Phosphorus method
Test solution
Use a volumetric flask with a suitable volume for preparation of a solution containing about 5 mg per millilitre of dry polysaccharide.
Transfer the contents of a container quantitatively to the flask and dilute to volume with water R.
Dilute the solution so that the volume used in the test (1 mL) contains about 6 µg of phosphorus.
Transfer 1.0
mL of the solution to a 10 mL ignition tube.
Reference solutions
Dissolve 0.2194 g of potassium dihydrogen phosphate R in 500 mL of water R to give a solution containing the equivalent of 0.1 mg of phosphorus per millilitre.
Dilute 5.0 mL of the solution to 100.0 mL
with water R. Introduce 0.5 mL, 1.0 mL
and 2.0 mL of the dilute solution into 3 ignition tubes.
Prepare a blank solution using 2.0 mL of
water R in an ignition tube.
To all the tubes add 0.2 mL of sulfuric acid R and heat in an oil bath at 120 °C for 1 h and then at 160 °C until white fumes appear (about 1 h). Add 0.1 mL of perchloric acid R and heat at 160 °C until the solution is decolorised (about 90 min).
Cool and add to each tube 4 mL of water R and 4 mL of ammonium molybdate reagent R.
Heat in a water-bath at 37 °C for 90 min and cool.
Adjust the volume to 10.0 mL with water R. The blue colour is stable for several hours.
Measurements
Measure the absorbance of each solution at 760 nm using the blank as the compensation liquid.
Draw a calibration curve from the absorbances of the 6 reference solutions and the corresponding protein contents and read from the curve the content of protein in the test solution.
References
Ph. Eur. method 2.5.18