URINE 567 



cipitate of benzidine suphate being filtered off and the sulphuric 

 acid of the compound titrated with N/io KOH using phenolphthalein 

 as an indicator. This is possible because the benzidine is a very weak 

 base and its sulphate readily dissociates. It is necessary that excess 

 of HC1 be avoided in the precipitation process. 



Procedure. (a) Inorganic Sulphates. Preparation of the benzidine solu- 

 tion. Rub 4 grams of benzidine (Kahlbaum) into a fine paste with about 10 

 c.c. of water and transfer to a 2 -liter flask with the aid of about 500 c.c. of water. 

 Add 5 c.c, of concentrated HC1 (sp. gr. 1.19) and make up to 2 liters with distilled 

 water. One hundred and fifty c.c. of this solution, which keeps indefinitely, 

 are sufficient to precipitate o.i gram H 2 SO 4 . 



Measure 25 c.c. of urine into a 250 c.c. Erlenmeyer flask and acidify with 

 dilute hydrochloric acid (i : 4) until the reaction is distinctly acid to Congo red 

 paper. Usually 1-2 c.c. of dilute acid are required. One hundred c.c. of the 

 benzidine solution, as prepared above, are then run in and the precipitate, 

 which forms in a few seconds, allowed to settle^for ten minutes. Filter with 

 suction and wash the precipitate with 10-20 c.c. of water saturated with benzidine 

 sulphate. 1 Transfer the precipitate and filter paper to the original precipitation 

 flask with about 50 c.c. of water and titrate hot with N/io KOH, after first 

 adding a few drops of saturated alcoholic solution of phenolphthalein. 



Calculation. One c.c. of N/io KOH corresponds to 4.9 mg. H 2 SO 4 or 4.0 mg. 

 of SO 3 . Multiply the number of cubic centimeters of N/io KOH required by 

 4.9 and by 4 to get the amount of H 2 SO4 in 100 c.c. of the urine analyzed. 



(b) Total Sulphates (Inorganic and Ethereal). Measure 25 c.c. of urine 

 into an Erlenmeyer flask, add 2-2.5 c.c. of dilute HC1 (i : 4) and 20 c.c. of water 

 and boil for 15-20 minutes. The ethereal sulphates are hydrolized. 2 Allow the 

 solution to cool and then precipitate the sulphate with benzidine as in the deter- 

 mination of inorganic sulphates. The titration and calculation are also carried 

 out hi the same way. 



(c) Ethereal Sulphates. Determine the total sulphates and inorganic sul- 

 phates as indicated above. Subtract the amount of inorganic sulphate from 

 that of the total sulphate and obtain the amount of ethereal sulphate present. 



(d) Total Sulphur. According to Rosenheim and Drummond 3 the benzidine 

 method may be employed for the estimation of total sulphur in the solution ob- 

 tained on the oxidation of urine by the Wolf-Osterberg 4 modification of Bene- 

 dict's method. This modification involves the use of larger quantities of urine 

 than the Benedict method or a reduction in accuracy and hence probably has 

 no advantages over Benedict's original procedure. 



1 In order to obtain accurate results it is most important that the precipitate should be 

 finely suspended in water before titration and this again entails certain precautions during 

 filtration so as to prevent the caking together of the precipitate. The authors use a funnel 

 of 6 



or with 



2 A larger amount of HC1 may be used (20 c.c. of the dilute acid) if desired. In this 

 case it is necessary to neutralize the solution carefully after boiling and again add dilute 

 HC1 until the reaction is acid to Congo red. 



3 Rosenheim and Drummond: Bioch. Jour., 8, 143, 1914* 



4 Wolf and Osterberg: Bioch. ZeiL, 29, 429, 1910. 



