The document describes the process of forming osazone crystals from sugars through reaction with phenylhydrazine. Osazones are formed when reducing sugars react with phenylhydrazine at boiling point. Different sugars form characteristic crystal shapes that allow identification under a microscope. The procedure involves adding phenylhydrazine mixture to the sugar solution and observing crystal formation with boiling. Time of crystal formation varies between sugars but can be used to help identify the type of sugar.

2. Introduction
 The technique was developed by Emil Fischer, , a
German chemist.

3. Introduction
 Phenylhydrazine is the chemical compound with the
formula C6H5NHNH2.

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

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

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

7. Procedures
 2. Add 5 ml of the test solution

8. Procedures
 3. shake vigorously to insure complete solution

9. Procedures
 4. Stopper the mouth of the tube loosely with cotton

10. Procedures
 5. Place the tube immediately in a boiling water bath

11. Procedures
 6. Record the time

12. Procedures
 7. Observe the tube

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

14. Procedures
 9. Cool off the tube

15. Procedures
 10. Examine the crystals under low power objective

16. Results and Discussions
 The 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.
Fructose Mannose Glucose

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

19.  Making Osazone Crystals
 The 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 Form
 The 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;  Actual Time:
 glucose, four to five minutes;  Xylose – 20 mins
 xylose, seven minutes;  Galactose – 25 mins
 galactose, 15-19 minutes;  Glucose – 30 mins
 maltose, osazone soluble in hot  Sucrose – 47 mins
water  Fructose – 57 mins
 Maltose – 59 mins

27. Application
 For identifying sugars esp. Reducing sugars.
 Osazones are used as dyes

28. References
 http://www.ehow.com/info_8392949_different-
osazone-crystals.html#ixzz1Rx9lFGBJ
 http://www.pua.edu.eg/Version2/Courses2/Dentistry
%20Courses/Freshmen/Spring/BCM101/Practical/Wee
k%202%20practical%20_Chemistry%20of%20carbohy
drates_.pdf
 http://www.chemistry.ccsu.edu/glagovich/teaching/31
6/qualanal/tests/osazone.html