Experiment 3

Introduction

Proteins are polymers of amino acids. They are complex organic compounds containing nitrogen, hydrogen, carbon and oxygen. Proteins are abundant in our everyday food e.g. egg, soya bean, pulses, fish, milk etc. Presence of proteins can be confirmed qualitatively by several tests. Due to the presence of characteristic side chains in them, certain amino acids exhibit typical color reactions that form the basis for their identification. In this experiment, the following tests will be performed for the qualitative analysis of proteins.

Materials required

Glasswares: Test tubes, Beakers

Reagents: Distilled water, Concentrated Sulphuric acid, Concentrated Nitric acid

Other requirements: Test tube holder, Test Tube stand, Bunsen burner, Boiling water Bath, Pipettes/Dropper

Instructions:

1. Read entire procedure carefully before starting the experiment.

2. Always use clean and dry glasswares.

3. Standard protein samples provided are Standard Protein 1, Standard Protein 2 and Standard Protein 3 (BSA, Casein and Gelatin).

4. Protein samples 1 and 2 are provided as test samples.

5. Perform all the experiments very carefully as the concentrated acids are very hazardous.

NOTE: Take 5 test tubes and label it as Standard Protein 1, Standard Protein 2, Standard Protein 3, Protein sample 1 and 2, individually for all the below mentioned tests.

1.Biuret Test

Principle:

The Biuret test for proteins qualitatively detects the presence of proteins in solution with the development of a deep violet colour. In alkaline condition, Biuret (NH2-CO-NH-CO-NH2) reacts with compounds containing two or more peptide bonds to form complexes of violet colour. The name of this test comes from the name of the compound, Biuret which gives this test.

2. Ninhydrin Test

 Principle:

 Amino acids contain a free amino and carboxyl group which react together with ninhydrin to produce a coloured product. In the pH range of 4-8, all α- amino acids react with ninhydrin, a powerful oxidizing agent to give a purple colored product termed Rhuemann’s purple.  Amino acids that have secondary amino group attachments also react with ninhydrin and forms yellow coloured reaction product instead of purple colored product.  Peptides, peptones and proteins also react positively when subjected to the ninhydrin reaction.  

Procedure:

1. Add 1 ml of each standard and protein samples to separate test tubes. 

2. Add 200 μl of Ninhydrin reagent and boil for 1 minute and and observe the results.

3. Suphur test

Principle:

 Sulphur is present in proteins as cystine, cysteine or methionine. In alkaline media, lead acetate reacts to form black or brown coloured precipitate of PbS. 

Procedure:

1. To 1 ml of each standard and protein sample, add 1 ml of lead acetate solution. Mix well. 

2. Add 0.2 ml of 1 M NaOH solution dropwise till precipitate dissolves. Boil for 1 minute and observe the results.

Observation and Result: 

After performing each test, record the results of the standard and test proteins as in the following table:

Positive result is indicated by the following observations: 

1. Biuret Test: The formation of violet coloured product 

2. Ninhydrin Test: The formation of dark blue color 

3. Sulphur Test: The formation of brown or black colour 

Interpretation: 

The different types of proteins are identified by the characteristic colour observed after performing each test and characterized by using various qualitative methods of protein estimation. 

Reference Material

1. Himedia protocol

2. Qualitative Analysis of Proteins - MeitY OLabs

3. Qualitative Analysis of Proteins - Animation

4. Simulation

Questions

1. What qualitative reaction can be used to prove the presence of a peptide bond? 

2. Do you think free amino acids will give a positive result with biuret test ? why? 

3. What is the least number of amino acids bonded together by peptide bonds that will respond positively to biuret test? 

4. What qualitative test can be used for proteins having cysteine and cystine aminoacids ?