URINE: QUANTITATIVE ANALYSIS. 383 



VII. Hippuric Acid. 



Dakin's Methods. 1 Preliminary Procedure. Place 150 c.c. (or 

 more) of the urine under examination in a porcelain evaporating 

 dish and evaporate almost to dryness upon a water-bath. Add 

 about i gram of sodium dihydrogen phosphate, about 25 grams of 

 gypsum (CaSO 4 , 2H 2 O) and rub up with a pestle and stir with a 

 spatula until a uniform mixture results. Dry the powder thus pro- 

 duced in a water-oven for about two hours, at the end of which 

 period it should be rubbed up a second time, to remove lumps, and 

 transferred to a Schleicher and Schiill "extraction shell" and ex- 

 tracted in a Soxhlet apparatus in the usual way (see p. 410). The 

 extraction medium is ethyl acetate and the flask containing the acetate 

 should be strongly heated over a sand-bath 2 for about two hours. 

 The ethyl acetate extract is now transferred to a separatory funnel, 

 and the original flask rinsed with sufficient fresh ethyl acetate to 

 make the total volume in the separatory funnel 3 about 100 c.c. 

 Wash the ethyl acetate solution five times with a saturated solution 

 of sodium chloride, using 8 c.c. of the sodium chloride solution at 

 each extraction, shaking vigorously and removing the sodium 

 chloride extract in each case before adding fresh sodium chloride 

 solution. The sodium chloride removes the urea completely and the 

 hippuric acid is then determined in the urea-free solution by the fol- 

 lowing volumetric or gravimetric procedure : 



i. Volumetric Determination. Transfer the urea-free ethyl 

 acetate solution, prepared as described above, to a Kjeldahl flask, 

 add about 25 c.c. of water, a small piece of pumice stone to prevent 

 bumping, attach a condenser and distil off the ethyl acetate 4 over 

 a free flame. After practically all of the ethyl acetate has been dis- 

 tilled off the nitrogen in the remaining solution should be deter- 

 mined by means of the Kjeldahl method (see p. 381). 



The main source of error in this method is the fact that any 

 nitrogen present in the form of phenaceturic acid or indole acetic 

 acid is determined as hippuric acid nitrogen. The error from this 

 source is, however, usually trifling. 



Calculation. Calculate as usual for nitrogen determinations, re- 



1 Private communication to the author from Dr. H. D. Dakin. 



2 A water-bath cannot be substituted inasmuch as the resultant extraction 

 would be too slow. 



3 This ethyl acetate solution contains hippuric acid, urea and other substances. 

 *The ethyl acetate after separation from the watery layer of the distillate 



may be dried over calcium chloride and used again. 



