Osazone Test- Definition, Principle, Procedure, Result, Uses

Osazone test is a chemical test that is used to detect and differentiate reducing sugars from non-reducing sugars. Reducing sugars are carbohydrates that can reduce other compounds by donating electrons or hydrogen atoms. Non-reducing sugars are carbohydrates that cannot do so. Examples of reducing sugars are glucose, fructose, lactose, maltose, etc. Examples of non-reducing sugars are sucrose, trehalose, etc.

Osazone test is based on the reaction of reducing sugars with phenylhydrazine, a chemical compound that contains nitrogen and hydrogen atoms. Phenylhydrazine reacts with the carbonyl group (C=O) of the reducing sugar and forms a yellow-colored crystalline compound called osazone. Osazone has a characteristic shape, solubility, melting point, and time of formation that can be used to identify the type of reducing sugar.

Osazone test is also known as phenylhydrazine test because phenylhydrazine is the main reagent used in this test. Osazone test was first developed by German chemist Emil Fischer in 1884. He used this test to determine the structure and configuration of different sugars.

Osazone test is a simple and inexpensive test that can be performed in a laboratory with basic equipment and materials. Osazone test can be used for various purposes such as identifying unknown sugars, locating sugars in plant tissues, studying the metabolism of sugars, etc. Osazone test is one of the oldest and most widely used tests for carbohydrate analysis.

The main objectives of the osazone test are:

• To detect reducing sugars in a given sample. Reducing sugars are those that can reduce other substances by donating electrons. Examples of reducing sugars are glucose, fructose, lactose, maltose, etc.
• To differentiate reducing sugars from non-reducing sugars. Non-reducing sugars are those that cannot reduce other substances by donating electrons. Examples of non-reducing sugars are sucrose, trehalose, etc.
• To distinguish different reducing sugars between each other based on their osazone formation. Osazones are yellow-colored crystals that have characteristic shapes and melting points. Different reducing sugars form different osazones with phenylhydrazine in a specific time period.

The osazone test is a simple and useful test for the identification and characterization of different sugars in clinical and biochemical settings.

The principle of osazone test is based on the fact that carbohydrates with free or potentially free carbonyl groups react with phenylhydrazine to form osazone. Phenylhydrazine is a chemical compound that acts as a reducing agent and reacts with the aldehyde or ketone group of the sugar molecule. The reaction involves three steps:

• The first step is the formation of a phenylhydrazone by the condensation of one molecule of phenylhydrazine with the carbonyl group of the sugar.
• The second step is the oxidation of the phenylhydrazone by another molecule of phenylhydrazine, resulting in the loss of water and the formation of a 1,2-diphenylhydrazone.
• The third step is the condensation of a third molecule of phenylhydrazine with the 1,2-diphenylhydrazone, leading to the formation of an osazone.

The osazone is a yellow-colored crystalline compound that has a characteristic shape, solubility, melting point, and time of formation. The osazone is different for different sugars because it depends on the configuration of the sugar molecule at carbon 3 and beyond. Since both carbon 1 and 2 are involved in the reaction, sugars that have the same configuration at these two positions will produce the same osazone. For example, glucose and fructose produce glucosazone, while lactose and maltose produce lactosazone and maltosazone respectively. The osazone crystals can be observed under a microscope and identified by their shape and structure.

The following figure shows a typical reaction of osazone formation. In this example, D-glucose reacts with phenylhydrazine to form glucosazone, which is a type of osazone. The reaction involves three molecules of phenylhydrazine and two carbons of the sugar. The first molecule of phenylhydrazine reacts with the carbonyl group of the sugar to form a hydrazone. The second molecule of phenylhydrazine oxidizes the hydrazone to form a phenylosazone. The third molecule of phenylhydrazine condenses with the phenylosazone to form an osazone. The osazone has a characteristic yellow color and crystalline structure.

Figure: Formation of an osazone. D-glucose reacts with phenylhydrazine to give glucosazone. Image Source: Shoyrudude555.

