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Biuret test

Biuret test
n., [ˈbju.rɛt tɛst]
Definition: A chemical test that detects any compound containing two or more peptide bonds

In this article we will answer the following three questions: What is a Biuret Test?  What does biuret test for? What is biuret protein test? The biuret test mechanism is utilized to identify the existence of peptide bonds in an analyte. It can be employed to quantify the quantity of protein present in the sample. During the test, the occurrence of peptides triggers the creation of light purple (or mauve) coordination complexes between copper (II) ions in an adequately alkaline solution.

What is the Biuret Test?

The Biuret test is used to detect the presence of proteins in a sample. The test is founded on the idea that copper ions in an alkaline solution can form a complex with the peptide bonds in proteins, resulting in a change in color,  from blue to violet.


The biuret reaction mechanism is a chemical test used to determine peptide bonds and the quantity of protein in an analyte. When the solution is sufficiently alkaline, the inclusion of peptides results in the creation of coordination complexes of the copper (II) ion that are pale purple.

According to the Beer-Lambert rule, the intensity of the purple color and the absorption at 540 nm is directly proportional to the number of proteins in the given analyte. The bonds in biuret molecules are analogous to peptide bonds, a positive biuret test result is also achieved when the analyte contains biuret molecules.

Biuret solution is a blue-colored reagent used in biochemistry to test for the presence of proteins in a sample. The reagent contains copper ions that react with peptide bonds in proteins, resulting in the formation of a complex that absorbs light at a wavelength of around 540 nm, which causes the blue solution to change color to violet or pink.

The intensity of the color change is directly proportional to the concentration of proteins present in the sample. Biuret solution is commonly used in laboratory experiments to quantitatively determine protein concentration in a given solution and is also used in clinical diagnosis to detect the presence of proteins in various bodily fluids.

Watch this vid about Biuret Test:


Biology definition:
A Biuret test is used for detecting compounds with peptide bonds. A biuret reagent may be used to test the aqueous sample. Thus, the biuret test is used to detect proteins. That is because proteins are made up of polypeptides, which in turn, are made of amino acids joined by peptide bonds.

  • A pale violet or pinkish color indicates shorter polypeptide chains or fewer peptide bonds.
  • The longer the polypeptide chain is, the more peptide bonds there are, and therefore, the more intense the violet color will be when the biuret test is applied.
  • A negative result (lack of violet color formation) may mean a lack of protein, or the presence of free amino acids (without peptide bonds).

Note that the test gives a positive result to any compound containing two carbonyl groups attached to a nitrogen or carbon atom. Thus, it may not be completely protein-specific. Performing other protein tests may be necessary.

Biuret Test Principle (How does the biuret test work?)

The mechanism copper (II) binds to protein peptide nitrogen atoms. This assay was used for protein determination in entire tissue samples without being affected by amino acids. Ammonium sulfate ((NH4)2SO4) precipitation purifies proteins, but buffers like ammonia interfere with biuret assay. Copper (II) ions react with peptide nitrogens to displace peptide hydrogens (as long as the environment is sufficiently alkaline).

A chelate complex is formed when four nitrogen atoms donate lone pairs to create coordinate covalent connections with the cupric ion. 540nm light absorbs this chelate complex, turning it purple. Proteins in the analyte generate a purple complex. The purple color comes from the analyte’s peptide bond concentration.

Principle of Biuret Test
Figure 1: Principle of Biuret Test Image Source: microbiologynote.com


Biuret Test Manual

Materials Required:
• 1% alanine with 5% albumin or egg white (for positive control)
• De-ionized water (as negative control)
• Biuret reactants
• Water bath
• Pipettes
• Dry test tubes

Biuret Reagents

What is biuret reagent? Biuret reagent is made up of Copper sulfate (CuSO4), sodium tartrate (NaK), and sodium hydroxide (NaOH) (known as Rochelle salt as well). This reagent is not based on Biuret ((H2N-CO-)2NH) despite its name. This is a crucial part of the Biuret test.

