Principle Procedure Reactions and Results of Protein Tests
Proteins are important biological compounds that have a higher molecular weight. They are probably nature’s most complex organic materials. Plants tend to build their proteins from minerals, carbon dioxide, and water in the presence of sunlight. These compounds are the most abundant organic compounds found in nature. In every living organism, they are present. Animals tend to get their share of proteins from plants. Life is linked with the existence of proteins.
The proteins are identified by some laboratory tests. These tests are discussed below along with its theory, required material for tests, and observation. The aim of the protein laboratory test is discussed below.
Aim
The aim of this article to perform a total protein lab test for identifying the presence of proteins in the samples given.
Theory
Proteins possess a higher molecular mass of long-chain polymers that consist of α-amino acids. Cells contain proteins and they are therefore present in all living bodies. Proteins consist of carbon, oxygen, nitrogen, hydrogen, and often sulphur and phosphorus. There are a few tests that are carried out for finding the presence of proteins in the samples given.
These laboratory tests of proteins are:
Biuret test
Millions test
Xanthoproteic test
Ninhydrin test
Each of the tests is discussed below along with the chemical reaction involved in the test.
Biuret Test
The compounds having peptide linkage undergo the Biuret test. Proteins are known as the polypeptides of amino acids that are linked together by the peptide bonds. A protein alkaline solution is treated with one drop of aqueous copper sulphate and a bluish violet colour is seen.
(Image will be uploaded soon)
The Biuret test is useful in the identification of proteins and protein estimation. Biuret is a kind of chemical that is formed when urea gets heated to 180 ℃. During this reaction, two molecules of urea tend to condense and form a biuret or a bi-urea molecule. The biuret reagent forms a complex of violet colour in the presence of copper ions.
Note: The formation of the violet colour confirms that the proteins are present.
Xanthoproteic Test
When proteins are treated with nitric acid, it gives an orange or yellow colour. The concentrated nitric acid gets used for the nitration process. When proteins are treated with nitric acid, it gives a yellow precipitate that later turns to orange colour when it is treated with an alkali.
(Image will be uploaded soon)
Note: The appearance of the yellow coloured solution confirms that proteins are present.
Millon’s Test
The phenolic group of the tyrosine compound of proteins tends to react to the mercuric sulphate in the presence of sulphuric acid and sodium nitrate and gives a red colour. The Million’s test is done on the proteins that have phenolic amino acids. However, gelatin does not show this test. Initially, a white precipitate is formed when the proteins are treated with the Million’s reagent and they then turn into brick red in colour when boiled. This confirms that proteins are present.
(Image will be uploaded soon)
Note: When a brick red solution appears, it confirms the presence of the proteins.
Ninhydrin Test
Proteins undergo a reaction with the pyridine solution of ninhydrin to give a coloured solution. This colour ranges from deep blue to violet-pink and even red in a few cases. The ninhydrin solution is prepared when 0.1g of ninhydrin is dissolved in 100ml distilled water. However, this ninhydrin is highly unstable and can only be kept for 2 days. The chemical reaction is as follows.
(Image will be uploaded soon)
Note: The appearance of the violet coloured solution confirms that the proteins are present.
Materials Required for the Protein Lab Test
The list of materials are given below that are required for the protein lab test.
Sodium hydroxide
Pyridine solution
Ninhydrin reagent
Copper sulfate solution
Nitric acid
Sodium nitrite
Sulfuric acid
Mercuric sulfate
Distilled water
Dropper
Test tubes
Test tube holder
Stirrer
Water bath
Apparatus Setup of the Protein Total Spot Urine Test
Given below is the setup for the test to give low protein lab results.
(Image will be uploaded soon)
Procedure
The procedures of the laboratory test of proteins are as follows:.
Biuret Test:
Take the sample given that is to be tested in a test tube.
Then add 2ml of the sodium hydroxide solution to the sample.
To this add about 5 to 6 drops of the copper sulfate solution.
If a bluish violet colour appears, it indicates the presence of the proteins.
Xanthoproteic Test:
Take 2ml of the sample in a clean test tube.
Add a few drops of the concentrated sulfuric acid to it and then heat.
If a yellow precipitate is seen to be formed, it confirms the presence of the proteins.
Millions Test:
Take 2ml of sample solution given in a test tube.
To it add about 2–3 drops of the Millon’s reagent and then shake well.
Observe any changes.
If a white precipitate is seen which then changes to brick red when heated, then it confirms the presence of the proteins.
Ninhydrin Test:
Take the given sample solution that is to be tested in a test tube.
Add about 1–2ml of the ninhydrin solution to it.
Then boil the mixture and observe any changes.
If the solution turns blue in colour, then it confirms the presence of the proteins.
Observation and Inference from the Protein Laboratory Test
Results and Discussions
The given sample contains _________ (proteins).
Precautions
Handle the chemicals with care when performing the experiments and tests.
Wear lab aprons and gloves while performing the experiments.
Use only the droppers for taking the reagents from the bottles.
Viva Questions for the Total Protein Lab Test
1. What are proteins?
Answer: Proteins are made of smaller units known as amino acids which are attached in the form of longer chains to one another. Twenty different types of amino acids can be combined together to form a protein molecule.
