544 PHYSIOLOGICAL CHEMISTRY 



acid silver lactate solution (from 2 to 20 c.c. of a 3 per cent solution of silver 

 lactate in 3 per cent lactic acid) until no further precipitate is obtained. Add 

 a few drops of colloidal iron, shake the flask, dilute to mark with distilled water, 

 shake again, and filter the contents through a dry filter. Phenols are not pre- 

 cipitated by this procedure but are recovered quantitatively hi the filtrate. Trans- 

 fer 25 c.c. of the filtrate to a 50 c.c. volumetric flask, and add a sufficient quantity 

 of saturated sodium chloride solution, containing 10 c.c. of strong hydrochloric 

 acid per liter, to precipitate all the silver. Fill the flask to the mark with dis- 

 tilled water, mix thoroughly, and filter through a dry filter. This filtrate, which 

 contains half the phenol from the urine taken for analysis, is used for the deter- 

 mination of free and total phenols. 



Free Phenols. Place 20 c.c. of the nitrate mentioned above in a 50 c.c. 

 volumetric flask, add 5 c.c. of the phosphotungstic-phosphomolybdic acid re- 

 agent 1 and 15 c.c. of a saturated solution of sodium carbonate. Dilute to volume 

 with luke warm water (3O-35C.), mix thoroughly and after allowing to stand 

 for 20 minutes compare the deep blue color in the Duboscq colorimeter (see 

 Fig- JSS* page 486) against a standard solution of phenol (see below) similarly 

 treated. 



Total Phenols (Free and Conjugated). Place 20 c.c. of the same filtrate 

 used for the determination of free phenols in a large test-tube, add 10 drops 

 of concentrated hydrochloric acid, cover the tube with a small funnel, heat 

 rapidly to boiling over a free flame, and then place in a boiling water-bath 

 for ten minutes. This process serves to decompose the conjugated phenols. 

 At the end of the ten minutes, remove the tube, cool, and transfer the contents 

 to a 100 c.c. volumetric flask. Add 10 c.c. of the phosphotungstic-phospho- 

 molybdic reagent, 25 c.c. of saturated sodium carbonate solution, dilute to mark 

 with luke warm water (3O-35C.), mix thoroughly, allow to stand for 20 minutes, 

 and read in the Duboscq colorimeter (see page 486) against a standard solution 

 of phenol (see below). 



Standard Solution of Phenol. The standard used is a solution of pure phenol 

 in N/ioo hydrochloric acid containing i mg. of phenol in 10 c.c., standardized by 

 means of the iodometric titration. The preparation is carried out as follows: Make 

 a phenol solution in N/io hydrochloric acid, which contains approximately i mg. of 

 crystallized phenol per cubic centimeter. Transfer 25 c.c. of this solution to a 

 250 c.c. flask, add 50 c.c. of N/io sodium hydroxide, heat to 65C., add 25 c.c. of 

 N/io iodine solution, stopper the flask, and let stand at room temperature 30 or 

 40 minutes. Add 5 c.c. of concentrated hydrochloric acid and titrate the excess 

 of iodine with N/io thiosulphate solution. Each cubic centimeter of N/io iodine 

 solution corresponds to 1.567 mg. of phenol. On the basis of the result dilute the 

 phenol solution so that 10 c.c. contain i mg. of phenol. Five c.c. of this solution 

 (equivalent to 0.5 mg. of phenol), when 10 c.c. of the phosphotungstic phospho- 

 molybdic reagent and 25 c.c. of saturated sodium carbonate solution are added, and 

 the whole made up with water at about 3oC. to 100 c.c., give when set in the 

 colorimeter at 20 mm. a convenient standard. 



Calculation. The filtrate used for the determination of free and total phenols 



1 This reagent is prepared as follows: Boil together for two hours (using a reflux con- 

 denser) 100 grams of sodium tungstate, 20 grams of phosphomolybdic acid (or an equiva- 

 lent of molybdic acid), 50 c.c. of phosphoric acid (85 per cent.), and 75 c.c. of distilled 

 water. After the period of heating, cool, dilute to i liter with distilled water, and filter 

 if necessary. 



