488 PHYSIOLOGICAL CHEMISTRY 



into a volumetric flask (100 c.c.) containing about 20 c.c. of ammonia-free water 

 and 2 c.c. of N/io hydrochloric acid (see Figs. 154 and 155, page 487). The 

 air current should be only moderately rapid for the first two minutes but at the 

 end of this two-minute period the current should be run at its maximum speed 

 for an interval of eight minutes. 



Disconnect the flask, dilute the contents to about 60 c.c. with ammonia- 

 free water and dilute similarly i mg. of nitrogen in the form of ammonium sul- 

 phate 1 in a second volumetric flask. Nesslerize both solutions as nearly as 

 possible at the same time with 5 c.c. of Nessler-Winkler solution 2 diluted, imme- 

 diately before using, with about 25 c.c. of ammonia-free water to avoid turbidity. 

 Immediately fill the two flasks to the mark with ammonia-free water, mix well 

 and determine the relative intensity of the two colors by means of a Duboscq 

 colorimeter. 3 



The color of the unknown should be adjusted to that of the standard both from 

 above and below the level of the latter. The matching of the colors is ordinarily 

 very easy. It is desirable to make the readings by- diffused daylight if possible. 

 If electric light must be used, a sheet of smooth white paper should be interposed 

 between the colorimeter and the source of light. 



Calculation. The reading of the standard divided by the reading of the un- 

 known gives the nitrogen in milligrams in the volume of the urine taken. Calcu- 

 late the total nitrogen output for the 24-hour period. 



Interpretation. See page 484. 



3. Bock arid Benedict's Modification of the Folin-Farmer Procedure. 

 Bock and Benedict 4 have found distillation of the ammonia more accurate than 



not large enough to permit of the use of a two-hole rubber stopper when suction is used, 

 the ammonia should be collected in one of the Jena test-tubes previously described which 

 contains 2 c.c. of N/io hydrochloric acid and about 5 c.c. of ammonia-free water. The am- 

 monium salt is then transferred to the volumetric flask with 40-50 c.c. of water and Nes- 

 slerized as described. 



1 Care should be taken to secure the pure salt. All ammonium salts contain pyridine 

 bases which titrate like ammonia but^do not react with Nessler's reagent. Pure ammonium 

 sulphate may be prepared by decomposing a high-grade ammonium salt with sodium hy- 

 droxide and passing the liberated ammonia into pure sulphuric acid. The salt is then pre- 

 cipitated by means of alcohol, then brought into solution in water and re-precipitated by 

 alcohol. The final product should be dried in a desiccator over sulphuric acid. Dr. H. L. 

 Emerson of Boston prepares a salt which is very satisfactory for use in this method. 

 According to Bock and Benedict, Kahlbaum's "Zur Analyse" ammonium chloride is 

 satisfactory. 



2 Chem. Zeit., 1899, p. 541. The Nessler-Winkler solution has the following formula: 



Mercuric iodide 10 grams. 



Potassium iodide 5 grams. 



Sodium hydroxide 20 grams. 



Water 100 c.c. 



The mercuric iodide is rubbed up in a small porcelain mortar with water, then washed 

 into a flask and the potassium iodide added. The sodium hydroxide is dissolved in the 

 remaining water and the cooled solution added to the above mixture. The solution cleared 

 by standing is preserved in a dark bottle. 



The 25 c.c. portion of the diluted reagent should be added about one-third at a time to 

 the contents of the flask. It is very essential that the dilution of the reagent takes place 

 immediately preceding its use, inasmuch as the diluted reagent deteriorates in a few minutes as 

 is indicated by the formation of a brick-red precipitate. Fortunately the reagent does not 

 decompose in this manner in the presence of the ammonium salt. 



3 The standard may be set at any desired depth but a very satisfactory depth is 20 mm. 

 The depth should be uniform throughout any series of comparative tests. 



4 Bock and Benedict: Jour. Biol. Chem., 20, 47, 1915. 



