414 



PHYSIOLOGICAL CHEMISTRY 



melting-point, and as a further and more accurate means of identifica- 

 tion it may be recrystallized and identified by the determination of its 

 melting-point and nitrogen content. The reaction taking place in the 

 formation of phenylglucosazone is as follows : 



CH 2 OH CH 2 OH 



(CHOH) 3 

 CHOH 



\H 



Glucose 



CH 2 OH 



(CHOH); 



I 



c=o 



\^- 



+C 6 H 5 NH-NH 2 - 



Phenylhydrazine 



+C 6 H 5 NH-NH 2 



(CHOH) 3 



+C 6 H 5 NH-NH 2 -> 

 CHOH 

 ^N-NHC 6 H 5 +H 2 



C 

 \H 



Phenylhydrazone 



CH 2 OH 



(CHOH) 3 



I 



C = N-NHC 6 H 6 +H 2 O 



\ 



H 



Aniline 



C 

 \H 



Glucosazone 



(b) Place 5 c.c. of the urine in a test-tube, add i c.c. of phenylhydrazine- 

 acetate solution furnished by the instructor, 1 and heat on a boiling water-bath 

 for one-half to three-quarters of an hour. Allow the liquid to cool slowly and 

 examine the crystals microscopically (Plate III, opposite page 22). 



The phenylhydrazine test has been so modified by Cipollina as to 

 be of use as a rapid clinical test. The directions for this test are given 

 in the next experiment. 



2. Cipollina's Test. Thoroughly mix 4 c.c. of urine, 5 drops of phenylhydrazine 

 (the base) and % c.c. of glacial acetic acid in a test-tube. Heat the mixture for 

 about one minute over a low flame, shaking the tube continually to prevent loss of 

 fluid by bumping. Add 4-5 drops of potassium hydroxide or sodium hydroxide 

 (sp. gr. 1. 1 6), being certain that the fluid in the test-tube remains acid; heat the 

 mixture again for a moment and then cool the contents of the tube. Ordinarily 

 the crystals form at once, especially if the urine possesses a low specific gravity. 

 If they do not appear immediately allow the tube to stand at least 20 minutes before 

 deciding upon the absence of sugar. 



Examine the crystals under the microscope and compare them with those shown 

 in Plate III, opposite page 22. 



3. Riegler's Reaction. 2 Introduce o.i gram of phenylhydrazine-hydrochloride 

 and 0.25 gram of sodium acetate into a test-tube, add 20 drops of the urine under 



1 This solution is prepared by mixing one part by volume, in each case of glacial acetic 

 acid, one part of water and two parts of phenylhydrazine (the base). 



2 Riegler: Compt. rend. soc. biol., 66, p. 795. 



