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