Objective
The Biuret test provides a qualitative assessment of protein presence by detecting the peptide bonds between amino acids, which are the building blocks of proteins. The test relies on the reaction between proteins and copper(II) ions under alkaline conditions. In the Biuret test, the sample is mixed with a reagent containing copper(II) sulfate (CuSO4) and sodium hydroxide (NaOH).
The peptide bonds in proteins react with the copper ions in the reagent, forming a complex of copper ions coordinated by the nitrogen atoms in the peptide bonds. This results in a color change from blue to purple in the presence of proteins. The intensity of the purple color is proportional to the concentration of proteins in the sample.
Principle of Biuret Test
The biuret test also referred to as Piotrowski’s test, is a chemical test used for detecting the presence of peptide bonds. Biuret test is based on the biuret reaction in which a peptide structure containing at least two peptide links produces a violet color when treated with alkaline copper sulphate. In the presence of an alkaline solution blue-colored copper II ions can form a complex with the peptide bonds (CO-NH group) since the peptide has unshared electron pairs in nitrogen and oxygen of water.
Four nitrogen atoms donate lone pairs to four covalent bonds with cupric ion resulting in the formation of a chelate complex. The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the displacement of the peptide hydrogen atoms under the alkaline conditions. Once this complex has been formed, the solution turns from blue to purple. The deeper the purple color, the more the number of peptide-copper complexes that have been formed.
Since all proteins and peptides possessing at least two peptide linkage i.e tripeptide gives positive biuret test, the principle of biuret test is conveniently used to detect the presence of proteins in biological fluids.
The Biuret test is used in biochemical analysis, such as in the fields of biology, biochemistry, and food science, to quickly determine the presence of proteins in various samples, including biological fluids, food products, and laboratory cultures. It is a simple and rapid qualitative test that provides valuable information about the protein content of a sample, aiding in various analytical and diagnostic applications.
Reagent
Biuret reagent is an aqueous solution of potassium sodium tartrate treated with cupric sulfate and sodium hydroxide. In the presence of peptide bonds (protein), this blue solution will change color to pink-purple.
- Rochelle salt/Potassium Sodium Tartrate (KNaC4H4O6.4H2O)- It acts as a chelating agent and stabilizes the copper II ions.
- Potassium Hydroxide Solution (KOH) does not get involved in the reaction but provides the alkaline medium.
- Hydrated Copper Sulphate. This provides the Copper III ions which form the chelate complex. Copper II ions give the reagent its characteristic blue color.
Preparation Of 1000ml Of Biuret Reagent
Biuret reagent is prepared by adding NaoH in Copper II Sulphate solution, making it alkaline.
Reagent Preparation Procedure
- Take 1.5 gram of pentavalent copper sulphate (CuSO4) and 6 gram of Sodium potassium tartarate and dissolve them in 500 ml of distilled water. Sodium potassium tartarate acts as a chelating agent and stabilizes the copper II ions.
- Take 400 ml of 2 molar sodium hydroxide
- Mix both solutions in volumetric flask and make it final volume to 1000 ml by adding distilled water.
Biuret Test Procedure
- Take 3 dry clean test tubes
- Add between 1-2ml of the test solution, egg albumin and deionized water in the respective test tubes.
- Add between 1-2 ml of Biuret reagent to all the test tubes.
- Shake well and allow the mixtures to stand for at least 5 minutes.
- Observe for any color change.
Result Interpretation
| OBSERVATION | INFERENCE |
| No color change i.e the solution remains blue | Proteins are absent (Negative Biuret Test) |
| The solution turns from blue to deep purple | Proteins are present (Positive Biuret Test) |
Limitations of Biuret Test
- The Biuret test is not as sensitive as some other protein assays, such as the Bradford assay or the Lowry assay. It may not detect proteins at very low concentrations accurately.
- Certain substances, such as reducing agents (e.g., ascorbic acid) and high concentrations of certain ions (e.g., phosphate ions), can interfere with the Biuret reaction, leading to false-positive or false-negative results.
- The color intensity produced in the Biuret reaction may not be linearly proportional to the concentration of proteins in the sample over a wide range of concentrations. This non-linearity can make quantitative analysis challenging.
- While the Biuret test is relatively simple to perform, it requires several steps, including the preparation of reagents and incubation periods, which can make it more time-consuming compared to some other protein assays.
- The Biuret test may not be suitable for all types of samples, such as those containing detergents, lipids, or certain organic solvents, which can interfere with the reaction or produce false results.