Experiment 4

Introduction

To identify and characterise the different types of carbohydrates by commonly used qualitative methods: 

1. Benedict’s Test 

2. Barfoed’s test 

3. Iodine Test 

Carbohydrates are organic molecules which contain carbon, hydrogen and oxygen and are the most abundant class of organic compounds found in living organisms. It is the main energy source for animals as they breakdown carbohydrates during the process of metabolism to release energy. Carbohydrates also serve as a structural material (cellulose), a component of the energy transport compound ATP, recognition sites on cell surfaces, and one of three essential components of DNA and RNA. 

All carbohydrates are made up of units of sugar or saccharide units. They are classified according to their molecular size and solubility. Carbohydrates are classified into groups according to the number of individual simple sugar units: 

1) Monosaccharides: Carbohydrates that contain only one sugar unit are called monosaccharides. These are the simplest form of sugar and are usually colourless, water soluble and crystalline solids, e.g. glucose, fructose, galactose, etc. 

2) Disaccharides: Carbohydrates that contain two sugar units are called disaccharides. It is formed when two monosaccharides undergo condensation reaction and are water soluble, e.g. sucrose, lactose, etc. 

3) Polysaccharides: Carbohydrates that contain more than two sugar units are called polysaccharides. It is formed when more than two monosaccharide units are bound together by glycosidic bonds, e.g. starch, glycogen, cellulose, etc. 

Presence of carbohydrates can be confirmed qualitatively by several tests. Due to the presence of different numbers of sugar units, specific carbohydrates exhibit typical colour reactions that form the basis for their identification.

1. Benidict's test

Principle: 

Benedict’s test is based upon the participation of the aldehyde and ketone groups in a chemical reaction. In this procedure, Benedict’s reagent, which contains a basic solution of cupric ions, is mixed with a sugar. The copper ions will attract the electrons from the aldehyde or ketone group changing the charged copper ions to a neutral metal. Copper ions are blue in color whereas copper metal is orange. Disaccharides will react with the Benedict’s reagent only if it contains an exposed aldehyde or ketone group. This type of sugar is known as a reducing sugar. 

Interpretation

Positive result shows the presence of reducing sugar. 

2. Barfoed's test

Principle

The Barfoed’s test is used to distinguish between reducing monosaccharides and disaccharides. Barfoed’s reagent contains cupric ions in an acidic solution. Milder conditions and careful control of the heating time allow monosaccharides, but not disaccharides, to be oxidized. The cupric ions are reduced in the presence of a monosaccharide resulting in a red precipitate. 

                                     RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+

Materials required

1. Preparation of standard sugars

• Standard  Sugar 1 (Glucose) – Dissolve 1.1 g of powder in 110 ml  of water, mix well to dissolve.

• Standard Sugar 2 (Starch) – Dissolve 550 mg of   powder in  55 ml of water, mix well to dissolve.

• Standard Sugar 3 (Fructose) – Dissolve 50 mg of powder in 5 ml of water, mix well to dissolve.

• Standard sugar 4 (Sucrose)-Dissolve 1.0 g of powder in 100 ml  of water, mix well to dissolve.

2. Preparation of Test Sugars:

2. • Test Sugar 1 – Dissolve 2 g of powder in 200 ml of water, mix well to dissolve.

   • Test tubes

   • test tubes racks

   • Pipettes and pipette tips

   • Barfoed's reagent

   • Boiling water bath

Interpretation

Positive result shows the presence of reducing monosaccharide 

3. Iodine Test 

Principle

Iodine forms colored complexes with polysaccharides. The color of the complex depends upon the three dimensional structure of the polysaccharide. Starch is a coiled structure which turns blue when bound to Iodine, whereas glycogen, which is a branched molecule, turns red-violet.

Materials required

1. Preparation of standard sugars

• Standard  Sugar 1 (Glucose) – Dissolve 1.1 g of powder in 110 ml  of water, mix well to dissolve.

• Standard Sugar 2 (Starch) – Dissolve 550 mg of   powder in  55 ml of water, mix well to dissolve.

• Standard Sugar 3 (Fructose) – Dissolve 50 mg of powder in 5 ml of water, mix well to dissolve.

• Standard sugar 4 (Sucrose)-Dissolve 1.0 g of powder in 100 ml  of water, mix well to dissolve.

2. Preparation of Test Sugars:

2. • Test Sugar 1 – Dissolve 2 g of powder in 200 ml of water, mix well to dissolve.

   • Test tubes

   • test tubes racks

   • Pipettes and pipette tips

   • Iodine's reagent

   • Boiling water bath

Interpretation

Positive result shows the presence of starch 

Reference Material

1. Experimental Biochemistry, A student Companion by Beedu Sashidhar Rao and Vijay Deshpande.

Questions

1. In Benedict’s test, reducing sugars reduce cupric hydroxide to what? 

2. Name other test for carbohydrate estimation

3. Function of carbohydrate bio-molecules in body?