Different sugars produce different osazones with different shapes and melting points. For example, fructose and glucose produce the same osazone because they have the same configuration below C-2. However, mannose and glucose produce different osazones because they differ in the configuration at C-2. The shape and time of appearance of the osazones can be used to identify and differentiate various sugars.

The osazone test requires the following reagents, materials, and equipment:

• Reagent: Osazone mixture, which is prepared by mixing 0.5 g of phenylhydrazine hydrochloride and 0.1 g of sodium acetate. Glacial acetic acid is also needed as a solvent and catalyst for the reaction.
• Test sample: The test sample can be any solution containing reducing sugars, such as glucose, fructose, maltose, lactose, etc. The concentration of the sugar should be high enough to produce visible crystals of osazone.
• Materials: Test tubes, test tube stand, pipettes, and slides are required for holding and transferring the solutions and observing the crystals.
• Equipment: A vortex mixer is used to mix the solutions well. A water bath is used to heat the solutions and accelerate the reaction. A microscope is used to examine the shape and structure of the crystals under low magnification.

• Take 5 ml of test solution in a clean, dry test tube.
• Add 0.3 g of osazone mixture and five drops of glacial acetic acid to the test tube.
• Mix it well and warm the test tube gently in the water bath if required to dissolve all the elements.
• Keep the test tube in boiling water and observe the formation of crystals at various time points.
• Observe the shape of the crystal under low magnification under a microscope.

The procedure is simple and straightforward. You just need to add the reagent and the acid to the test solution and heat it up. Then you can watch for the appearance of yellow crystals that indicate the presence and type of reducing sugar. The shape and structure of the crystals vary depending on the sugar. You can use a microscope to see them more clearly.

Some additional sentences to conclude the point are:

• The osazone test is a useful method to identify and differentiate reducing sugars based on their chemical structure and reaction time.
• The osazone crystals have distinctive shapes that can be recognized by comparing them with a reference chart or table.

The result of the osazone test is based on the appearance of yellow-colored crystals of osazone under the microscope. The shape, size, solubility, melting point, and time of formation of the crystals vary depending on the type of reducing sugar present in the sample. By comparing these characteristics with a standard chart, different sugars can be identified and differentiated.

The following table summarizes the typical features of osazone crystals for some common reducing sugars:

Figure: Examples of osazone crystals for different sugars. Image Source:

Thus, by observing the time and shape of the osazone crystals, one can distinguish between fructose and glucose, galactose and maltose, and lactose and sucrose. However, this test cannot differentiate between C-2 epimers or ketoses with a configuration identical to aldoses below C-2, as they produce the same osazone. For example, glucose and mannose form glucosazone, while fructose and sorbose form fructosazone.

• This test is the only test that can be used to distinguish lactose from maltose during the identification of unknown sugars .
• This is a simple, cheap, and relatively less time-consuming test for the identification and differentiation of different sugars encountered in clinical practice .
• This test can also be used for locating sugars in plant tissues.

• The osazone test has some limitations that should be considered before using it for the identification and differentiation of reducing sugars. Some of the limitations are:

• The test is not specific for reducing sugars, as some non-reducing sugars like sucrose can also give a positive result if boiled for a long time. This can lead to false-positive results and confusion.

• The test is not sensitive enough to detect small amounts of reducing sugars in a sample. A large quantity of sugar is required for the formation of visible crystals. This can limit the applicability of the test in some cases.

• The test is not reliable for samples that contain a mixture of different reducing sugars. The osazone crystals formed by different sugars may have similar shapes and colors, making it difficult to distinguish them. Moreover, some sugars may interfere with the reaction of others, resulting in incomplete or delayed formation of crystals.

• The test requires careful observation and interpretation of the crystal shape and time of appearance. These factors can vary depending on the temperature, concentration, and purity of the reagents and the sample. Therefore, the test results should be confirmed by other methods or tests.

• The test involves the use of phenylhydrazine, which is a toxic and carcinogenic compound. It can cause skin irritation, eye damage, and respiratory problems if inhaled or ingested. Therefore, proper precautions and safety measures should be followed while performing the test.

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