Theory & Procedure, Qualitative Analysis of Proteins | Additional Study Material for NEET PDF Download

Objective

Our objective is to study some simple tests of proteins.

The Theory

Food is a necessary material which must be supplied to the body for its normal and proper functioning. It is the main source of energy and promotes growth. It regulates body processes like assimilation and digestion and sustains life. Every good food contains some important nutrition like proteins, carbohydrates, fats, vitamins, minerals and water. All these nutrients are important for health and they work together to build new cells in our body and keep the body working properly.

What are Proteins?

Protein is an important macronutrient essential for survival. They are constituent of calls and hence are present in all living bodies. 10-35% of calories should come from protein. Protein is found in meats, poultry, fish, meat substitutes, cheeses, milk etc.

Proteins are large biological molecules composed of α-amino acids (Amino acid in which amino group is attached to α-carbon, which exist as zwitter ions and are crystalline in nature). They contain carbon, hydrogen, oxygen, nitrogen and sometimes phosphorous and sulphur.

Amino acids are molecules contain both amino (NH₂) and carboxylic (COOH) group. Amino acid molecules undergo condensation reaction to form a specific type of linkage known as peptide linkage.

Depending on the number of amino acid molecules involved in the condensation reaction, the products formed are classified as;

Dipeptide

They are the products formed by the condensation of two α-amino acid molecules.

Tripeptide

They are formed by the condensation of three α-amino acid molecules.

If large number of amino acid molecules combine, the product formed is called polypeptide. A polypeptide having molecular mass greater than 10000 is called a protein. Proteins differ from one another primarily in their sequence of amino acid. There are about more than 20 amino acids. Some amino acids are not made by the body and are supplied through diet. They are called essential amino acids.

Some Important Tests for the Detection of Proteins

Biuret test

This test is used to detect the presence of peptide bond. When treated with copper sulphate solution in presence of alkali (NaOH or KOH), protein reacts with copper (II) ions to form a violet coloured complex called biuret.

Xanthoproteic test

It is an identification test of protein and it gives a positive result with those proteins with amino acid carrying aromatic group. When protein is treated with hot concentrated nitric acid, a yellow coloured substance is formed. The yellow colour is due to xanthoproteic acid which is formed by the nitration of certain amino acids present in protein such as tyrosine and tryptophan.

Ninhydrin test

This is a test for amino acids and proteins with free –NH₂ group. When such an –NH₂ group reacts with ninhydrin, an intense blue coloured complex is formed.

Millon’s test

When egg albumin is treated with Millon’s reagent, it first gives a white coloured precipitate which then changes to brick red on boiling. Gelatin does not give this test.

Learning Outcome

• Students understand various identification tests for proteins.
• Students acquire skill to perform the experiment in the real lab.

Materials Required

Samples

• Egg albumin
• Gelatin

Reagents

• NaOH solution
• 1% CuSO₄ solution
• Conc. HNO₃
• Ninhydrin solution
• Millon’s reagent

Apparatus

• Test tube
• Dropper

Procedure

Real Lab Procedure

Biuret test

Take a small quantity of the dispersion of the sample in a test tube and add 2 ml of NaOH solution into it. Now add 4-5 drops of 1% CuSO₄ solution and warm the mixture for about 5 minutes.

Observation

Inference

Bluish violet colouration indicates the presence of protein.

Xanthoproteic test

Take about 2 ml of the sample in a test tube and add few drops of conc. HNO₃ into it and heat the test tube.

Observation

Inference

Yellow precipitate indicates the presence of protein.

Ninhydrin test

Take 2 ml of the sample in a test tube and add 3-4 drops of Ninhydrin solution and boil the contents.

Observation

Inference

Intense blue colouration confirms the presence of protein.

Millon’s test

Take 1-2 ml of the sample in a test tube and add 2 drops of Millon’s reagent.

Observation

Inference

White precipitate which changes to brick red on boiling indicates the presence of protein.

Simulator Procedure (as performed through the Online Labs)

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

Biuret Test

• Drag the dropper to the test tubes to drop NaOH solution into tubes A and B respectively.
• Drag the dropper to the test tubes to drop 1% copper sulphate solution into tubes A and B respectively.
• Click on the knob of the burner to turn it on.
• Drag test tube A to the burner to heat the contents of the test tube.
• Click on the inference icon to see the inference.
• Drag test tube B to the burner to heat the contents of the test tube.
• Click on the inference icon to see the inference.

Xanthoproteic Test

• Drag the dropper to the test tubes to drop Conc. Nitric acid into tubes A and B respectively.
• Click on the knob of the burner to turn it on.
• Drag test tube A to the burner to heat the contents of the test tube.
• Click on the inference icon to see the inference.
• Drag test tube B to the burner to heat the contents of the test tube.
• Click on the inference icon to see the inference.

Ninhydrin Test

• Drag the dropper to the test tubes to drop Ninhydrin solution into tubes A and B respectively.
• Click on the knob of the burner to turn it on.
• Drag test tube A to the burner to heat the contents of the test tube.
• Click on the inference icon to see the inference.
• Drag the test tube B to the burner to heat the contents of the test tube.
• Click on the inference icon to see the inference.

Millon’s Test

• Drag the dropper to the test tube A to drop Millon’s reagent into it.
• Click on the inference icon to see the inference.
• Drag the dropper to the test tube B to drop Millon’s reagent into it.
• Click on the knob of the burner to turn it on.
• Drag the test tube A to the burner to heat the contents of the test tube.
• Click on the inference icon to see the inference.
• Drag the test tube B to the burner to heat the contents of the test tube.
• Click on the inference icon to see the inference.

To redo the experiment, click on the ‘Reset’ button.

Note: Click on the ‘HELP’ button to see the instruction.

Precautions

• Handle the chemicals with care.
• Heating and Boiling should be done very carefully.
• Use droppers to take reagents from the bottles.