The biuret test is a biochemical test commonly used to identify the presence of protein in the given same. This colorimetric based test is based on the reaction between peptide bonds and copper ions, resulting in a distinctive violet or purple color.
Proteins are polymers of amino acids. A peptide bond is formed by the combination of amino acids in which the amine group (–NH₂) of one amino acid reacts with the carboxylic acid (–COOH) of another amino acid. During this reaction a water molecule is removed, forming an amide group (CO-NH). The result of the above reaction was one water molecule removed and two amino acids joined together.
Biuret test needs testing the sample with the biuret reagent. The reagent is a mixture of potassium sodium tartrate (KNaC₄H₄O₆⋅ 4H₂O), copper (II) sulfate or cupric sulfate (CuSO4), and sodium hydroxide (NaOH). Sodium hydroxide in the solution makes the solution alkaline and potassium sodium tartrate acts as the chelating agent.
Under alkaline conditions, the copper (II) ions (Cu²⁺) binds to the nitrogen atoms of the peptide bonds. The nitrogen atoms donate their lone pairs of electrons to the copper ion to form covalent bonds. One copper ion joins with four nearby peptides. Together this reaction forms a colored chelate complex. This complex absorbs light at 540 nm and this appears violet color.
The main objective of the biuret test is to detect the protein in the given sample or a solution. To detect the presence of the peptide pond.
The copper (II) present in reagent binds itself to the nitrogen atoms that are the present in the protein peptides. Now four nitrogen atoms donate their lone pairs of electrons to form covalent bonds with the copper (II) ions. This reaction results in the formation of a chelate complex, this complex has the ability to absorb light with a wavelength of 540 nm, which gives a purple or violet colour to it.
Dissolve 1 gram of Copper sulfate (CuSO4) in 100 milliliter of distilled water. Add 1.2 grams of Sodium potassium tartrate to the copper sulfate solution. Adding sodium potassium tartrate acts as a chelating agent and it helps stabilize the copper ions.
Dissolve 10 grams of Sodium hydroxide (NaOH) in 90 ml of distilled water to make the 10% NaOH solution. Add 10 ml of the 10% NaOH solution to the 100 ml of 1% copper sulfate solution.
Take the three clean test tubes and mark them as test, positive control and negative control.
In the first tube, add 1 - 2 ml of test sample. Likewise add 1 - 2 ml of albumin solution in the positive control tube, in the third tube add 1 - 2 ml of distilled water.
Then add 1 - 2 ml of biuret reagent in all three tubes. After that, shake all the tubes to mix the reagent and samples.
After mixing the samples, leave the tubes in the stand for at least 5 minutes. Observe the tubes for the development of violet color in the tubes.
Positive Biuret Test Result: Violet or purple color appears denotes presence of proteins (peptide bonds).
Negative Biuret Test Result: No color change (solution remains blue), sample lacks significant protein content.
The biuret test is used mainly for diagnostic purposes, like determining serum proteins. Here are some other applications of these tests;
Biuret test helps in finding the proteins in the given unknown sample.
It is used for quantification of protein by using a spectrophotometer.
Biuret test help determine proteins in the urine, CSF, and other body fluids.The test helps to find the presence of specific proteins during food analysis.
Use sample and reagent in the recommended 1:1 volume, 1:1 ratio always gives a better result.
Avoid adding excessive Biuret reagent, too much reagent keeps the solution blue instead of purple giving false negative.
Do not read the result before 5 minutes. Reading before 5 minutes might give you false negative results.
Samples with high amounts of amino acid histidine can give a false positive result.
If the buffer used to purify protein has an ammonium or magnesium it can affect the test.
This test alone is not enough for quantifying the amount of protein in the given sample, further spectrophotometric analysis required.
Proteins are polymers of amino acids. A peptide bond is formed by the combination of amino acids in which the amine group (–NH₂) of one amino acid reacts with the carboxylic acid (–COOH) of another amino acid. During this reaction a water molecule is removed, forming an amide group (CO-NH). The result of the above reaction was one water molecule removed and two amino acids joined together.
Biuret test needs testing the sample with the biuret reagent. The reagent is a mixture of potassium sodium tartrate (KNaC₄H₄O₆⋅ 4H₂O), copper (II) sulfate or cupric sulfate (CuSO4), and sodium hydroxide (NaOH). Sodium hydroxide in the solution makes the solution alkaline and potassium sodium tartrate acts as the chelating agent.
Under alkaline conditions, the copper (II) ions (Cu²⁺) binds to the nitrogen atoms of the peptide bonds. The nitrogen atoms donate their lone pairs of electrons to the copper ion to form covalent bonds. One copper ion joins with four nearby peptides. Together this reaction forms a colored chelate complex. This complex absorbs light at 540 nm and this appears violet color.
Objectives of Biuret Test
The main objective of the biuret test is to detect the protein in the given sample or a solution. To detect the presence of the peptide pond.
Principle of Biuret Test
The copper (II) present in reagent binds itself to the nitrogen atoms that are the present in the protein peptides. Now four nitrogen atoms donate their lone pairs of electrons to form covalent bonds with the copper (II) ions. This reaction results in the formation of a chelate complex, this complex has the ability to absorb light with a wavelength of 540 nm, which gives a purple or violet colour to it.
Preparation of Biuret Reagent
Dissolve 1 gram of Copper sulfate (CuSO4) in 100 milliliter of distilled water. Add 1.2 grams of Sodium potassium tartrate to the copper sulfate solution. Adding sodium potassium tartrate acts as a chelating agent and it helps stabilize the copper ions.
Dissolve 10 grams of Sodium hydroxide (NaOH) in 90 ml of distilled water to make the 10% NaOH solution. Add 10 ml of the 10% NaOH solution to the 100 ml of 1% copper sulfate solution.
Procedure of Biuret Test
Take the three clean test tubes and mark them as test, positive control and negative control.
In the first tube, add 1 - 2 ml of test sample. Likewise add 1 - 2 ml of albumin solution in the positive control tube, in the third tube add 1 - 2 ml of distilled water.
Then add 1 - 2 ml of biuret reagent in all three tubes. After that, shake all the tubes to mix the reagent and samples.
After mixing the samples, leave the tubes in the stand for at least 5 minutes. Observe the tubes for the development of violet color in the tubes.
Interpretation of Biuret Test
Positive Biuret Test Result: Violet or purple color appears denotes presence of proteins (peptide bonds).
Negative Biuret Test Result: No color change (solution remains blue), sample lacks significant protein content.
Applications of Biuret Test
The biuret test is used mainly for diagnostic purposes, like determining serum proteins. Here are some other applications of these tests;
Biuret test helps in finding the proteins in the given unknown sample.
It is used for quantification of protein by using a spectrophotometer.
Biuret test help determine proteins in the urine, CSF, and other body fluids.The test helps to find the presence of specific proteins during food analysis.
Precautions for Biuret Test
Use sample and reagent in the recommended 1:1 volume, 1:1 ratio always gives a better result.
Avoid adding excessive Biuret reagent, too much reagent keeps the solution blue instead of purple giving false negative.
Do not read the result before 5 minutes. Reading before 5 minutes might give you false negative results.
Limitations of Biuret Test
Samples with high amounts of amino acid histidine can give a false positive result.
If the buffer used to purify protein has an ammonium or magnesium it can affect the test.
This test alone is not enough for quantifying the amount of protein in the given sample, further spectrophotometric analysis required.
