URINE 537 



2. Determination by Difference. Method of Plimmer and Skelton. 1 Allan- 

 toin is transformed into urea and determined as such by the acid-magnesium 

 chloride method of Folin for urea in urine. 2 Urease, however, has no effect upon 

 allantoin. Therefore, determine the urea + allantoin of the urine according to Fo- 

 lin's procedure, and also determine the true urea according to the urease method 

 (see Urea). The difference between the results thus obtained represents allantoin. 

 If sugar is present it must be removed before applying Folin's procedure. 



Hippuric Acid 



i. Method of Folin and Flanders. 3 Principle. The hippuric acid 

 is hydrolyzed to benzoic acid in alkaline solution and then the solution 

 is boiled with strong nitric acid to remove pigments and emulsifying 

 substances. The benzoic acid is extracted with chloroform and ti- 

 trated with sodium ethylate. 



Procedure. Measure 100 c.c. of urine into a porcelain evaporating dish by 

 means of a pipette. Add 10 c.c. of 5 per cent NaOH and evaporate to dryness 

 on the steam-bath. Transfer the residue to a 500 c.c. Kjeldahl flask by means of 

 25 c.c. of water and 25 c.c. of concentrated HNOs. Add 0.2 gram of copper 

 nitrate, a couple of pebbles or glass pearls and boil very gently for four and one- 

 half hours over a micro-burner. Fit the necks of the flasks with condensers of 

 the Hopkins type made from large test-tubes fitted with two-hole rubber stoppers, 

 the inlet tubes extending near the bottom of the test-tubes while the outlet tube 

 is shorter. These condensers should fit rather loosely. A good current of water 

 flowing through the condensers prevents loss of benzoic acid or change in con- 

 centration of the nitric acid. 



After cooling, rinse the condensers down with 25 c.c. of water and transfer the 

 contents of the flask to a 500 c.c. separatory funnel, with the aid of 25 c.c. more 

 of water. The total volume of the solution is now 100 c.c. Add to the solution 

 sufficient ammonium sulphate to just saturate it (about 55 grams). Make four 

 extractions with freely washed chloroform, using 50, 35, 25, and 25 c.c. portions. 

 The first two portions may be used to further rinse out the Kjeldahl flask. 



Collect the successive portions of chloroform in another separatory funnel. 

 Add to the combined extracts 100 c.c. of a saturated solution of pure sodium chlo- 

 ride, to each liter of which has been added 0.5 c.c. of concentrated HC1. Shake 

 well, draw the chloroform into a dry 500 c.c. Erlenmeyer flask and titrate with 

 N/io sodium alcoholate, 4 using 4 or 5 drops of phenolphthalein as an indicator. 

 The first distinct end point should be taken, although it may fade on standing a 

 short time. 



Calculation. Multiply the number of cubic centimeters of alcoholate used by 

 the factor for hippuric acid as determined by standardization to obtain the 

 amount of hippuric acid in the 100 c.c. of urine used. One c.c. of exactly N/io 



1 Plimmer and Skelton: Bioch. J., 8, 70 and 641, 1914. 

 1 Mathews Physiological Chemistry, 2d. Ed., p. 953. 



3 Folin and Flanders: Jour. Biol. Chem., n, 257, 1912. 



4 The sodium alcoholate is made by dissolving 2.3 grams of cleaned metallic sodium in i 

 liter of absolute alcohol. It is advisable that it be slightly weaker rather than stronger 

 than tenth-normal. It may be standardized against pure benzoic acid in washed chloro- 

 form. It may also be standardized against N/io HC1 provided the alcoholate solution 

 contains not more than traces of carbonate. 



