55 PHYSIOLOGICAL CHEMISTRY 



To the clear solution add 5 c.c. of very dilute alcohol (about 18-20 per cent) and 

 continue the boiling for a few minutes. The alcohol is added to remove the 

 chlorine which was formed when the solution was acidified. Add 10 c.c. of a 

 10 per cent solution of barium chloride, slowly, drop by drop, 1 to the liquid. 

 Allow the precipitated solution to stand in the cold two days and then filter and 

 continue the manipulation according to the directions given under Total Sul- 

 phates, page 546. 



Calculation. Make the calculation according to directions given under Total 

 Sulphates, page 546. Calculate the quantity of sulphur, expressed as SO 3 or S, 

 present hi the twenty-four-hour urine specimen. 



Interpretation. See page 549. 



(b) Volumetric Procedures 



6. Volumetric Determination of Ethereal and Inorganic Sulphates. 

 Method of Roseriheim and Drummond. 2 Principle. The sulphates 

 of the urine are precipitated by means of benzidine solution, the pre- 

 cipitate of.benzidine sulphate 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 SO4. 



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. 3 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. 



1 See note (3) at the bottom of p. 546. 



2 Rosenheim and Drummond: Biochem. J., 8, 143, 1914. 



3 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 cm. diameter and a perforated porcelain plate (5-7 mm.) covered either with paper pulp 

 or with a well-fitting filter paper. Do not allow the precipitate to be sucked dry on the fil- 

 ter. The final filtrate should show no acid reaction to Congo red. 



