This document describes the osazone test, which is used to identify sugars. The test involves reacting sugars with phenylhydrazine at boiling point to form osazone crystals. Different sugars form characteristic crystal shapes that can be examined under a microscope to identify the type of sugar. The time it takes for crystals to form also helps in identification, with fructose forming crystals within two minutes while galactose takes 15-19 minutes. The test is useful for identifying reducing sugars.

1. OSAZONE TEST

2. IntroductionThe technique was developed by Emil Fischer, , a German chemist.

3. IntroductionPhenylhydrazine is the chemical compound with the formula C6H5NHNH2.

4. IntroductionOsazones are formed when the sugars react with a compound known as phenylhydrazine at boiling point.General Reaction:

5. IntroductionThese sugars are reducing ones which have either a free aldehyde or a ketone group to react with the phenylhydrazine.

6. Procedures1. Pipette 1 ml of the phenylhydrazine mixture to a test tube.

7. Procedures2. Add 5 ml of the test solution

8. Procedures3. shake vigorously to insure complete solution

9. Procedures4. Stopper the mouth of the tube loosely with cotton

10. Procedures5. Place the tube immediately in a boiling water bath

11. Procedures6. Record the time

12. Procedures7. Observe the tube

13. Procedures8. If a precipitate is formed, record the time and remove the tube from the bath

14. Procedures9. Cool off the tube

15. Procedures10. Examine the crystals under low power objective

16. Results and DiscussionsThe reaction is stepwise;

17. Osazone formation involves hydrazone formation at C-1 of an aldose (or C-2 of a ketose) and oxidation of C-2 (or C-1) of an alcohol group to a ketone (or an aldehyde). The new carbonyl group is also converted to a hydrazone. MannoseGlucoseFructose

18. Osazone crystals have a characteristic shape under the light microscope and help in the identification of the sugar type.Monosaccharides - like needle-shaped or broomstickDisaccharides - like sunflowersLactose - like tight balls of needles

19. Making Osazone CrystalsThe difference in the structure of the monosaccharides is caused by the diverse groups attached to the first and second carbons of the sugar molecules. Their needle-shaped crystals show that the position of the first and second carbons do not matter in the crystal formation.

20. Viewed under the microscope: Glucosazone

21. Viewed under the microscope: Fructosazone

22. Viewed under the microscope: Galactosazone

23. Viewed under the microscope: Maltosazone

24. Viewed under the microscope: Xylosazone

25. Viewed under the microscope: Sucrose

26. Time to FormThe time needed to create osazone crystals varies among the various sugars involved, but helps to identify the sugars being tested. For an osazone crystal to be presented from a hot solution will take as long as follows:fructose, two minutes;

27. glucose, four to five minutes;

28. xylose, seven minutes;