Osazone Test – Principle, Uses, Protocol, Results (Observation and Interpretation)

Osazone is an essential biochemical test to detect reducing sugars in a given sample. The time taken to form a specific complex varies for each reducing sugar.

So, you can differentiate them accordingly. The other term used for the osazone test is the Phenyl Hydrazine test because Phenyl hydrazine is the reagent used in this test.

Uses of Osazone Test

Uses of this biochemical test are:

• Detection of the reducing sugars in the sample
• Differentiate between different types of reducing sugars.

Principle of Osazone Test

It is an oxidation condensation reaction. Phenyl Hydrazine is used as a reagent with an acetate buffer. The reaction occurs between the phenyl hydrazine molecules and carbon one and two of the carbohydrate, and it yields 1,2-diphenylhydrazone, which is called osazone.

Only those carbohydrates can form an osazone that has a free carbonyl group. It is only present in the reducing sugars, so they give a positive osazone test.

Osazones are crystalline compounds, and their shapes and characteristics vary for each reducing sugar. Only the first two carbons of a reducing sugar are involved in this reaction. So, carbon is two epimers from the same type of osazone compound.

Laboratory Requirements

Following are the equipment and reagents required for Osazone Test.

 Apparatus

• Test tubes
• Test tubes holders
• Test tubes stand
• Pipette
• Pipette suckers

Equipment

• Vortex Mixer
• Compound microscope
• Water Bath

Reagents

• A mixture of 0.5g Phenylhydrazine hydrochloride and 0.1g of sodium acetic acid
• The glacial acetic acid is its anhydrous form
• Sample solution

Protocol

1. Take a clean and dry test tube. Add 5ml of the sample under study in it.
2. Add 0.3 gram of prepared mixture and five drops of acid in it.
3. Shake gently and put it in the water bath to form a uniform solution.
4. Now keep it in the water bath and observe when the crystals appear.
5. Separate the crystals on a glass slide and observe their shape under a light microscope.

Interpretation of Test

Only reducing sugars give the positive osazone test. The type and shape of the crystal are the basis of identifying different reducing sugars.

The following table shows some different possible results obtained from various reducing sugars.

Carbohydrate	Time of formation (min)	Crystalline structure
Fructose	2	Needle shape
Glucose	5	Needle or broomstick shape
Galactose	20	Thorny ball or rhombic plate shape
Maltose	30-45	Sunflower/ petal shaped
Lactose	30-45	puff shaped powder or cotton balls

Uses of Osazone Test

• This test is the less expensive, easy to perform and interpret, distinguishing reducing sugars from non-reducing sugars.
• This test can identify sugars in different plant tissues. 
• Different reducing sugars form different osazone compounds. So, it is a significant way to separate them from each other.

Limitations of Osazone Test

• For identification, reducing sugars must be present in the larger quantity in the sample.
• It can also give a false-positive result for sucrose, a non-reducing sugar, if heated for more than 30 minutes.
• Detection is not possible if more than one reducing sugar is present in the sample.

Frequently Asked Questions

References

1. Tiwari A. (2015). Practical Biochemistry. LAP Lambert Academic Publishing.
2. Shah, Tejas. (2016). Utility of Osazone Test to Identify Sugars. Journal of Medical Science And clinical Research. 04. 14361-14365. 10.18535/jmscr/v4i12.14.
3. Mester, L.; El Khadem, H.; Horton, D. (1970). “Structure of saccharide osazones.” Journal of the Chemical Society C: Organic (18): 2567. doi:10.1039/J39700002567.
4. Ramakrishnan, S. (2004).Textbook of Medical Biochemistry. Orient Blackswan. ISBN 9788125020714.
5. https://en.wikipedia.org/wiki/Osazone
6. https://microbenotes.com/osazone-test/
7. https://www.slideshare.net/namarta28/qualitative-tests-for-carbohydrates-35884145
8. https://medicalstudyzone.com/osazone-test/
9. https://dx.doi.org/10.18535/jmscr/v4i12.14