PRACTICAL EXERCISES 511 



for a minute or two ; a bluish colour appears if, as is generally the case, 

 indoxyl is present, indigo (C 16 H 10 N 2 O 2 ) being formed by the oxidizing 

 action of the ferric chloride on the indoxyl, the compound of which 

 with sulphuric acid has been broken up by the hydrochloric acid. The 

 urine must be free from albumin. In performing the test in human 

 urine, which contains a smaller quantity of the indigo-forming sub- 

 stance, 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 obtaining the reaction, the urine 

 may first be decolorized 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. 510), and the indigo-blue dissolved by means 

 of 5 c.c. of chloroform. The chloroform solution is then compared 

 colorimetrically 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. of 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. 8). Some proteins peptones and 

 albumo'ses in the presence of the same reagents, give a similar colour, 

 the so-called biuret reaction. 



(2) Quantitative Estimation Folin s Method. Put 3 c.c. of urine, 

 20 grammes of magnesium chloride, and 2 c.c. of concentrated hydro- 

 chloric acid into an Erlenmeyer flask ol 200 c.c. capacity fitted with a 

 short backflow tube (200 mm. long and 10 mm. in diameter). Add a 

 small piece of paraffin to prevent foaming. Boil briskly, and then 

 continue boiling moderately for forty-five to sixty minutes. Now 

 cautiously dilute the mixture with water and wash it into a litre flask. 

 Add about 7 c.c. of a 20 p2r cent, solution of sodium hydroxide, and 

 distil off into decinormal acid. Usually about 350 c.c. of water should 



