URINE. 283 



boil for a few moments and filter while hot. Concentrate the fil- 

 trate, over a burner, to a small volume. Cool the solution, acidify 

 it strongly with concentrated hydrochloric acid and stand it in a 

 cool place for 24 hours. Filter off the crystals of hippuric acid 

 which have formed and wash them with a little cold water. Re- 

 move the crystals from the paper, dissolve them in a very small 

 amount of hot water and percolate the hot solution through thor- 

 oughly washed animal charcoal, being careful to wash out the last 

 portion of the hippuric acid solution with hot water. Filter, con- 

 centrate the filtrate to a small volume and stand it aside for crys- 

 tallization. Examine the crystals under the microscope and com- 

 pare them with those in Fig. 92, page 282. This method is not as 

 satisfactory as Roaf's method (see below). 



(b) Roafs Method. Place 500 c.c. of urine of the horse or 

 cow 1 in a casserole or precipitating jar and add an equal volume 

 of a saturated solution of ammonium sulphate 2 and 7.5 c.c. of con- 

 centrated sulphuric acid. Permit the mixture to stand for twenty- 

 four hours and remove the crystals of hippuric acid by filtration. 

 Purify the crystals by recrystallization according to the directions 

 given above under First Method. Examine the crystals under the 

 microscope and compare them with those given in Fig. 92, p. 282. 



If sufficient urine is not available to permit the use of 500 c.c. 

 a smaller volume may be used inasmuch as it is possible, by the 

 above technique, to isolate hippuric acid in crystalline form from as 

 small a volume as 25-50 c.c. of herbivorous urine. The greater 

 the amount of ammonium sulphate added the more rapid the 

 crystallization until at the saturation point the crystals of hippuric 

 acid sometimes form in about ten minutes. 



2. Melting-Point. Determine the melting-point of the hip- 

 puric acid prepared in the above experiment (see p. 269). 



3. Solubility. Test the solubility of hippuric acid in the ordi- 

 nary solvents (page 23) and in alcohol, and ether. 



4. Dehn's Reaction. Introduce about 5 c.c. of the urine or the 

 solution under examination into a test-tube and add sufficient hypo- 

 bromite solution 3 to impart to the mixture a permanent yellow color. 

 In the case of urine enough hypobromite should be added to decom- 

 pose the urea. Heat the mixture to boiling and note the forma- 

 tion of an orange or brown-red precipitate if hippuric acid is pres- 



1 See note on p. 282. 



2 125 grams of solid ammonium sulphate may be substituted. 



3 See note on p. 375. 



