluorine. Rinse the glass tube with 30 mL of dehydrated alcohol, and titrate immediately with 0.1 N Sodium hydroxide in dehydrated alcohol, using the Mixed indicator, to the appearance of the first green color. Each mL of 0.1 N Sodium hydroxide in dehydrated alcohol is equivalent to 100.8 µg of hydrogen ion (acidity). The limit is 0.01%.

Sodium methoxide solution— Dissolve 10 g of sodium methoxide in dehydrated alcohol to make 500 mL, and mix.

Buffer solution— Dissolve 9.55 g of monochloroacetic acid and 2 g of sodium hydroxide in water to make 100 mL. If necessary, adjust by the addition, of either reagent to obtain a solution having a pH of 3.0.

Standard fluoride solution— Dissolve 2.2105 g of sodium fluoride in water to make 1000.0 mL. Each mL is equivalent to 1.00 mg of fluoride ion.

Thorium nitrate solution— Dissolve 9 g of thorium nitrate in water to make 1000.0 mL, and mix. Standardize as directed under Standard curve.

Standard curve— Into each of four 180-mL beakers pipet 50.0 mL of Sodium methoxide solution and 0.25, 0.50, 1.0, and 2.0 mL, respectively, of Standard fluoride solution. Treat each beaker in the same manner, as follows. Add 2 drops of phenolphthalein TS, and render just acid with 6 N hydrochloric acid. Add 1.0 mL of sodium alizarinsulfonate solution (1 in 2000), and add 6 N hydrochloric acid dropwise until the pink color is discharged. Dilute with water to 100 mL, and add Buffer solution (approximately 4 mL) until the pH is 3.1. Titrate with Thorium nitrate solution, while stirring constantly and rapidly, to a permanent pink color. During the titration, maintain the solution at a pH between 2.9 and 3.1 by adding small volumes of Buffer solution, if necessary, but not more than a total of 10 mL. Plot the mg of fluoride ion versus the mL of Thorium nitrate solution consumed.

Test preparation— Prepare as directed in the Assay, except to draw 1.0 mL instead of 0.5 mL of Isoflurophate into the bulb.

Procedure— Place 50.0 mL of Sodium methoxide solution in a 180-mL beaker, and add 2 drops of phenolphthalein TS. Acidify, dropwise, with 6 N hydrochloric acid. Add 1.0 mL of sodium alizarinsulfonate solution (1 in 2000), then add 6 N hydrochloric acid dropwise until the pink color disappears. Add 0.5 N sodium hydroxide until a faint pink color appears, then add 0.05 N hydrochloric acid until the pink color just disappears. Add 4 mL of the Buffer solution. Carefully place the Test preparation in the beaker with the stem of the bulb inserted in a suitable length of glass tubing. Break the bulb by pressing down on the glass tubing, making sure that the bulb is beneath the surface of the liquid and that the bottom of the beaker is properly supported so that it will not break when the bulb is broken. Wash down the glass tube with water, and dilute with water to 100 mL. Titrate immediately with the Thorium nitrate solution. Determine the mg of ionic fluorine present in the Test preparation directly from the thorium nitrate standardization curve. Not more than 0.15% of ionic fluorine is found.

Solvent— Use dry carbon disulfide, chromatographic grade.

Internal standard solution— Pipet 1.0 mL of chromatographic grade cyclohexanone into a 100-mL volumetric flask, dilute with Solvent to volume, and mix. Pipet 3.0 mL of the resulting solution into a 100-mL volumetric flask, dilute with Solvent to volume, and mix. Each mL of the Internal standard solution contains 0.30 µL of cyclohexanone.

Standard preparation— Dissolve a suitable quantity of Isoflurophate, previously subjected to the Assay, in peanut oil, and dilute quantitatively and stepwise with peanut oil to obtain a solution having a known concentration of about 0.8 mg of isoflurophate per g of solution. Transfer about 1.2 g of this Isoflurophate solution in peanut oil, accurately weighed, to a 10-mL volumetric flask, pipet 1.0 mL of Internal standard solution into the flask, dilute with Solvent to volume, and mix.

Assay preparation— Tare an unsealed, thin-walled glass bulb with a thin, long stem, having a capacity of 1 to 2 mL. Under a hood, open the Isoflurophate container and place it in a firmly based container in a suitable vacuum-filtration flask. Insert the stem of the bulb under the surface of the liquid, and insert the stopper in the filtration flask. Allow about 0.5 mL of liquid to be drawn up into the bulb, and release the vacuum. Remove the bulb from the container, wipe the stem clean, fire-seal it without loss of any glass, cool, and again weigh.

Chromatographic system (see Chromatography 621)—Under typical conditions, the gas chromatograph is equipped with a flame-ionization detector, and contains a 1.8-m × 4-mm glass column packed with 5% phase G33 on 80- to 100-mesh support S1AB, utilizing either a glass-lined sample introduction system or on-column injection. The column is maintained isothermally at a temperature between 75 and 80, and the injector port and detector block are maintained at 200 and 250, respectively; dry, oxygen-free helium is used as the carrier gas at a flow rate adjusted to obtain a cyclohexanone peak about 6 minutes after sample introduction.

Procedure— Inject 6 µL of the Standard preparation into a suitable gas chromatograph, and record the chromatogram. Measure the areas under the first (cyclohexanone) and second (isoflurophate) peaks, and record the values as AD and AS, respectively. Calculate the factor F taken by the formula: in which WS is the weight, in mg, of Isoflurophate solution in peanut oil in the Standard preparation, and C is the weight, in mg, of isoflurophate per g of Isoflurophate solution in peanut oil. Similarly inject 6 µL of the Assay preparation, and record the chromatogram. Measure the areas under the first (cyclohexanone) and second (isoflurophate) peaks, and record the values as aD and aU, respectively. Calculate the percentage of C6H14FO3P in the portion of Isoflurophate taken by the formula: in which WU is the weight, in mg, of Isoflurophate in the Assay preparation; and F is the factor as determined above.