Theory & Procedure, Detection of Carbohydrates, Proteins and Fats | Additional Study Material for NEET PDF Download

Objective

Our objective is to detect the presence of carbohydrates, protein and fats and to show their presence in suitable plant and animal materials.

Theory

The food we eat is one of the necessary factors in our daily life that provides nutritional support for the human body. Food consists of both organic and inorganic substances. Carbohydrates, fats and proteins are the main organic substances present in the food, which provide energy.

Let’s start with carbohydrates.

What are Carbohydrates?

One of the main components of our daily diet is carbohydrates. This type of foods includes sugars, starch and fibres. They are composed of sugar molecules that contain carbon, hydrogen and oxygen.

Carbohydrates are classified into simple carbohydrates and complex carbohydrates.

Simple Carbohydrates

Simple carbohydrates are composed of one or two sugar units. Because of their smaller size, simple carbohydrates can be broken down and can be absorbed by the body easily. So they act as the quickest source of energy. They are found in fruits in the form of fructose, milk in the form of lactose and table sugar in the form of sucrose.

There are two types of simple carbohydrates: Monosaccharides and Disaccharides.

Monosaccharides are the simplest carbohydrates, consisting of only one sugar unit.  Glucose, fructose and galactose are examples of monosaccharides. They have the capability of reducing cupric (Cu²⁺) ions into cuprous (Cu⁺) ions due to the presence of free aldehydic and ketonic groups  and are called reducing sugars. These reducing sugars give positive results in Benedict’s test and Fehling’s test because they reduce the cupric ion present in the Benedict’s and Fehling’s solution and form a precipitate of cuprous oxide. Depending upon the concentration of sugar, green, orange or brick red precipitates are obtained.

Disaccharides are composed of two chemically-linked monosaccharide units. Sucrose, lactose and maltose are examples of disaccharides. Sucrose is a non-reducing disaccharide. When it is boiled with HCl, sucrose undergoes hydrolysis to form glucose and fructose, which gives positive result with Benedict’s and Fehling’s solutions.

Complex Carbohydrates

Complex carbohydrates are composed of long chains of simple carbohydrate units. Because of their larger size, they can be broken down into simple carbohydrates.

Complex carbohydrates can be classified as Oligosaccharides and Polysaccharides.

Oligosaccharides consist of less than 10 monosaccharide units. Disaccharides are also a class of oligosaccharides. Raffinose and stachyose are examples of oligosaccharides.

Carbohydrates, made up of large number of monosaccharide units, are called polysaccharides. Starch, glycogen and cellulose are example of polysaccharides. Starch gives a blue-black complex with iodine.

What are Proteins?

Proteins are large biological molecules made up of large number of amino acid units. Amino acids are molecules consisting of both the amino (-NH₂) group and carboxylic group (-COOH). In proteins, the amino acid units are linked together by specific linkages called peptide linkages. Because of the complex nature of protein, our body takes a longer time to break down protein molecules. Compared to carbohydrates, proteins are a much slower and longer-lasting source of energy. Most proteins are soluble in acidic or alkaline solutions, but insoluble in water.

Proteins give colour reactions due to one or more radicals or groups present in the complex protein molecules. All proteins do not give all colour reactions because all of them do not contain the same amino acid units. This property of protein can be used for the detection of protein in a given sample.

Biuret test is a method used for the detection of peptide bond in a protein molecule. In the Biuret test, the nitrogen atoms in the peptide chain react with copper ions in the reagent to form a violet coloured complex.

Xanthoproteic test is used for the identification of protein containing aromatic amino acid units.  By heating with nitric acid, the benzene ring in the amino acid unit is nitrated and forms a yellow coloured nitro-compound which turns to orange colour with alkali.

What are Fats?

Fats are complex molecules made up of fatty acids and glycerol. Our body needs fats for growth and energy. Fats contain carbon, hydrogen and sometimes oxygen. Phosphorous, nitrogen and sulphur are also present in some fats. They are insoluble in water, but soluble in non polar s like chloroform and benzene. They are found stored in many oil seeds and some animal tissues. They produce translucent spots on paper due to the diffraction of light. They also give a pink colour with azo dye, sudan III.

Learning Outcomes:

• Students understand the characteristics of carbohydrates, proteins and fats.
• Students learn the tests to detect the presence of carbohydrates, proteins and fats.
• Students will be able to do the experiment more accurately in the real lab once they understand the steps through the animation and simulation.

Real Lab Procedure

Test for Carbohydrates

1. Test For Glucose
   1. Benedict's Test

Materials Required:

Procedure:

• Using a dropper, take a small quantity of Benedict’s reagent.
• Add the reagent to the test tube containing banana extract.
• Boil the sample over a burner for 2 minutes holding the test tube firmly with a test tube holder.
• Keep shaking the test tube as it is being heated.
• A brick red precipitate appears, indicating the presence of glucose in the banana extract.

