Qualitative Analysis of Carbohydrates

Title: Qualitative Analysis of Carbohydrates

Aim: To study and Perform simple Qualitative tests of carbohydrates in the given sample.

Materials Required:

1. Molisch’s reagent
2. Fehling’s reagent
3. Benedict’s reagent
4. Tollen’s reagent
5. Iodine solution
6. Concentrated sulfuric acid
7. Sodium hydroxide solution
8. Test tubes
9. Test tube holder
10. Test tube stand
11. Bunsen burner
12. Water bath
13. Dropper

 

Principle:

Introduction of Carbohydrate:

Generally carbohydrates are defined as polyhydroxy aldehydes or polyhydroxy ketones or the compounds which produces such products on hydrolysis. Carbohydrates are called saccharides. Some of them have sweet taste and are called sugars.

Based on the reactivity with Chemical reagent, carbohydrates are classified as;

Reducing sugars:

Carbohydrates that can reduce Tollen’s, Benedict’s or Fehling’s reagents are called reducing sugars (sugar with free aldehyde or ketone group). All monosaccharides and most of the disaccharides are reducing sugars. Some examples are Maltose and Lactose.

Non-reducing sugars:

Carbohydrates that cannot reduce Tollen’s, Benedict’s or Fehling’s reagents are called non-reducing sugars. Sucrose is a non-reducing sugar.

The carbohydrates (saccharides) are divided into four chemical groups: Monosaccharides, Disaccharides, Oligosaccharides and Polysaccharides.

Procedure: Some Important Tests for the Detection of Carbohydrates.

1. Molisch’s test
2. Fehling’s test
3. Benedict’s test
4. Tollen’s test
5. Iodine test
6. Seliwanoff’s Test:
7. Osazone Test

(1) Molisch’s Test:

Molisch’s test is a general test for carbohydrates. This test is given by almost all of the carbohydrates. In this test, concentrated sulfuric acid converts the given carbohydrate into furfural or its derivatives, which react with α-naphthol to form a purple coloured product.

1. Take 2ml of the given sample solution in a clean test tube.
2. Add 2-3 drops of Molisch reagent slowly.
3. Now add concentrated sulfuric acid along the sides of the test tube.
4. The acid layer forms a layer at the bottom.
5. Note the junction of the two layers.
6. If there is a formation of the violet ring then the presence of carbohydrate is confirmed.

Note: The appearance of purple or violet ring confirms the presence of carbohydrate.

(2) Fehling’s Test:

This test is given by reducing sugars. To the aqueous solution of carbohydrate fehling’s solution is added and heated in water bath. The formation of red precipitate confirms the presence of reducing sugars.

1. Take 2ml of given sample solution in a clean test tube.
2. Add 2 ml of Fehling’s solution A and Fehling’s solution B to it.
3. Keep the solution in a boiling water bath for about 10 minutes.
4. If there is the formation of red precipitate then the presence of carbohydrate is confirmed.

Note: The appearance of red precipitate confirms the presence of carbohydrates.

(3) Benedict’s Test:

This test is given by reducing sugars. in an alkaline medium, sodium carbonate converts glucose to enediol and this enediol reduces cupric to cuprous forming cuprous hydroxide. This solution is kept in sodium citrate and on boiling, red precipitate of cuprous oxide is formed.

1. Take the given sample solution to be tested in a clean test tube.
2. Add 5ml of Benedict’s reagent to it.
3. Boil the solution for about 2 minutes.
4. Cool the solution and observe the solution.
5. If there is formation of green, red or yellow precipitate then there is presence of reducing sugars.

Note: The appearance of red precipitate confirms the presence of carbohydrates.

(4) Tollen’s Test:

This test is given by reducing sugars. Carbohydrates react with Tollens reagent and forms a silver mirror on the inner walls of the test tube. This confirms the presence of reducing sugars. Silver ions are reduced to metallic silver.

1. Take the given sample solution in a clean test tube.
2. Add 2-3ml of tollens reagent to it.
3. Keep the test tube in a boiling water bath for 10 minutes.
4. If there is the appearance of shiny silver mirror confirms the presence of reducing sugars.

Note: The appearance of silver mirror confirms the presence of reducing sugars.

(5) Iodine Test:

This test is only given by starch. Starch reacts with iodine solution forms complex blue colour solution. On heating the blue colour disappears and on cooling the blue colour reappears.

1. Take the sample solution to be tested in a clean test tube.
2. Add 2-3 drops of iodine solution.
3. Observe the change in colour.
4. If there is the appearance of a blue colour then the presence of starch is confirmed.

Note: The appearance of blue colour solution confirms the presence of starch.

 

Confirmatory Tests:

Osazone Test (Phenyl hydrazine test): 

The ketoses and aldoses react with Phenylhydrazine to produce a phenylhydrazone which further reacts with another two molecules of phenylhydrazine to yield osazone.

Take about 300 mg of phenyl hydrazine mixture (discussed below), to it add a few drops of glacial acetic acid and then 5 ml of sugar solution. Shake well and heat in a boiling water bath for 30 to 45 minutes. Take the tube out of the water bath and allow it to cool slowly. Yellow crystals of osazones will appear. Examine the crystals under the microscope and describe the nature of crystals.

Phenyl hydrazine mixture is prepared by mixing equal weights of phenyl hydrazine hydrochlo­ride and anhydrous sodium acetate. The mixing is to be done thoroughly in a mortar. Glucose and Fructose give identical osazones called Glucosazones and Fructosazones.

Precautions:

1. Handle the acids like concentrated sulfuric acid with care.
2. Always use droppers to take reagents from the reagent bottles.
3. While heating the reaction mixture do it carefully.

Report:

The given organic compound is a _________ (reducing sugar/starch/carbohydrate) compound.

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