MOLONEY— shutt] SOURCE OF BENZOIC ACID 151 



puric acid, of the urine in the several experiments being that of Steen- 

 bock.i (J. Biol. Chem. XI. 204. 1912). 



Benzoic Acid from Cow's Urine 



1. 100 cc. fresh urine, total 0-64 gram 



2. 100 cc. fresh urine evaporated to one half original 



volume, acidified with HCl and cooled to room 

 temperature and the benzoic acid content 

 determined in the precipitated hippuric acid. . . 0-35 " 



3. 100 cc. fresh urine, acidified with HCl, cooled to 



0°C and the benzoic acid determined in the 

 precipitated hippuric acid 0-35 " 



From these results it will be seen that a method for obtaining 

 benzoic acid from urine by cooling the fresh, acidified urine to 0°C is as 

 efificient as evaporating the fresh urine to one half its original volume 

 and precipitating the hippuric acid at room temperature. 



Using the above data as a basis of calculation and assuming 

 20 pounds of urine daily per 1,000 pounds weight of cow, the total 

 daily benzoic acid output would be approximately 1 pound per 8 

 cows. By acidifying and cooling to 0°C, as described above, and 

 assuming that all the urine is collected, the amount of benzoic acid 

 available daily would be approximately 1 pound per 15 cows. 



In the case of putrefied urine, i.e. urine in which the hippuric 

 acid had been hydrolyzed by bacterial action to benzoic acid and glyco- 

 coll, it was found that very little benzoic acid separated out after 

 acidification and cooling to 0°C. However, if the putrified urine is 

 first clarified with milk of lime, allowed to stand until clear and the 

 supernatant liquid poured or siphoned off, the benzoic acid readily 

 separates out on acidification and cooling, the percentage of efficiency 

 being practically that obtained in working on fresh urine. 



400 cc. fresh urine is boiled with 10 grams NaOH for 2 hours under a reflux 

 condenser in order to hydrolyze the hippuric acid to benzoic and then acidified with 

 50 per cent H2SO4. Bromine water is then added to precipitate phenols — and the 

 solution cooled and made up to 250 cc. A 50 cc aliquot is extracted with 4 portions 

 — 50 cc — 40 cc — 20 cc — 20 cc of sulphuric ether. The combined ether solution 

 is slowly dropped into a U tube through which a current of air is drawn, the U tube 

 being kept in a water-bath which is kept at a temperature of 40°C. 



The benzoic acid is then sublimed from the U tube into a tared condensing tube 

 — the condensing tube consisting of a glass tube 25 cm. long, 9 mm. bore, 25 grams in 

 weight, with 3 glass bulbs blown on it, the bulbs being filled with glass wool. 



The sublimation was carried out at a temperature below 130°C. 



