Use method E for substances containing 2 mg or less of nitrogen.
Weigh accurately the quantity of the substance under examination specified in the monograph or a quantity equivalent to about 35 mg of nitrogen into a 200-ml long necked flask, add 20 ml of nitrogen-free sulphuric acid, unless otherwise specified in the monograph, and heat for 15 minutes. Add 3 g of anhydrous sodium sulphate and 0.3 g of nitrogenfree mercuric oxide and complete Method A, beginning at the words "Heat the mixture ...". 1 ml of 0.1 M sulphuric acid is equivalent to 0.002802 g of N.
Weigh accurately the quantity of the substance under examination specified in the monograph or a quantity equivalent to about 15 mg of nitrogen into a 200-ml long necked flask, add 10 ml of nitrogen-free sulphuric acid in which 0.2 g of salicylic acid has been previously dissolved and mix. Allow the mixture to stand for 30 minutes with frequent shaking and add 1 g of a powdered mixture of 10 parts of anhydrous sodium sulphate or potassium sulphate and 1 part of cupric sulphate, mix and carefully add 1 ml of hydrogen peroxide solution (100 vol) down the wall of the flask. Complete Method C beginning at the words "Heat until the solution ....". 1 ml of 0.1 M hydrochloric acid is equivalent to 0.001401 g of N.
Apparatus:A unit of the type generally known as semi-micro Kjeldahl apparatus.
Collect the distillate in 25.0 ml of 0.01 M sulphuric acid, continue the distillation until the distillate measures about 100 ml. Titrate the distillate with 0.01 M sodium hydroxide using methyl red-methylene blue solution as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the ammonia liberated by the substance under examination. 1 ml of 0.01 M sulphuric acid is equivalent to 0.0002802 g of N.
For dried blood products prepare a solution of the preparation as directed in the monograph. To a volume expected to contain about 0.1 g of protein add sufficient saline solution to produce 20 ml. To 2 ml of the resulting solution, in a 75-ml boiling tube, add 2 ml of a solution containing 75.0 per cent v/v of nitrogen-free sulphuric acid, 4.5 per cent w/v of potassium sulphate and 0.5 per cent w/v of copper (II) sulphate, mix and loosely stopper the tube. Heat gradually to boiling, boil vigorously for 5 hours and cool. If the solution is not clear add 0.25 ml of hydrogen peroxide solution (20 vol), continue heating until a clear solution is produced and cool. During heating, take precautions to ensure that the upper part of the tube is not overheated. Transfer the solution to a distillation apparatus using three 3-ml quantities of water, add 10 ml of 10M sodium hydroxide and distil rapidly for 4 minutes, collecting the distillate in a mixture of 5 ml of a saturated solution of boric acid and 5 ml of water and keeping the tip of the condenser below the level of the acid. Lower the collection flask so that the condenser can drain freely and continue the distillation for a further] minute. Titrate with 0.02 M hydrochloric acid using methyl red mixed solution as indicator (1 ml).
To a further volume of the preparation under examination, or of the solution prepared from it, expected to contain about 0.1 g of protein, add 12 ml of saline solution, 2 ml of a 7.5 per cent w/v solution of sodium molybdate and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid and 30 volumes of water. Shake, allow to stand for 15 minutes, add sufficient water to produce 20 ml, shake again and centrifuge. Using 2 ml of the resulting clear supernatant liquid repeat the procedure described above beginning at the words 'in a 75-ml boiling tube ... ' (V2 ml). Calculate the protein content in mg per ml of the preparation under examination, using the expression 6.25 x 0.280 (V1-V2) and taking into account the initial dilution.
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