4QO PHYSIOLOGICAL CHEMISTRY 



procedure gives satisfactory results. The method should be checked 

 up carefully by each new learner of the method, using pure solutions. 

 Outside air is better than laboratory air for aspiration purposes. 

 Care is needed in using the pipettes, which should be of the Ostwald 

 type and accurate. In using them allow the pipette to drain against 

 the side of the vessel for 10 seconds and then blow out clean so that 

 nothing is left behind in the tip. The reagents used must be as free 

 as possible from ammonia and must be checked up, particularly the 

 sulphuric acid and potassium sulphate. Those who have trouble in 

 using a colorimeter may substitute titration with N/5O hydrochloric 

 acid using alizarin, or better methyl red, as an indicator. 



4. Gulick's Modification of the Folin-Farmer Colorimetric Method. 1 Prin- 

 ciple. By using small amounts of sulphuric acid and potassium sulphate it is possi- 

 ble to Nesslerize the products of the digestion directly 

 without the necessity of previous aspiration of the 

 ammonia. 



Procedure. Dilute the sample of urine to an exact 

 multiple of its original volume (about 4-10 times) with 

 acid mixture, 2 so that 0.5 c.c. of the dilution will contain 

 between 0.4 and 0.7 mg. of nitrogen. With an Ostwald 

 pipette introduce 0.5 c.c. of this mixture into the bulb 

 of a micro-oxidation flask. The type illustrated in Fig. 

 157, with a bulb capacity of about 15 c.c., is the most 

 convenient. Add also a small spherical glass bead or 

 scrap of platinum or, better, a piece of platinum wire 4-5 

 mm. long bent into a tight spiral. Agitate continually 

 while boiling off the water over a micro-burner (about 

 one minute). Set up to heat over a very small flame well 

 guarded against the wind. A luminous flame 6-7 mm. 



hi 8 h comin g from a burner tube of about 3 mm - outside 

 diameter may be used. The digestion bulb is set at a 



slant so that the mouth is directed upward and the heat applied at the tip. 

 Heat for at least one minute after the acid becomes clear white. The boiling and 

 oxidation require about 6-10 minutes. As soon as the glass is cool enough to bear 

 water add sufficient ammonia-free water to dissolve the contents, and rinse quanti- 

 tatively into' a 50 c.c. volumetric flask. 



-. Introduce into a second 50 c.c. flask 0.5 mg. of nitrogen in the form of pure 

 ammonium sulphate. Fill both flasks to about 40 c.c. with ammonia-free water. 

 Into each of the flasks then inject 5 c.c. of the modified Winkler solution 3 in a vigor- 

 ous stream from a pipette. Fill to the mark and mix thoroughly. Compare 



1 Gulick: Jour. Biol. Chem., 18, 541, 1914. 



2 Acid Oxidizing Mixture. To 125 c.c. of ammonia-free water add 40 c.c. of sulphuric 

 acid, 5 c.c. of a saturated solution of mercuric chloride and 20 gm. of potassium sulphate. 

 Then make up to 200 c.c. with ammonia-free water. 



3 Modified Winkler Solution. Dissolve 40 grams of sodium hydroxide in about 200 c.c. 

 of ammonia-free water. Mix 15 grams of mercuric iodide and 10 grams of potassium iodide 

 and dissolve in about 15 c.c. of water. Transfer with the aid of the alkali to a 500 c.c. 

 volumetric flask and make up to 500 c.c. with ammonia-free water. Transfer to an Erlen- 

 meyer flask and let stand 24 hours to settle. 



