494 PHYSIOLOGICAL CHEMISTRY 



2. Benedict's Method. 1 Principle. The urea is decomposed by 

 heating with a mixture of potassium bisulphate and zinc sulphate. The 

 fact that the hydrolyzing agent is a salt and that the digestion takes 

 place in the practical absence of water seem to insure less decomposition 

 of substances other than urea. The ammonia formed is distilled off 

 and determined in the usual manner. 



Procedure. Five c.c. of urine are introduced into a rather wide Jena glass 

 test-tube, about 3 grams of potassium bisulphate and 1-2 grams of zinc sulphate 2 

 added, a small quantity of powdered pumice and a bit of paraffin are introduced 

 and the mixture boiled almost to dryness either over a free flame or by immersion 

 in a sulphuric acid bath at about 130. The tubes are then weighted (a screw 

 clamp is convenient) and immersed for three-fourths of their length in a bath of 

 sulphuric acid at a temperature of 162-165 (not lower) for one hour. 



The contents of the tube are then washed into an 800 c.c. Kjeldahl distillation 

 flask, diluted to about 400 c.c. with water, made alkaline by the addition of 15-20 

 c.c. of 10 per cent KOH (or 25 c.c. 15 per cent Na 2 CO 3 ) and distilled as usual in the 

 Kjeldahl method (page 483 ) . The value obtained must be corrected for ammonia 

 by a separate determination of the latter. 



Welker 3 has suggested an electrical bath for use in the first part of this method. 



3. Microchemical Method of Folin and Pettibone. 4 Principle 

 The urine is heated with potassium acetate and acetic acid to hydro- 

 lyze the urea. The desired temperature is maintained with the aid 

 of a temperature indicator. The ammonia formed is aspirated off 

 and determined colorimetrically with Nessler-Winkler reagent. 



Procedure. Dilute the urine so that i c.c. contains 0.75-1.5 mg. of urea 

 nitrogen. Generally dilutions of i : 20 or i : 10, depending on the concentration, 

 are satisfactory. By means of an Ostwald pipette (see page 487) introduce i c.c. 

 of the diluted urine into a large dry Jena test-tube (20-25 mm- by 200 mm.) which 

 already contains 7 grams of dry ammonia-free potassium acetate 5 (free from 

 lumps), i c.c. of 50 per cent acetic acid, a small sand pebble or a little powdered zinc 

 (not zinc dust) to prevent bumping during boiling and a temperature indicator. 6 



1 Benedict: Jour. Biol. Ghent. , 8, 405, 1911. 



2 An excess of zinc salt is to be avoided as too large quantity tends to cause slight frothing 

 during the final distillation. 



3 Welker: Biochem. Bull., i, 439, 191:2. 



4 Folin and Pettibone: Jour. Biol. Chem., n, 513, 1912. 



6 A satisfactory preparation containing less than i per cent of moisture and free from 

 ammonia may be obtained from J. T. Baker Chemical Co., Phillipsburg, N. J. 



6 "This temperature indicator consists of powdered chloride-iodide of mercury (HglCl) 

 inclosed in a sealed glass bulb not over i mm. in diameter. This salt is bright red at ordi- 

 nary temperatures. At n8C. it turns yellow and melts to a clear dark red liquid at i55C. 

 It solidifies again at about i48C. and resumes its red color gradually only in the course of 

 about 24 hours. The melting-point temperature, i53C., is fortunately a temperature 

 very readily obtained and maintained by means of potassium acetate and as the acetate 

 begins to cake and solidify at i6o-i6iC., there is no danger in this combination of having 

 either too high or too low a temperature without its being unmistakably apparent. 



The HglCl may be prepared by heating, in a dry state, intimately mixed mercuric 

 chloride and mercuric iodide in molecular proportions at i5o-i6oC. for 6-8 hours. At the 

 end of the heating the product should be powdered and used as it is for it cannot be purified 



by the use of solvents. It should be kept dry until sealed up as indicated." These tern* 

 perature indicators may be obtained ready prepared in tube 



New York. 



>erature indicators may be obtained ready prepared in tubes from Eimer and Amend 