2. What are the two different types of proteins?
Answer: The two different kinds of proteins are fibrous proteins and globular proteins.
3. What happens when a protein undergoes hydrolysis?
Answer: When proteins undergo the process of hydrolysis, they form α-amino acids.
4. How can amino acids together form a polypeptide?
Answer: Two amino acids get linked together when a water molecule is lost. The amino acids that are joined together by the peptide bonds tend to form a chain of polypeptides and every unit of the amino acids in the polypeptide is referred to as the residue.
5. What do you mean by the monomer and polymer of the proteins?
Answer: A monomer is referred to as a molecule that forms bigger polymer molecules. It is known to be the building block of proteins. For example, amino acids are the building blocks of proteins. A polymer is known as a protein of the monomer series.
Conclusion
There are many important functions that are performed by proteins which are essential for all forms of life. They are important structural molecules. In this article, we learn all the necessary information of laboratory tests of proteins.
FAQs on Laboratory Test Of Proteins in Chemistry
1. What are the laboratory tests for proteins?
The main laboratory tests for proteins are the Biuret test, Xanthoproteic test, Millon’s test, Ninhydrin test, and heat coagulation test. These qualitative tests detect proteins based on their chemical properties.
- Biuret test: Detects peptide bonds (violet color).
- Xanthoproteic test: Detects aromatic amino acids (yellow/orange color).
- Millon’s test: Detects tyrosine (red color).
- Ninhydrin test: Detects free amino groups (purple color).
- Heat coagulation test: Shows protein denaturation on heating.
2. What is the Biuret test for proteins?
The Biuret test is a chemical test that detects peptide bonds in proteins by producing a violet color in alkaline copper sulfate solution. When a protein solution is treated with sodium hydroxide (NaOH) and a few drops of copper(II) sulfate (CuSO4), a violet or purple complex forms.
- Reagents: Dilute NaOH + CuSO4
- Observation: Violet or purple color
- Inference: Presence of two or more peptide bonds
3. How does the Biuret test detect peptide bonds?
The Biuret test detects peptide bonds because Cu2+ ions form a violet-colored coordination complex with nitrogen atoms in peptide linkages under alkaline conditions. In the presence of NaOH, peptide bonds (–CO–NH–) bind with copper(II) ions to produce a colored complex.
- Alkaline medium is essential.
- At least two peptide bonds are required.
- Color change: Blue → Violet.
4. What is the Xanthoproteic test for proteins?
The Xanthoproteic test is a chemical test that detects aromatic amino acids in proteins by forming a yellow or orange color with concentrated nitric acid (HNO3). When protein is treated with conc. HNO3, nitration of aromatic rings occurs.
- Reagent: Concentrated HNO3
- Observation: Yellow color (turns orange on adding NaOH)
- Amino acids detected: Tyrosine, tryptophan, phenylalanine
5. What is Millon’s test and what does it detect?
The Millon’s test is a qualitative test that detects the amino acid tyrosine in proteins by producing a red color. When protein containing tyrosine is treated with Millon’s reagent (mercuric nitrate in nitric acid) and heated, a red precipitate or solution forms.
- Reagent: Millon’s reagent
- Observation: Red coloration
- Specific for: Phenolic –OH group of tyrosine
6. What is the Ninhydrin test for proteins?
The Ninhydrin test detects free amino groups (–NH2) in amino acids and proteins by producing a purple or blue color. When ninhydrin reacts with free amino acids upon heating, it forms a colored complex called Ruhemann’s purple.
- Reagent: Ninhydrin solution
- Observation: Purple/blue color
- Special case: Proline gives yellow color
7. What is the heat coagulation test for proteins?
The heat coagulation test identifies proteins based on their ability to denature and coagulate when heated. When a protein solution (such as egg albumin) is heated, it forms a white coagulum due to structural denaturation.
- Observation: Formation of white precipitate
- Cause: Breakdown of secondary and tertiary structure
- Example: Boiling egg white
8. What is the difference between the Biuret test and the Ninhydrin test?
The main difference is that the Biuret test detects peptide bonds in proteins, while the Ninhydrin test detects free amino groups in amino acids.
- Biuret test: Requires two or more peptide bonds; gives violet color.
- Ninhydrin test: Reacts with free –NH2 groups; gives purple color.
- Biuret does not detect free amino acids effectively.
- Ninhydrin is commonly used in chromatography detection.
9. Why does protein give a violet color in the Biuret test?
Proteins give a violet color in the Biuret test because Cu2+ ions form a colored coordination complex with nitrogen atoms of peptide bonds in alkaline medium. The interaction between copper(II) ions and –CO–NH– groups produces the characteristic purple color.
- Alkaline condition provided by NaOH
- Complex formation with peptide nitrogen
- Intensity increases with protein concentration
10. How can you confirm the presence of protein in a laboratory sample?
The presence of protein in a laboratory sample can be confirmed by performing the Biuret test, which produces a violet color in alkaline copper sulfate solution.
- Add NaOH to make the solution alkaline.
- Add a few drops of CuSO4 solution.
- Observe violet coloration.
