92 PHYSIOLOGICAL CHEMISTRY. 



and mix thoroughly. In a second tube place 5 c.c. of concentrated 

 sulphuric acid. Incline the tube containing the sulphuric acid 

 and by means of a pipette allow the albumin-glyoxylic acid 

 solution to flow carefully down the side. When stratified in this 

 manner a reddish-violet color forms at the zone of contact of the 

 two fluids. This color is due to the presence of the tryptophane 

 group. Gelatin does not respond to this test. For formula for 

 tryptophane see page 73. 



5. Biuret Test. To 2-3 c.c. of egg albumin solution in a test- 

 tube add an equal volume of concentrated potassium hydroxide solu- 

 tion, mix thoroughly, and add slowly a very dilute (2-5 drops in a 

 test-tube of water) cupric sulphate solution until a purplish- violet or 

 pinkish-violet color is produced. The depth of the color depends 

 upon the nature of the protein, proteoses and peptones giving a de- 

 cided pink, while the color produced with gelatin is not far removed 

 from a blue. This reaction is given by those substances which con- 

 tain two amino groups in their molecule, these groups either being 

 joined directly together or through a single atom of nitrogen or 

 carbon. The amino groups mentioned must either be two CONH 2 

 groups or one CONH 2 group and one CSNH 2 , C(NH)NH 2 or 

 CH 2 NH 2 group. It follows from this fact that substances which 

 are non-protein in character but which contain the necessary groups 

 will respond to the biuret test. As examples of such substances 

 may be cited oxamide, 



CONH 2 



CONH 2 ' 



and biuret, 



CONH 2 



NH . 

 CONH 2 



The test derives its name from the fact that this latter sub- 

 stance which is formed on heating urea to 180 C. (see page 269), 

 will respond to the test. Protein material responds positively since 

 there are two CONH 2 groups in the protein molecule. 



According to Schiff the end-reaction of the biuret test is depend- 

 ent upon the formation of a copper-potassium-biuret compound 

 (cupri-potassium biuret or biuret potassium cupric hydroxide). 

 This substance was obtained by Schiff in the form of long red 

 needles. It has the following formula : 



