1. Lab Activity 3 Formation of Osazones (Phenyl Hydrazine Reaction)
IUG, 2016
Dr. Tarek Zaida

2. Background Phenyl hydrazine reacts with monosaccharides.
Thus monosaccharides can be determined by the reaction with phenyl hydrazine.
It is possible to isolate the hydrazone of an aldose or ketose.
During reaction with monosaccharides, additional phenyl hydrazine is consumed in oxidizing the adjacent OH-group to carbonyl group which then forms a second phenyl hydrazone.
Such bisphenyl hydrazones are called osazones.

3. Osazones crystals have characteristic shapes & melting point which assist in the identification of the reducing sugars. The following reaction is known as phenyl hydrazine reaction:

4. Since only C1 & C2 of a saccharide are involved in osazones, sugars with the same configuration at the remaining carbon atom gives the same osazone.
Osazones are insoluble yellow compounds.
Depending on the time required to form the insoluble yellow osazone, different sugars can be classified into the following:
Mannose: 1-5 min
Fructose: 2 min
Glucose:5 min
Xylose: 7 min
Arabinose: 10 min
Galactose: 20 min

5. The mechanism of the osazone formation is not fully understood yet.
Initially the sugar and phenylhydrazine form a phenylhydrazone (glucose →glucose phenyl-hydrazone).
Upon addition of a second and a third equivalent of phenylhydrazine , the osazone is formed as
a yellow solid.

6. Experiment 1: Formation of Osazones
Reagents:
1% solutions of glucose, galactose, lactose, maltose, mannose, and xylose
Phenyl hydrazine mixture (2 parts phenyl hydrazine hydrochloride are mixed with 3 parts sodium acetate).

7. Procedure
To 300 mg of phenyl hydrazine mixture add 5 ml of the tested solution,
Shake well, and heat on a boiling water bath for 30 – 45 min.
Allow the tubes to cool slowly (not under tap) and examine the crystals microscopically, draw the shapes of the crystalls and compare their shapes with those drawn in figure1 (manual).
Carefully note the time when the osazone is precipitated and also whether it is formed in hot or cold solution.

8. Tests for Individual Carbohydrates

9. 1. Bial's (Orcinol) Test for pentoses ( for the detection of pentoses)
Principle
Pentoses are converted to furfural by this reagent, which forms a blue green color with orcinol.

10. Set up 1 % solutions of: xylose, arabinose, glucose, fructose,
Reagents
Set up 1 % solutions of:
xylose,
arabinose,
glucose,
fructose,
maltose.
Bial's reagent (0.1 % orcinol in concentrated HCl containing 0.1 % FeCl3.6H2O).

11. Procedure
1. Add about 2 ml of 1% xylose, glucose, fructose, maltose, arabinose, and xylose solution to their respective labeled test tubes.
2. Add 3 ml of Bial's reagent to each tube and mix well.
3. Carefully heat each tube (with some agitation) directly over the burner flame. Hold the tube at a diagonal and heat along the sides of the tube rather than at the bottom to prevent eruption of the liquid from the tube. Move the tube diagonally in and out of the flame, until the mixture just begins to boil. Stop heating when the mixture begins to boil.
A blue-green color indicates a positive result. Prolonged heating of some hexoses yields hydroxymethyl furfural which also reacts with orcinol to give colored complexes.

12. 2. Aniline Acetate Test for Pentoses
The furfural produced by the reaction of hot dilute HCl on pentoses forms a bright red color with aniline acetate in a test paper held over the mouth of the reaction flask.

13. Reagents
1% solution of glucose, fructose, lactose, xylose, & arabinose.
Aniline solution is prepared as follows:
Shake 5 ml of aniline with 5 ml of water and add 5 ml of glacial acetic acid to adjust clear the emulsion.

14. Procedure
1. Place 5 ml of solution to be tested and 20 ml water in 250 ml Erlenmeyer flask. 2. Add 20 ml of conc. HCl and boil gently for about 1 min. 3. Cease heating, hold a filter paper moistened with a few drops of aniline acetate over the mouth of the flask. 4. Bright red color on a filter paper indicates a positive result.

15. 3. Seliwanoff's (Resorcinol) Test (used for detection of Ketoses)
Principle Ketohexoses (such as fructose) and disaccharides containing a ketohexose (such as sucrose) form a cherry-red condensation product. Other sugars (e.g. aldose) may produce yellow to faint pink colors.

16. Reagents
Set up 1 % solution of:
glucose,
sucrose,
fructose,
lactose,
maltose.
Seliwanoff's reagent (0.5 % resorcinol in 3N HCl).

17. Procedure
1. Add about 3 ml of Seliwanoff's reagent to each labeled test tube.
2. Add 3 drops of the respective sugar solution to the appropriate test tubes, and mix well.
3. Place all the test tubes in the boiling water bath at the same time and heat for 3 min after the water begins to boil again. Record your observations.
A positive result is indicated by the formation of a red color with or without the separation of a brown-red precipitate.