Conclusion:

On boiling banana extract with the Benedict’s reagent, the cupric ion present in the Benedict’s reagent is reduced by the reducing agent, sugar, to form a brick red coloured precipitate of cuprous oxide.

2. Fehling's Test

Materials Required:

Procedure:

• Using a dropper, take a small quantity of Fehling’s solution A.
• Add the Fehling’s solution A to the test tube containing banana extract.
• Using a dropper, take a small quantity of Fehling’s solution B.
• Add Fehling’s solution B to the test tube containing banana extract.
• Boil the sample over a burner for 2 minutes, holding the test tube firmly with a test tube holder.
• Keep shaking the test tube while heating.
• A brick red precipitate appears, indicating the presence of glucose in the banana extract.

Conclusion:

The cupric ion present in the Fehling’s solution is reduced on boiling by the reducing substance, sugar, to form the brick red coloured precipitate of cuprous oxide.

2. Test for Sucrose

Materials Required:

Procedure:

• Using a dropper, take a small quantity of concentrated HCl.
• Add 2 to 3 drops of concentrated HCl to the test tube containing sugarcane extract.
• Boil the sample over a burner for 2 minutes, holding the test tube firmly with a test tube holder.
• This hydrolyses sucrose into glucose and fructose.
• Using a dropper, take a small quantity of NaOH solution.
• Add few drops of NaOH solution to the test tube to make the solution alkaline.
• Now we can perform Benedict’s test to this solution to test for the presence of glucose.
• Using a dropper, take a small quantity of Benedict’s reagent.
• Add the reagent to the test tube containing the sample.
• Boil the sample over a burner for 2 minutes, holding the test tube firmly with a test tube holder.
• The colour changes from blue to green and finally to orange or brick red, indicating the presence of glucose.

3. Test for Starch

Materials Required

Procedure

• Using a dropper, take a small quantity of iodine solution.
• Add 5 drops of iodine solution to the test tube containing potato extract.
• Blue black colour indicates the presence of starch in potato extract.

Test for Proteins

1. Biuret Test

Materials Required

Procedure

• Using a dropper, take a small quantity of 40% NaOH solution.
• Add a few drops of NaOH solution to the test tube containing egg albumin.
• Using a dropper, take a small quantity of 1% CuSO4 solution.
• Add 2-3 drops of CuSO4 solution to the test tube containing egg albumin.
• Shake the solution to mix it well.
• A violet colour appears in the test tube, which indicates the presence of proteins.

2. Xanthoproteic Test

Materials required

Procedure

• Using a dropper, take a small quantity of concentrated HNO3.
• Add 5 drops of Concentrated HNO3 to the test tube containing egg albumin.
• Boil the sample over a burner for 2 minutes, holding the test tube firmly with a test tube holder. Yellow precipitate appears in the test tube.
• Using a dropper, take a small quantity of ammonia solution.
• Add a few drops of ammonia solution to the sample.
• Shake the solution to mix it well.
• Yellow ppt. changes to orange in colour, which indicates the presence of protein.

3. Million’s Test

Materials Required

Procedure

• Using a dropper, take a small quantity of Million's regent.
• Add few drops of Million's reagent to the test tube containing egg albumin.
• Wait for some time.
• Pink colour appears in the test tube, which indicates the presence of protein.

Test for Fats

1. Sudan III Test

Materials Required:

Procedure:

• Using a dropper, take a small quantity of Sudan III reagent.
• Add few drops of Sudan III reagent to the test tube containing egg albumin.
• Shake the solution to mix it well.
• Pink droplets appear indicating the presence of fat in the sample.

2. Paper Spot Test

Materials Required:

Peanut seeds and piece of white paper.

Procedure:

• Take a peanut seed from the watch glass.
• Crush the peanut seed and rub it on a piece of white paper.
• Paper becomes translucent at the spot, which indicates the presence of fat.

Simulator Procedure (as performed through the Online Labs)

You can select the type of test from the ‘Select the food substance’ drop down list.

Carbohydrates

You can select the type of test from the ‘Select the carbohydrate’ drop down list.

1. Glucose

You can select the type of test from the ‘Select the Test” drop down list.

1. Benedict’s Test

• Click and drag the dropper from the Benedict’s reagent bottle and move it into the test tube containing banana extract to drop the Benedict’s reagent into it.
• Drag the test tube towards the beaker to place it in the water bath.
• Click on the switch of the hot plate to turn it on.
• Click on the inference icon to see the inference.

2. Fehling’s Test

• Click and drag the dropper from Fehling’s solution A and move it into the test tube containing banana extract to drop the Fehling’s solution A into it.
• Click and drag the dropper from the Fehling’s solution B bottle and move it into the test tube containing banana extract to drop the Fehling’s solution B into it.
• Drag the test tube towards the beaker to place in the water bath.
• Click on the switch of the hot plate to turn it on.
• Click on the inference icon to see the inference.

