OSAZONE Phenylhydrazine Test

Principle

Compounds containing the -CO-CHOH- group from crystalline osazone with phenyl hydrazine. Emil Fischer in 1884 discovered reactions of sugars with phenylhydrazine to form crystalline “osazone“.

phenylhydrazine reacts with the carbonyl group of the sugar to give the phenylhydrazine, which then reacts with further two molecules phenylhydrazine to from osazone.

The formation of osazone from an aldose is outlined as follows:

Aldose + phenylhydrazine ===Phenylhydrazone + H20

Phenylhydrazone + phenylhydrazine === Osazone + C6H5NH2 + NH3

* In osazone formation only the first two carbon atoms of the sugar are involved. Hence the sugar which have the same arrangement on the last four carbon atoms will yield the same osazone.

Thus glucose, fructose, and mannose, all form of the same osazone  since the configuration on the carbon atoms e, 4, 5, and 6 is the same for these sugars.

Although mannose forms the osazone as glucose, but the hydrazone is different forming white precipitate at room temperature.

Similarly the osazone of galactose is different because the arrangement of group on 4th carbon atom.

Osazone crystals have characteristics shapes and melting points which helps in the Identification of the reducing sugar . Further evidence for identification is obtained by observing the time of formation of the crystals and whether the osazone precipitates from the hot solution or cold solution.

The following tables give the time of osazone formation, its melting point and it’s shape.

SugarTime of osazone formation (min)Melting point (C)
Arabinose10166
Fructose02205
Galactose20206
Glucose05205
Xylose07163
LactoseSoluble in hot water200
MaltoseSoluble in hot water208
Mannose1 – 5205
SucroseDoes’t form osazone

Requirements

0.1 M solution of test sugar (glucose, mannose, fructose, galactose, etc.) phenylhydrazine-HCL aolids, sodium acetate, water bath, non absorbent cotton.

Procedure

  1. Take a test tube and add into 1 ml of test sugar solution.
  2. Add 2 ml of hot phenylhydrazine mixture in the tube containing sugar solution, plug with cotton and mix it well.
  3. Place the tube in a boiling water bath and record the time of formation of osazone.
  4. After the crystals have formed, remove the tubes from the waterbath and cool at room temperature.
  5. Spread the suspension of the derivative on a clean, dry microscopic slide and carefully draw a diagram of the crystals observed under the low power of microscope.
  6. Measure the meltingpoint of the osazone obtained.
  7. Identify the sugar on the basis of shape of the crystal, time of crystal formation and its melting point.
TestXyloseGlucoseMannoseFructoseGalactoseLactoseMaltoseSucrose
Molich’s++++++++
Benedict’s++++++++
Barfoed’s+(2)+(2)+(2)+(2)+(2)+(slow)+(slow)
Bial’s+(blue)+(brown)+(brown)+(brown)+(brown)
Seliwanoff’sbg(7)pinkpinkpinkpink+(15-17)+(15-30)+(1-2)
phlorogluc+
Mucic acid++
Osazone(7)(4-5)(6-7)(2)(15)(25)(25)(30)
Carbohydrate Review Chart

Identification of unknown sugar

If the test solution is alkaline, neutralize or make faintly acidic with HCL and add few drops of iodine;

note down the formation of colour as follows:

  • Starch ➜ Blue color.
  • Dextrin/Glycogen ➜ Reddish color.
  • Sugar ➜ No color (identify as follows). 
carbohydrat test reaction

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