Biuret Reagent Preparation

Making it involves adding NaOH to a CuSO4 solution, which turns the solution alkaline. The process to make one thousand milliliters of Biuret reagent is as follows.

  • Mix 1.5g pentavalent CuSO4 and 6g of C4H4O6KNa·4H2O (sodium potassium tartrate) in 500ml pure water.
  • The chelating chemical sodium potassium tartrate is used to keep the copper ions in place.
  • Then, fill a volumetric flask with 375 ml of 2 molar hydroxides.
  • Pour enough distilled water into the container until the volume reads 1000 ml.

Biuret Test Procedure

Following is the procedure for the Biuret test.

  1. Collect three freshly cleaned test tubes.
  2. Now mix in some albumin, distilled water, and 1-2ml of the test solution.
  3. Put 1-2 mL of Biuret reagent within every test tube.
  4. After 5 minutes, give the mixture another good shake.
  5. Finally, take note of the color variation.
Procedure of Biuret Test
Figure 2: Procedure of Biuret Test. Image Source: microbiologynote.com
  • Precaution

The test is frequently impeded by ammonium and magnesium ions. But, it can be eliminated by applying an abundance of alkali or carboxyl.

  • Observation

Below are the Biuret test Observation and Results for each sample:

  • If the solution does not turn from blue to another color during the biuret reaction, then means there are no peptide bonds in the sample.
  • The presence of peptide bonds can be detected by observing a color change from blue to violet in the solution.
  • Biuret Test Results

As amino acids are linked together by a peptide bond. A change from blue to pink in the solution act as an indicator for protein and peptide bonds in the sample.

Result of Biuret Test
Figure 3: Result of Biuret Test. Image source: Microbiologynote.com
    • Positive Result

  • The Color changes to purple.
  • Biuret test positive results are obtained with all proteins and peptides.
  • Only the Histidine and amino acids give a positive result.
    • Negative Result

  • · No change in color.
Biuret Test For Protein Principle
Figure 4: Biuret Test For Protein Principle. Image Source: Microbiologynote.com.

Add a few drops of a 5% sodium hydroxide solution to each test tube to make sure the sample is alkaline. In addition, a Biuret reagent is composed of 5% sodium hydroxide and 1% Copper II sulfate, (NaOH+CuSO4), therefore it must be carefully mixed before use.

Increasing Biuret Test Sensitivity

The Folin-Ciocalteu test utilizes Cu+ as a reducing agent to produce molybdenum blue, which is used to detect proteins within a concentration range of 0.005-2 mg/mL. Molybdenum blue can also bind specific organic dyes, resulting in further signal amplification. Another technique that utilizes Cu+ is the BCA or bicinchoninic acid assay, which produces a deep purple solution compound to identify proteins within a concentration range of 0.0005-2 mg/mL. This assay is also known as the “Pierce assay” in honor of the reagent kit creator.

  • Advantages

    • This approach is the quickest and easiest way to determine whether or not a sample contains protein. The Kjeldahl test is more costly than this one.
    • Except for protein, the test is affected by very few other substances. The biuret test for starch, biuret test for carbohydrates, biuret test for lipids, and glycine biuret test will be negative. The biuret test for urea and biuret test for protein is positive due to peptide bonds.
    • It can only determine the presence of N in peptide or protein links. The ninhydrin test shows the presence of amines.
  • Disadvantages

    • An enormous amount of bile pigments and ammonium salts can affect the process.
    • Different Biuret solution colors represent different proteins like purple egg test and gelatine creates a pink-purple tint. In addition, the purity of the solution is diminished by the presence of fats and carbs.
    • For a chemical test for proteins to be detected, they need to be soluble.
    • This is not an all-encompassing test. It is necessary to standardize the biuret test colors for use with recognized proteins such as BSA.

Biuret Test Uses

The Biuret test shows the presence of proteins in a wide variety of samples.