2. Sucrose
   1. Benedict’s Test

• Click and drag the dropper from the concentrated HCl bottle and move it into the test tube containing sugarcane extract to drop the HCl into it.
• Click on the knob of the burner to turn it on.
• Drag the test tube towards the burner to heat it.
• Click on the inference icon to see the inference.
• Click and drag the dropper from the NaOH solution bottle and move it into the test tube containing sugarcane extract to drop the NaOH into it.
• Drag the dropper towards test tube B to drop NaOH slowly along the side of the test tube.
• Click on the next button to continue.
• Click and drag the dropper from the Benedict’s reagent bottle and move it into the test tube to drop Benedict’s reagent into it.
• Drag the test tube towards the beaker to place in the water bath.
• Click on the switch of the hot plate to turn it on.
• Click on the inference icon to see the inference.

2. Fehling’s Test

• Click and drag the dropper from the concentrated HCl bottle and move it into the test tube containing sugarcane extract to drop the HCl into it.
• Click on the knob of the burner to turn it on.
• Drag the test tube towards the burner to heat it.
• Click on the inference icon to see the inference.
• Click and drag the dropper from the NaOH solution bottle and move it into the test tube containing sugarcane extract to drop the NaOH into it.
• Click on the next button to continue.
• Click and drag the dropper from Fehling’s solution A and move it into the test tube to drop the Fehling’s solution A into it.
• Click and drag the dropper from the Fehling’s solution B bottle and move it into the test tube to drop Fehling’s solution B into it.
• Drag the test tube towards the beaker to place in the water bath.
• Click on the switch of the hot plate to turn it on.
• Click on the inference icon to see the inference.

3. Starch
   1. Iodine Test

• Click and drag the dropper from the Iodine bottle and move it into the test tube containing potato extract to drop the Iodine solution into it.
• Click on the inference icon to see the inference.

2. Benedict’s Test

• Click and drag the dropper from the concentrated HCl bottle and move it into the test tube containing potato extract to drop the HCl into it.
• Click on the knob of the burner to turn it on.
• Drag the test tube towards the burner to heat it.
• Click on the inference icon to see the inference.
• Click and drag the dropper from the NaOH solution bottle and move it into the test tube to drop the NaOH into it.
• Click on the next button to continue.
• Click and drag the dropper from the Benedict’s reagent bottle and move it into the test tube to drop the Benedict’s reagent into it.
• Drag the test tube towards the beaker to place in the water bath.
• Click on the switch of the hot plate to turn it on.
• Click on the inference icon to see the inference.

3. Fehling’s Test

• Click and drag the dropper from the concentrated HCl bottle and move it into the test tube containing potato extract to drop the HCl into it.
• Click on the knob of the burner to turn it on.
• Drag the test tube towards the burner to heat it.
• Click on the inference icon to see the inference.
• Click and drag the dropper from the NaOH solution bottle and move it into the test tube to drop the NaOH into it.
• Click on the next button to continue.
• Click and drag the dropper from Fehling’s solution A and move it into the test tube to drop Fehling’s solution A into it.
• Click and drag the dropper from the Fehling’s solution B bottle and move it into the test tube to drop Fehling’s solution B into it.
• Drag the test tube towards the beaker to place in the water bath.
• Click on the switch of the hot plate to turn it on.
• Click on the inference icon to see the inference.

Proteins

1. Biuret Test

• Click and drag the dropper from the NaOH solution bottle and move it into the test tube containing egg albumin to drop the NaOH into it.
• Click and drag the dropper from the CuSO4 solution bottle and move it into the test tube containing egg albumin to drop the CuSO4 into it.
• Click on the inference icon to see the inference.

2. Xanthoproteic Test

• Click and drag the dropper from the concentrated HNO3 solution bottle and move it into the test tube containing egg albumin to drop the HNO3 into it.
• Click on the knob of the burner to turn it on.
• Drag the test tube towards the burner to heat it.
• Click and drag the dropper from the NH4OH solution bottle and move it into the test tube containing egg albumin to drop the NH4OH into it.
• Click on the inference icon to see the inference.

3. Million’s Test

• Click and drag the dropper from the concentrated Million’s reagent bottle and move it into the test tube containing egg albumin to drop the Million’s reagent into it.
• Click on the inference icon to see the inference.

Fats

1. Sudan III Test

• Click and drag the dropper from the concentrated Sudan III reagent bottle and move it into the test tube containing coconut oil to drop the Sudan III reagent into it.
• Click on the inference icon to see the inference.

2. Paper Spot Test

• Drag the hand towards the watch glass to crush the peanut.
• Drag the hand towards the paper to press it.
• Click on the inference icon to see the inference.
• You can repeat the experiment by clicking on the ‘Reset’ button.