480 A MANUAL OF PHYSIOLOGY 



of the indigo-forming substance, the faint blue liquid should be shaken 

 up with a few drops of chloroform. The latter takes up the colour, 

 which is thus rendered more evident. If there is difficulty in obtain- 

 ing the reaction, the urine may first be decolourized by precipitating it 

 with acetate of lead, avoiding "excess. The precipitate is filtered off, 

 and the test then applied to the clear filtrate. The skatoxyl of urine 

 can also be oxidized to indigo, but it is present in far smaller amount. 

 The average quantity of indigo obtained from a litre of horse's urine 

 is about 150 milligrammes ; from a litre of human urine, not a 

 twentieth of that amount. 



For comparative quantitative determinations the method of 

 Folin may be used. One-hundredth of the twenty-four hours' urine 

 is taken. In this the indigo is developed by the addition of an equal 

 volume of Obermayer's reagent (p. 479), and the indigo-blue dissolved 

 by means of 5 c.c. of chloroform. The chloroform solution is then 

 compared coiorimetrically with Fehling's solution. This can be 

 done by putting the indigo solution and 5 c.c. of the Fehling's 

 solution respectively into small test-tubes of equal calibre, and 

 comparing the depth of tint. If the Fehling's solution is stronger 

 than the indigo solution, run water into the former from a pipette, 

 graduated in tenths of a c.c., shaking up after each addition, till 

 equality of tint has been reached. If the indigo solution has a 

 stronger blue colour than the Fehling's solution, dilute a measured 

 amount of it first of all with such a quantity of chloroform (say an 

 equal volume) as will make its tint distinctly weaker than that of the 

 Fehling's solution. Then dilute the Fehling's solution with water, as 

 before, till the tint is the same. From the amount of dilution the 

 quantity of indigo can be expressed in arbitrary units, taking Fehling's 

 solution as 100. Thus, if i c.c. of water must be added to the 5 c.c. ot 



Fehling's solution, the indican can be expressed as =83. 



The comparison can be made more accurately by a colorimeter, if 

 one is available. To determine the absolute amount of indigo 

 obtained, comparison must be made with a standard solution of 

 indigo. 



7. Urea (i) Decomposition of Urea. Heated dry in a test-tube, 

 it gives off ammonia. The residue contains biuret, which, when 

 dissolved in water, gives a rose colour with a trace of cupric sulphate 

 and excess of sodium hydroxide (or of the hydroxides of certain other 

 metals of the alkalies and alkaline earths, p. 7). Some proteins 

 peptones and albumoses in the presence of the same reagents, give 

 a similar colour, the so-called biuret reaction. 



(2) Quantitative Estimation The Hypobromite Method. The urea 

 is split up by sodium hypobromite (p. 440), and the carbon dioxide 

 being absorbed by the excess of sodium hydroxide used in preparing 

 the hypobromite, the nitrogen is collected over water in an inverted 

 burette. It is easy to calculate the weight of urea corresponding 

 to a given volume of nitrogen measured at a given temperature and 

 pressure. The nitrogen of urea is f, or ^ of the whole molecular 

 weight. Now, i c.c. of N weighs, at 760 millimetres of mercury 

 and o C., 0*00125 gramme. Therefore, i c.c. of N corresponds to 

 0-00125 x- 1 /= ' 268 gramme urea. Suppose, now, that i c.c. of 

 urine was found to yield 10 c.c. of N measured at 17 C. and 750 milli- 

 metres barometric pressure. Since a gas expands ..i., part of its 

 volume at o for every degree above o^we must correct the apparent 



