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6.7 DETERMINATION OF NITROGEN​

      Some alkaloids and other nitrogen-containing organic compounds fail to yield all of their nitrogen upon digestion with sulfuric acid therefore these methods cannot be used for the determination of nitrogen in all organic compounds. 

Method I

      NITRATES AND NITRITES ABSENT Place about 1 g of the substance, accurately weighed, in a 500-ml Kjeldahl flask of hard borosilicate glass. The material to be tested, if solid or semisolid, may be wrapped in a sheet of nitrogen-free filter paper for convenience in transferring it to the flask. Add 10 g of powdered potassium sulfate or anhydrous sodium sulfate, 500 mg of powdered copper(II) sulfate, and 20 ml of sulfuric acid. Incline the flask at an angle of about 45º, and gently heat the mixture, keeping the temperature below the boiling point until frothing has ceased. Increase the heat until the acid boils briskly, and continue the heating until the solution has been clear green in colour or almost colourless for 30 minutes. Allow to cool, add 150 ml of water, mix the contents of the flask, and again cool. Add cautiously 100 ml of a 40 per cent w/v solution of sodium hydroxide, in such a manner as to cause the solution to flow down the inner side of the flask to form a layer under the acid solution. Immediately add a few pieces of granulated zinc, and without delay connect the flask to a Kjeldahl connecting bulb (trap), previously attached to a condenser, the delivery tube from which dips beneath the surface of 100 ml of a 4 per cent w/v solution of boric acid contained in a conical flask or a wide-mouth bottle of about 500-ml capacity. Mix the contents of the Kjeldahl flask by gentle rotation, and distill until about four-fifths of the contents of the flask has distilled over. Titrate with 0.25 M sulfuric acid VS, determining the end-point potentiometrically. Perform a blank determination, and make any necessary correction. Each ml of 0.25 M sulfuric acid is equivalent to 7.003 mg of N.

      When the nitrogen content of the substance is known to be low, the 0.25 M sulfuric acid VS may be replaced by 0.05 M sulfuric acid VS. Each ml of 0.05 M sulfuric acid is equivalent to 1.401 mg of N.

      NITRATES AND NITRITES PRESENT Place a quantity of the substance, accurately weighed, corresponding to about 150 mg of N, in a 500-ml Kjeldahl flask of hard borosilicate glass, and add 25 ml of sulfuric acid in which 1 g of salicylic acid previously has been dissolved. Mix the contents of the flask, and allow the mixture to stand for 30 minutes with frequent shaking. To the mixture add 5 g of powdered sodium thiosulfate, again mix, and add 500 mg of powdered copper(II) sulfate. Proceed as directed under Nitrates and Nitrites Absent, beginning with “Incline the flask at an angle of about 45º.”

      When nitrogen content of the substance is known to exceed 10 per cent, 500 mg to 1 g of benzoic acid may be added, prior to digestion, to facilitate the decomposition of the substance.

Method II (Determination of Protein in Blood Products)

      For dried blood products prepare a solution of the preparation as directed in the monograph.

      To an accurately measured volume of the preparation being examined expected to contain about 100 mg of protein add sufficient saline TS to produce 20.0 ml. To 2.0 ml of the resulting solution, in a 75-ml boiling tube, add 2 ml of a solution containing 75 per cent v/v of nitrogen-free sulfuric acid, 4.5 per cent w/v of potassium sulfate and 0.5 per cent w/v of copper(II) sulfate, mix and loosely stopper the tube. Heat gradually to boiling, boil vigorously for one and a half hours and cool. If the solution is not clear, add 0.25 ml of hydrogen peroxide TS (20 volumes), continue heating until a clear solution is produced and cool. During heating, caution should be taken to ensure that the upper part of the tube is not overheated.

      Transfer the solution to a distillation apparatus, with the aid of three 3-ml portions of water. Add 10 ml of 10 M 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 1 minute. Titrate with 0.02 M hydrochloric acid VS using methyl red-methylene blue TS as indicator (V₁ ml).

      To a further volume of the preparation being examined, or of the solution prepared from it, expected to contain about 100 mg of protein, add 12 ml of saline TS, 2 ml of a 7.5 per cent w/v solution of sodium molybdate and 2 ml of mixture of 1 volume of nitrogen-free sulfuric acid and 30 volumes of water. Shake, allow to stand for 15 minutes, add sufficient water to produce 20.0 ml, shake again, and centrifuge. 

      Using 2 ml of the clear supernatant liquid thus obtained repeat the procedure described above beginning with “in a 75-ml boiling tube...” (V₂ ml). Calculate the protein content in mg per ml of the preparation being examined, using the expression 6.25 × 0.280 (V₁ – V₂) and taking into account the initial dilution.

Method III (Lowry Method for Determination of Protein)

      Dilute the sample, if necessary, to obtain a solution containing 20 to 120 μg/ml of protein. Pipette 1 ml of the resulting solution into a centrifuge tube, add 1 ml of a 10 per cent w/v solution of trichloroacetic acid and heat for 15 minutes in a water-bath. Cool and centrifuge for 20 minutes at over 1400 × g. Add 2 ml of a 5 per cent w/v solution of trichloroacetic acid to the resulting precipitate, shake well and centrifuge. Decant the supernatant liquid and dissolve the precipitate in 2.5 ml of alkaline-copper TS, shake well and allow to stand for at least 10 minutes. To the solution, add 2.5 ml of water and 0.5 ml of sodium molybdotungstophosphate TS and allow to stand at 37º for 30 minutes. Measure the absorbance of the resulting solution at the maximum at about 750 nm (Appendix 2.2) against the reagent blank. Calculate the protein content from the calibration curve of standard solutions, containing 50 μg per ml, 100 μg per ml and 150 μg per ml of albumin simultaneously treated in the same manner as the sample.

      Reagent

      ALBUMIN Use a suitable grade of bovine serum albumin for protein standard.

      ALKALINE-COPPER TS Mix 0.5 ml of a 2 per cent w/v solution of copper(II) sulfate with 0.5 ml of a 4 per cent w/v solution of sodium tartrate, add 50 ml of a 4 per cent w/v solution of sodium hydrogencarbonate in 0.2 M sodium hydroxide, and shake throughly.

      CITRATE BUFFER pH 5.0, 0.75 M Dissolve 223.6 g of trisodium citrate in sufficient water to produce 1000 ml and adjust the pH with hydrochloric acid.

      CITRATE BUFFER pH 5.0, 0.375 M Mix equal volumes of 0.75 M citrate buffer pH 5.0 and water and adjust the pH, if necessary, with hydrochloric acid.

      The activity (potency) of antibiotics may be demonstrated under suitable conditions by their inhibitory effect on micro-organisms. A reduction in antimicrobial activity also will reveal subtle changes not demonstrable by chemical methods. Accordingly, microbial or biological assays remain generally the standard for resolving doubt with respect to possible loss of activity.