URINE 521 



about 25 grams of gypsum (CaSO 4 , 2H 2 0) and rub up with a pestle and stir with a 

 spatula until a uniform mixture, results. Dry the powder thus produced 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 "extrac- 

 tion shell" and extracted in a Soxhlet apparatus in the usual way (see page 326). 

 The extraction medium is ethyl acetate and the flask containing the acetate should 

 be strongly heated over a sand-bath 1 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 2 

 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 com- 

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

 following volumetric or gravimetric procedure: 



1. 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 

 acetate 3 over a free flame. After practically all of the ethyl acetate has been dis- 

 tilled off, the nitrogen in the remaining solution should be determined by means of 

 the Kjeldahl method (see page 483). 



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, remembering 

 that i c.c. of N/io sulphuric acid is equivalent to 0.0179 gram hippuric acid. 



Interpretation. See page 520. 



2. Gravimetric Determination. The urea-free ethyl acetate solution contained 

 in the separatory funnel, after washing with sodium chloride solution, as described 

 under Preliminary Procedure, page 520, is washed with 5 c.c. of distilled water to 

 remove the major portion of the sodium chloride. Transfer the solution from the 

 separatory funnel to a round-bottomed flask and subject it to a steam distillation 

 in the usual way. A slow current of steam should be used while the ethyl acetate 

 is being distilled off and later a more rapid current may be employed. The dis- 

 tillation should be continued for 20 minutes. Now add about o.i gram of charcoal 

 to the aqueous solution which is heated to boiling and filtered hot. Evaporate the 

 solution in a weighed Jena glass dish on a water-bath until the volume of the solu- 

 tion is reduced to about 3 c.c. Stand the dish in a warm place until evaporation 

 is complete and a crystalline residue remains. Wash the residue, in turn, with 2 c.c. 

 of dry ether and i c.c. of water, dry it in an air-bath at iooC. and weigh. If it is 

 so desired the residue may be recrystallized from a little hot water and the melt- 

 ing-point determined. Pure hippuric acid melts at i87C. Contamination with 

 phenaceturic acid may be detected both by the melting-point and the microscopical 

 characteristics. 



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

 too slow. 



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



3 The ethyl acetate after separation from the watery layer of the distillate may be dried 

 over calcium chloride and used again. 



