A: Dissolve 10 g of Pullulan in 100 mL of water by adding in small portions with stirring: a viscous solution is produced.

B: Mix 10 mL of the viscous solution obtained in Identification test A with 0.1 mL of Pullulanase test solution prepared as described below, and allow to incubate at 25 for about 20 minutes: a substantial loss of viscosity is observed.

C: To 10 mL of a solution of Pullulan (1 in 50) add 2 mL of polyethylene glycol 600: a white precipitate is formed immediately.

Test stock solution— Transfer 0.8 g of Pullulan, previously dried and accurately weighed, to a 100-mL volumetric flask, dilute with water to volume, and mix.

Test solution— To 1.0 mL of the Test stock solution add 0.1 mL of saturated potassium chloride solution, and shake vigorously with 3 mL of methyl alcohol. Centrifuge, and use the supernatant.

Blank solution— Use water.

Procedure— Transfer 0.2 mL each of the Standard solution, the Test solution, and the Blank solution to a test tube containing 5 mL of a 1 in 500 solution of anthrone in 75% (v/v) sulfuric acid, with the test tube placed in ice water. Mix each tube immediately, and then heat the test tube at 90 for 10 minutes. Remove the tube, and allow it to cool in cold running water. Perform the test with the solutions so obtained using a suitable UV/visible spectrophotometer (see Spectrophotometry and Light-Scattering 851), and using water as a blank. Determine the absorbances AT, AB, and AS, at 620 nm, for the Test solution, the Blank solution, and the Standard solution, respectively. Determine the percentage of monosaccharide, disaccharide, and oligosaccharides in the portion of dried Pullulan taken by the formula: in which 4.1 is the dilution factor for the Test solution, and 50 is the dilution factor for the Standard solution. The total content of monosaccharide, disaccharide, and oligosaccharides is not more than 10.0%, corresponding to not less than 90% of glucan, on the dried basis.