Use method E for substances containing 2 mg or less of nitrogen.
Method A
Weigh accurately the quantity of the substance under examination specified in the individual monograph or a quantity equivalent to about 35 mg of nitrogen into a 200-ml long-necked flask and add 3 g of anhydrous sodium sulfate, 0.3 g of nitrogen-free mercuric oxide and 20 ml of nitrogen free sulphuric acid, unless otherwise specified in the monograph. Heat the mixture over a small flame until colorless and boil gently for a further 2 hours, unless otherwise directed in the monograph, care being taken to prevent the upper part of the flask from getting overheated. Cool, cautiously dilute to about 75 ml with water and add a piece of granulated zinc and a solution containing 1.5 g of sodium hydroxide per ml of the sulphuric acid used and 2 g of sodium thiosulphate in 25 ml of water.Method B
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 sulfate and 0.3 g of nitrogen free 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.Method C
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 longnecked flask and add 1 g of a powdered mixture of 10 parts of anhydrous sodium sulfate or potassium sulfate and 1 part of cupric sulfate. Add 10 ml of nitrogen-free sulphuric acid, mix, and carefully add 1 ml of hydrogen peroxide solution (l00 vol) carefully down the wall of the flask. Heat until the solution becomes clear green in color or almost colorless for 30 minutes. Cool, carefully add 20 ml of water, cool again and connect the flask to a distillation apparatus.Add 50 ml of 10 M sodium hydroxide and distill immediately by passing steam through the flask. Collect the distillate in 25.0 ml of 0.1 M hydrochloric acid and titrate the excess of acid with 0.1 M sodium hydroxide using methyl red-methylene blue solution as indicator. Repeat the operation using 25 mg of anhydrous dextrose in place of the substance under examination. The difference between the titrations represents the ammonia liberated by the substance under examination. 1 ml of 0.1 M hydrochloric acid is equivalent to 0.001401 g of N.
Method D
(When nitrates and nitrites are present)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 sulfate or potassium sulfate and 1 part of cupric sulfate, mix and carefully add 1 ml of hydrogen peroxide solution (100 vol) down the wall of the flask. Complete Method C beginning with the words "Heat until the solution ....". 1 ml of 0.1 M hydrochloric acid is equivalent to 0.001401 g of N.
Method E
Apparatus
A unit of the type generally known as semi-micro Kjeldahl apparatus.Method
Weigh accurately a quantity of the substance under examination equivalent to about 2 mg of nitrogen into the digestion flask of the apparatus. Add 1 g of a powdered mixture of 10 parts of anhydrous sodium sulfate or potassium sulfate and 1 part of cupric sulfate and wash down any adhering material from the neck of the flask with water. Add 7 ml of nitrogen-free sulphuric acid and 1 ml of hydrogen peroxide solution (100 vol) carefully down the wall of the flask. (Do not add hydrogen peroxide during the digestion). Heat until the solution has a clear blue color and the sides of the flask are free from carbonaceous matter. Cool, add carefully 20 ml of water, cool the solution and arrange for steam distillation. Add through the funnel 30 ml of 10 M sodium hydroxide, rinse the funnel with 10 ml of water, tightly close the apparatus and begin the distillation with steam immediately.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.
Method F
(Determination of Protein in Blood Products)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 percent v/v of nitrogen-free sulphuric acid, 4.5 percent w/v of potassium sulfate and 0.5 percent w/v of copper (II) sulfate, 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 percent 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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