A: The powdered mucilage stains red with ruthenium red TS and lead acetate TS.

B: It meets the requirements of the test for Swell volume.

Internal standard solution— Transfer 5.0 mL of n-propyl alcohol into a 500-mL volumetric flask containing approximately 450 mL of water. Dilute with water to volume, insert the stopper into the flask, and mix well.

Standard stock solution— Transfer 5.0 mL of absolute alcohol at 20 ± 2 into a 500-mL volumetric flask containing approximately 450 mL of water. Dilute with water to volume, insert the stopper into the flask, and mix well.

Standard solution— Transfer 10.0 mL of the Standard stock solution and 10.0 mL of Internal standard solution into a 100-mL volumetric flask. Dilute with water to volume, insert the stopper into the flask, and mix well.

Test solution— Transfer 0.5 g of Psyllium Hemicellulose, accurately weighed, into a 150-mL conical flask. Add about 90 mL of water, insert the stopper into the flask, and stir rapidly for 3 hours using a magnetic stirrer. Add 10.0 mL of the Internal standard solution, and mix well. Pass the sample through a filter having a 0.45-µm porosity.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm × 30-m fused silica analytical column coated with 3.0-µm G43 stationary phase. A 0.53-mm × 2-m fused silica guard column may be used. The chromatograph is programmed as follows. Initially, the column temperature is equilibrated at 40 for 5 minutes. The temperature is then increased at a rate of 10 per minute to 230, and is maintained at 230 for 3 minutes. The injection port temperature is maintained at 250, and the detector is maintained at 300. The carrier gas is helium. The split flow ratio is about 10:1, and the flow rate is maintained at about 4.0 mL per minute. Inject the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 0.5 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for all the peaks. Calculate the percentage of alcohol in the portion of Psyllium Hemicellulose taken by the formula: in which C is the concentration, in mg per mL, of alcohol in the Standard stock solution; W is the weight, in mg, of Psyllium Hemicellulose taken; and RU and RS are the ratios of the peak responses of alcohol to those of n-propyl alcohol from the Test solution and the Standard solution, respectively: not more than 12.0% (w/w) is found.

Alcohol solution— Transfer 82.0 mL of alcohol to a 100-mL volumetric flask, dilute with water to volume, and mix.

Buffer solution— Dissolve 1.95 g of 2-(N-morpholino)-ethanesulfonic acid and 1.22 g of tris(hydroxymethyl)aminomethane in 170 mL of water. Adjust with 6 N sodium hydroxide to a pH of 8.2, dilute with water to 200 mL, and mix. [note—It is important to adjust the pH to 8.2 at 24. If the Buffer solution temperature is 20, adjust the pH to 8.3; if the temperature is 28, adjust the pH to 8.1. For deviations between 20 and 28, adjust by interpolation.]

Acid solution— Prepare 0.561 N hydrochloric acid by dissolving 9.35 mL of 6 N hydrochloric acid in 70 mL of water. Dilute with water to 100.0 mL, and mix.

Phosphate buffer— Prepare a pH 6.0 phosphate buffer (see Buffer Solutions under Reagents, Indicators, and Solutions).

Protease solution— Dissolve 5 mg of protease in 0.1 mL of Phosphate buffer.

Enzyme purity— To ensure the absence of undesirable enzymatic activities and the presence of desirable enzymatic activities, proceed as directed for Test preparations and Procedure using the substrates listed in the following table in place of Psyllium Hemicellulose. The enzyme preparation is suitable if more than 90% of the original weight of pectin, arabinogalactan, and -glucan is recovered; not more than 2% of the original weight of casein and corn starch is recovered; and not more than 1% of the original weight of wheat starch is recovered. [note—Test the enzyme purity of every new lot of enzyme and at 6-month intervals thereafter.]

Blank preparations— Using two 400-mL tall-form beakers, appropriately labeled, proceed as directed for Procedure without Psyllium Hemicellulose.

Test preparations— Weigh accurately, in duplicate, approximately 0.2 g of Psyllium Hemicellulose, previously milled to very fine powder. [note—Duplicates should differ by less than 1 mg in weight.] Transfer duplicate samples to appropriately labeled 400-mL, tall-form beakers, and proceed as directed for Procedure.

Procedure— Treat each preparation in the following manner. Add 40 mL of Buffer solution to the beaker. [note—For the Test preparation, stir until Psyllium Hemicellulose is completely dispersed.] Add 125 µL of heat-stable -amylase solution, and stir to ensure uniform mixing. Cover the beaker with aluminum foil, and incubate over a water bath maintained at 95 to 100 for 15 minutes, with continuous agitation. [note—Start timing once the water bath temperature reaches 95; a total time of 35 minutes is usually sufficient.] Remove the beaker from the water bath, and cool to 60. Remove the aluminum foil, scrape any ring from inside the beaker, and disperse any gels in the bottom of the beaker with a spatula. Rinse the walls of the beaker and the spatula with 10 mL of water, collecting the rinsings in the beaker. Add 500 µL of Protease solution. Cover with aluminum foil, and incubate over a water bath maintained at 60 ± 3 for 30 minutes with continuous agitation. [note—Start timing when the bath temperature reaches 60.] Remove the foil, and transfer 5 mL of Acid solution while stirring. Adjust, if necessary, with 1 N sodium hydroxide or 1 N hydrochloric acid to a pH of 4.28 ± 0.07 at 60. [note—It is important to adjust the pH to 4.28 while the solution in the beaker is maintained at 60, otherwise the pH will increase at lower temperatures.] Add 150 µL of amyloglucosidase solution while stirring. Cover with aluminum foil, and incubate over a water bath maintained at 60 ± 3 for 30 minutes with constant agitation. [note—Start timing once the water bath reaches 60.] Transfer approximately 40 mL of the beaker contents to a 50-mL centrifuge tube, and sonicate the tube contents for 3 minutes.* Centrifuge at 10,000–14,000 rpm for 10 minutes. Carefully pour the supernatant into an appropriately labeled 600-mL tared beaker. Do not disturb any pellet in the bottom of the centrifuge tube. Add the remaining sample from the original 400-mL beaker into the centrifuge tube still containing the pellet. Rinse the 400-mL beaker with 15–20 mL of water, and add the rinsing to the 50-mL centrifuge tube. Centrifuge the sample at 10,000–14,000 rpm for 10 minutes. Carefully pour the supernatant into the 600-mL beaker containing the first supernatant. Add 390 mL (measured before heating) of alcohol at 60 to the 600-mL beaker. Cover the beaker, and allow to stand for at least 1 hour to form a precipitate.