CARBOHYDRATES 23 



CH 2 OH CH 2 OH 



I 

 (CHOH) 3 (CHOH) 3 



C = C = N-NHC 6 H 5 +H 2 



I ^N-NHCeHfi+CeHfiNHa+NHa ^N-NHC 6 H 6 



C C 



\H ^H 



Aniline Glucosazone 



(b) Place 5 c.c. of the sugar solution in a test-tube, add i c.c. of the phenyl- 

 hydrazine -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 p. 22). 



The phenylhydrazine test has been so modified by Cipollina as to 

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

 in the next experiment. 



4. Cipollina's Test. Thoroughly mix 4 c.c. of dextrose solution, 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 sodium hydroxide (sp. gr. 1.16), 

 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 sugar solution 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. 



5. 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 solution under 

 examination and heat the mixture to boiling. Now introduce 10 c.c. of a 3 per cent 

 solution of potassium hydroxide and gently shake the tube and contents. If the 

 solution under examination contains dextrose the liquid in the tube will assume a 

 red color. 



One per cent dextrose yields an immediate color, whereas 0.05 per cent yields 

 the color only after the lapse of a period of one-half hour from the time the alkali is 

 added. In urinary examination if the color appears after the thirty-minute interval 

 the color change is without significance, inasmuch as sugar-free urine will respond 

 thus. The reaction is given by all aldehydes and therefore the test cannot be safely 

 employed in testing urines preserved by formaldehyde. Albumin does not interfere 

 with the test. 



6. Bottu's Test. 3 To 8 c.c. of Bottu's reagent 4 in a test-tube add i c.c. of the 



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. 



3 Bottu: Compt. rend. soc. biol., 66, p. 972. 



4 This reagent contains 3.5 grams of o-nitrophenylpropiolic acid and 5 c.c. of a freshly 

 prepared 10 per cent solution of sodium hydroxide per liter. 



