39 PHYSIOLOGICAL CHEMISTRY 



the filtrate to a small volume and stand it aside for crystallization. Examine the 

 crystals under the microscope and compare them with those in Fig. 125, page 389. 

 This method is not as satisfactory as Roaf's method (see below). 



(b) Roaf's Method. Place 500 c.c. of urine of the horse or cow in a cas- 

 serole or precipitating jar and add an equal volume of a saturated solution of 

 ammonium sulphate 1 and 7.5 c.c. of concentrated sulphuric acid. Permit the 

 mixture to stand for 24 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. 125, page 389. 



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 technic, 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 crystal- 

 lization 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 hippuric acid pre- 

 pared in the above experiment (see page 375). 



3. Solubility. Test the solubility of hippuric acid in hot and cold water and 

 in alcohol, and ether. 



4. Formation of Nitro-Benzene (Liicke's Reaction). To a little hippuric acid 

 in a small porcelain dish add 1-2 c.c. of concentrated HNO 3 and evaporate to dry- 

 ness on a water-bath. Transfer the residue to a dry test-tube, apply heat, and 

 note the odor of the artificial oil of bitter almonds (nitrobenzene). 



5. Spiro's Reaction. 2 Warm the hippuric acid with acetic anhydride, anhy- 

 drous sodium acetate and benzaldehyde. After one-half hour permit the solution 

 to cool. Note the formation of crystals of the lactimide of phenylaminocinnamic 

 acid, melting-point 165-166. Heat some of the crystals with concentrated sodium 

 hydroxide until ammonia is given off. Acidify and note the formation of phenyl- 

 pyroracemic acid (C 6 H 6 CH 2 .CO.COOH). This acid is soluble in ether. 



6. Sublimation. Place a few crystals of hippuric acid in a dry test-tube and 

 apply heat. The crystals are reduced to an oily fluid which solidifies in a crystal- 

 line mass upon cooling. When stronger heat is applied the liquid assumes a 

 red color and finally yields a sublimate of benzoic acid and the odor of hydro- 

 cyanic acid. 



7. Formation of Ferric Salt. Render a small amount of a solution of hip- 

 puric acid neutral with dilute potassium hydroxide. Now add 1-3 drops of 

 neutral ferric chloride solution and note the formation of the ferric salt of hip- 

 puric acid as a cream-colored precipitate. 



8. Synthesis of Hippuric Acid. To some of the glycocoll prepared in the last 

 experiment or furnished by the instructor, add a little water, about i c.c. of benzoyl 

 chloride and render alkaline with potassium hydroxide solution. Stopper the tube 

 and shake it until no more heat is evolved. Now render strongly alkaline with 

 potassium hydroxide and shake the mixture until no odor of benzoyl chloride can be 

 detected. Cool, acidify with hydrochloric acid, add an equal volume of petroleum 

 ether, and shake thoroughly to remove the benzoic acid. (Evaporate this solution 

 and note the crystals of benzoic acid. Compare them with those shown in Fig. 127, 

 page 396.) Decant the ethereal solution into a porcelain dish and extract again 



1 125 grams of solid ammonium sulphate may be substituted. 

 2 Spiro: Zeit. physiol. Chem., 28, 174, 1899. 



