Tests of Carbohydrates

What are Carbohydrates?

Carbohydrates are one of the major classes of biomolecules besides proteins and lipids. From a tiny grain of rice to the thick woody barks of trees, carbohydrate is omnipresent in one form or the other, forming the structural backbone of most life forms on earth. A molecule that is so fundamental to our existence has been and is still studied extensively. In the process, the need for tests of carbohydrates arose and thus, came about the development of chemical tests for carbohydrates. 

The identification test for carbohydrates exploits the chemical structure of sugars to detect their presence or absence in the test solution. Our aim here is to study some carbohydrate test methods like Molisch's test, Benedict's test for carbohydrates, Iodine test for carbohydrates, and their underlying principles and the carbohydrate test procedures.

The tests for the presence of carbohydrates involve chemical tests of carbohydrates. The ones we will discuss here are:

  1. Molisch's test for carbohydrates

  2. Benedict's test for carbohydrates

  3. Iodine test for carbohydrates

  1. Molisch's Test

  • Principle: It is a general test for carbohydrates. In the Molisch's test experiment, carbohydrates are reacted with Molisch's reagent and concentrated sulphuric acid; the former dehydrates to form furfural and its derivatives. The products further react with sulfonated alpha-naphthol to give a purple coloured complex. All carbohydrates, that is, monosaccharides, disaccharides, and polysaccharides give a positive result for Molisch's test.

  • Requirements: Sample, distilled water, Molisch's reagent, concentrated sulphuric acid, sulfonated alpha-naphthol, Test-tubes, test-tube stand, test-tube holder, and dropper.

What is Molisch's Reagent?

The Molisch's reagent consists of α-naphthol dissolved in ethanol.

  • Molisch's Test Procedure

    • Take 2 mL sample in clean test-tube and 2 mL distilled water in another as control

    • Add 2-3 drops of Molisch's reagent to the tubes

    • Gradually, add concentrated sulphuric acid along the inner walls of the test-tubes

    • Formation of a violet ring at the junction of the liquid layers confirms the presence of carbohydrate

Image will be uploaded soon

  1. Benedict's Test

  • Principle: The principle is that when a solution of reducing sugar is heated with Benedict's reagent, the alkaline sodium carbonate converts the sugar into enediol* and this enediol further reduces the cupric ions of the reagent into cuprous ions. The resulting precipitate of cuprous oxide is brick-red in a colour that confirms the presence of reducing sugar. Lactose, maltose, and glucose give a positive reaction to this test.

  • Requirements: Sample, distilled water, Benedict's reagent, test-tubes, test-tube stand, test-tube holder, Bunsen burner, and dropper.

What is Benedict's Reagent?

Alkaline Benedict's solution is used to test the presence of reducing sugars. It has the following components:

  1. Anhydrous Sodium Carbonate – imparts alkalinity to the reaction medium

  2. Sodium Citrate – forms a complex with cupric ions so that they are not reduced to cuprous ions during storage

  3. Copper (II) Sulphate Pentahydrate – gives the cupric ions

  4. Distilled water- used as a solvent

  • Preparation of Benedict's Solution: To prepare a litre of Benedict's reagent, mix 173 g sodium citrate, 100 g anhydrous sodium carbonate, and 17.3 g copper (II) sulphate pentahydrate in 1000 mL of distilled water.

  • Benedict's Test Procedure

    • Add 2 mL of Benedict's reagent to 0.5-1 mL of the sugar solution (and distilled water as control)

    • The test-tubes are heated for 3 to 5 minutes

    • The appearance of brick-red precipitate confirms the presence of reducing sugar

Image will be uploaded soon

  1. Iodine Test

Principle: The test gives positive results only with polysaccharide starch. The principal is that on reacting with starch, Iodine gets trapped in the helical coils of the polysaccharide chain via a coordinate complex**. Due to the complex formation, a blue/black colour is observed, which confirms the presence of starch. The blue/black colour disappears on the addition of an alkali or on heating; this is due to the uncoiling of the polysaccharide network and release of the Iodine molecules.

Requirements: Sample, distilled water, Iodine solution, Test-tubes, test-tube stand, test-tube holder, Bunsen burner, and dropper.

Iodine Test Procedure

  • Take 2 mL sample in clean test-tube and 2 mL distilled water in another as control

  • Add five drops of Iodine solution to the test-tubes

  • A blue/black colour confirms the presence of starch and a yellow/brown colour its absence

FAQ (Frequently Asked Questions)

1. What are reducing and non-reducing sugars with examples?

Reducing sugars are the saccharides that contain a free aldehyde group or a ketone group. They are called 'reducing' sugars because they reduce Benedict's solution, Fehling's solution, and Tollen's reagent and themselves get oxidized to the corresponding carboxylic acid. All monosaccharides such as D-Glucose, D-Fructose and disaccharides like Lactose and Maltose are examples of reducing sugars.

Non-reducing sugars, by contrast, do not contain a free aldehyde or ketone group and therefore do not reduce Benedict's solution, Fehling's solution, or Tollen's reagent. All polysaccharides such as starch and cellulose are examples of non-reducing sugars. Disaccharide sucrose is also non-reducing because the carbonyl groups of its monosaccharide units (glucose and fructose) are not free.

2. What are the principles of Fehling's and Tollen's test for carbohydrates?

Fehling's test is given by reducing sugars. When Fehling's solution is added to the sugar solution and heated, the aldehyde group of the sugar gets oxidized by complexed copper ions to form an acid. During the process, the copper (II) ions are reduced to copper (I) ions, giving a red precipitate of cuprous oxide, confirming a positive Fehling's test. 

 Image will be uploaded soon

The Tollen's test is given by reducing sugars and is based on the principle that while aldehydes are readily oxidized, ketones are not. Tollen's reagent is a solution containing silver ions complexed with ammonia [Ag (NH3)2+]. On adding Tollen's reagent to a solution of reducing sugar, the aldehyde gets oxidized to the corresponding carboxylic acid, and the silver ions in Tollen's reagent are reduced to metallic silver which is visible as a mirror on the sides of the test-tube, indicating a positive result.

Image will be uploaded soon

*enediol – These are organic compounds with two hydroxyl groups adjacent to a double bond.

**Coordinate complex – a central atom/ion surrounded by a complex of molecules or ions.