  • The protein content of a product

The Biuret test is not only used to detect the existence of protein but can also be used to quantify the total protein content through spectrophotometric analysis. This involves adding a 1% concentration of sodium hydroxide or potassium hydroxide to an aqueous solution, followed by a few drops of CuSO4. If the solution turns purple, protein is present.

The Biuret method is based on the reaction between proteins (and compounds with multiple peptidic linkages) and copper, which forms a colorful complex. The intensity of the color, measured at a maximum absorbance of 454 nm, is proportional to the protein concentration in the sample. Thus, the biuret test for amino acids will be positive.

  • Medical Testing

It is frequently used as a method for estimating the amount of protein molecules present in urine.

This test quantitatively measures a protein in normal urine without medication or pigment interference, which can be used to monitor clinical trials for nephrotoxicity caused by new medications.

The Biuret test is commonly used in clinical chemistry to detect protein levels in blood and urine, which can provide diagnostic information about a patient’s kidney function and inflammation, among other health conditions. (Savory, Pu, & Sunderman Jr, 1968)

Frequently Asked Questions on Biuret Test

What reagent is used to test for protein?
Biuret solution is a reagent commonly used to test for the presence of proteins in a sample.

Biuret reagent contains what ions?
Biuret reagent contains copper ions (Cu2+) which react with peptide bonds present in proteins to form a complex that absorbs light at a wavelength of around 540 nm, causing the solution to change color.

What is Lugol’s test?
Lugol’s reagent is a commonly used solution in laboratory experiments to test for the presence of starch in a sample. It is composed of iodine and potassium iodide dissolved in water, and is named after the French physician Jean Lugol who first described the solution in the 19th century.

Lugol’s reagent works by reacting with the helical structure of starch molecules, causing a blue-black color to appear when it is present in the sample. The intensity of the color change is directly proportional to the concentration of starch present in the sample, and the test is highly sensitive, with the ability to detect starch at concentrations as low as 0.01%.

When there is no starch lugol’s reagent negative color is yellow, light yellow, or amber color.

What is B-PER Reagent?
The B-PER reagent and the Biuret test are two different types of chemical assays that are used for different purposes in biochemistry and molecular biology.

The Biuret test is a colorimetric assay that is commonly used to detect the presence of proteins in a sample. On the other hand, the B-PER reagent is a cell lysis reagent that is used to extract proteins from cells.

The reagent is added to cells to disrupt the cell membrane and solubilize the native or recombinant proteins without denaturation. The solubilized proteins can then be easily collected and used for downstream applications such as Western blotting, enzyme assays, or protein purification.

What is a Sodium hydroxide test positive result?
The sodium hydroxide (NaOH) test is a chemical test used to identify the presence of certain functional groups, such as carboxylic acids and phenols. When NaOH is added to a sample containing a carboxylic acid, it will react with the acid group to form salt and water.

The positive result of the NaOH test for a carboxylic acid is the formation of a salt, which may appear as a white precipitate or a clear solution depending on the solubility of the salt.

When NaOH is added to a sample containing a phenol, it will react with the phenol group to form an ionic compound called a phenoxide ion. The positive result of the NaOH test for phenol is the formation of a colored solution or precipitate, depending on the specific phenol being tested.

Take the Quiz on Biuret Test!


Choose the best answer. 

1. What is biuret test?
2. What is a positive biuret test?
3. What does a negative biuret test mean?
4. Which of the following sample will likely result in a negative biuret test?
5. Which of the following scenarios will indicate the presence of more proteins via a biuret test?

Send Your Results (Optional)

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  • Micro-biologynote. (2023). Biuret Test For Protein Principle, Procedure, Result, Application. Retrieved 25 Feb, 2023, from https://microbiologynote.com/biuret-test-for-protein-principle-procedure-result-application/#application-of-biuret-test
  • Savory, J., Pu, P. H., & Sunderman Jr, F. W. (1968). A biuret method for determination of protein in normal urine. Clinical chemistry, 14(12), 1160-1171.
  • Vedantu. (2023). Biuret Test. Retrieved 25 Feb, 2023, from https://www.vedantu.com/chemistry/biuret-test


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