Table of Contents
Osazone test for carbohydrates post we briefly summarises about: principle, reagents requirements, procedure, result, application and limitations of osazone test for carbohydrates.
Osazone Test for Carbohydrates
Osazone test for carbohydrates is a biochemical test for detecting reducing sugars. Osazone test can even distinguish between different reducing sugars based on the appearance time of the complex. Osazone test is also termed Phenyl hydrazine test based on the reagent used for this test.
Objectives
- To detect reducing sugars.
- To differentiate reducing sugars from non-reducing sugars.
- To distinguish different reducing sugars between each other.
Principle
Reducing sugars upon reaction with Phenylhydrazine produces osazones, which are the characteristic derivatives of carbohydrates. These osazone derivatives have definite crystalline shape. These crystals made it easy and possible to confirm the type of carbohydrate.
Sugars lacking free anomeric hydroxyl group (non-reducing sugars) do not respond to this test. Whereas glucose, fructose and mannose produces similar type of osazone, i.e., glucosazone. An osazone crystal differs from the other with respect to time of crystallization, crystal shape and melting point.
Figure: A typical reaction showing the formation of an osazone. D-glucose reacts with phenylhydrazine to give glucosazone. Image Source: Shoyrudude555.
Requirements
Reagent
1. Osazone mixture: 0.5 g of phenylhydrazine hydrochloride and 0.1 g of sodium acetate.
2. Glacial acetic acid
3. Test sample
Materials
1. Test tubes
2. Test tube stand
3. Pipettes
Equipment
1. Vortex
2. Water bath
3. Microscope
Procedure
1. Fill a clean, dry test tube with 5 mL of test solution. To the test tube, add 0.3 g of osazone mixture and five drops of glacial acetic acid.
2. To dissolve all of the ingredients, thoroughly mix everything together and gently warm the test tube in the water bath if necessary.
3. Keep the test tube in boiling water and watch the crystals form at different times. Examine the crystal’s form under a microscope at a modest magnification.
Results
Different sugars can be identified based on the shape and structure of the crystals as well as their appearance time. Maltose crystallises into distinct petal-shaped crystals in this test. Lactose, on the other hand, forms puff-shaped crystals. Galactose, another material, will produce phombi-plate shaped crystals.
Applications
1. During the identification of unknown sugars, osazone test for carbohydrates is the sole way to tell the difference between lactose and maltose.
2. Osazone test for carbohydrates is a simple, inexpensive, and time-consuming test for identifying and distinguishing various sugars found in clinical practise. Sugars can also be found in plant tissues with this assay.
Limitations
1. Even though sucrose is a non-reducing sugar, osazone test for carbohydrates
produces a positive result for it when cooked for 30 minutes or more.
2. If the sample contains a mixture of sugars, osazone test for carbohydrates
will not be accurate. Similarly, a positive outcome necessitates a huge amount of sugar.