Exams Prep Master
Biuret test, in chemistry, is used to identify whether the peptide bond is present in an analyte or not. Also, the amount of protein in the analyst can be calculated with the help of a biuret test. When the solution is sufficiently alkaline, the copper (II) ion forms the pale purple coloured coordination complexes which reflects the presence of peptide bonds in the analyte. The biuret test was rediscovered by the Polish physiologist Gustaw Piotrowski in 1857. Therefore, the test is also known as Piotrowski’s test. Let’s discuss the biuret test in detail along with some important questions.
| Table of Contents |
Key Terms: Biuret, Reagent, Reduction reaction, Analyst, Coordinate, Peptide bond, Concentration, Pale, Alkaline, Complex
What is the Biuret Test?
[Click Here for Sample Questions]
Biuret, the compound, produced by heating urea at 180 ℃ and Biuret test is the name of a chemical test that utilises the Biuret reagents. The test contains a 1% solution of Copper II sulphate (CuSO4). In the Biuret reagent, it is the Cu₂⁺ that forms a complex with the peptide bonds found in proteins.Therefore, this test helps to determine peptide bonds in any substance. The reagent in the biuret test does not contain the biuret itself but the test is named due to the positive reaction result to the peptide-like bonds in the biuret molecule. The Lowry test and BCA test are two of many advanced versions of the biuret test.
Read More:
Biuret Test Principle
[Click Here for Sample Questions]
The Biuret test is used to detect the peptide bond in the analyst. When the biuret reagent is mixed with the protein peptide in the alkaline solution then the pale purple colour substance is formed which is the by-product of formation of the copper coordinate complex.
Biuret Test Principle
The biuret reagent contains copper (II) which undergoes the reduction reaction with the nitrogen atoms of the protein peptides. This results in conversion to copper (I) from copper (II) and creates the copper coordination complex of pale purple colour. The purple colour of the copper coordination complex is directly proportional to the concentration of proteins in the given analyte i.e., the colour intensifies with the increase in peptide bonds in protein.
Biuret Test Manual
[Click Here for Sample Questions]
Materials Requirements for the test are as follows:
- 1% alanine and 5% albumin or egg white (as positive control)
- Biuret reagents
- Deionised water (as negative control)
- Dry test tubes
- Water bath
- Pipettes
Biuret Test Reagent
[Click Here for Sample Questions]
The biuret reagent is made from three solutions:
- Sodium hydroxide (NaOH)
- Hydrated copper (II) sulphate
- Potassium sodium tartrate
The following are the steps required to prepare the biuret reagent:
Step 1: For formation of 1000ml of biuret reagent, take 1.5 gram of hydrated copper (II) sulphate and 6 grams of potassium sodium tartrate.
Step 2: In 500 ml distilled water, make the aqueous solution of a mixture of hydrated copper sulphate and potassium sodium tartrate.
Step 3: Here, potassium sodium tartrate acts as a chelating agent which stabilizes the copper ions. Meanwhile, take 375 ml of 2 mol sodium hydroxide.
Step 4: Add both the solution in volumetric flask and pour the distilled water in an amount such that it equals to volume of 1000 ml.
Biuret Test Procedure
[Click Here for Sample Questions]
Things Required: 1% Alanine and 5% egg white, Dry Test tubes, Biuret reagent, Water bath and Pipettes.
The Biuret test can be performed by the following given steps:
Step 1: Take three test tubes and make sure they are dry and clean.
Step 2: Mix the three: test solution, albumin and deionised water in the test tube.
Step 3: Add 1-2 ml of biuret reagent in test tubes.
Step 4: After shaking, put the solution still for around 5 minutes.
Step 5: Observe the colour of the solution.
Precaution
[Click Here for Sample Questions]
Ammonium and magnesium ions often impede this test. However, by using excess alkali, it can be removed.
Observation
[Click Here for Sample Questions]
The observation and analysis of the result for each sample solution is tabulated below:
| Observation | Interpretation |
|---|---|
| Solution remains blue | Proteins are unavailable |
| Solution turns violet from blue | Proteins are available |
| Solution turns pink from blue | Proteins are available |
Positive Result
- Due to the test the colour turns purple.
- All proteins and peptides show positive results.
- Amino acid, Histidine, gives a positive result.
Negative Result
- No change in colour is observed.
- Also, a few drops of 5% sodium hydroxide solution can be added to each test tube to ensure that the test sample is alkaline. Moreover, if Biuret reagent is being used, ensure that you shake that well as it is prepared with a reagent of 1% Copper II sulphate and 5% sodium hydroxide.
It should be kept in mind to show your understanding of why 5% sodium hydroxide alkalises the sample and 1% Copper II sulphate forms a complex with peptide bonds with chemical reactions.
Things to Remember
- The Biuret test is also known as Piotrowski’s test.
- Pale purple colour indicates the presence of protein in analysts.
- Copper (II) ions act as a reducing agent.
- The BAC test and Lowry test are advanced variants of the Biuret test.
- The colour intensity is directly proportional to the amount of protein in the sample.
Read More:
| Related Articles | ||
|---|---|---|
| Acids and Bases | Uses of Carboxylic Acids | Tests for Carboxyl group |
Sample Questions
Ques: What are the precautions needed for performing the test? (2 marks)
Ans: While performing the biuret test, make sure of some precautions for the true result. Firstly, keep the test tube clean and dry. Also, sometimes the ions of ammonium and magnesium cause a hindrance in the result of the test, so to solve the problem, an individual can use excess alkali.
Ques: How can we increase the sensitivity of biuret test? (2 marks)
Ans: Biuret reagent contains copper ion which is a strong reducing agent as it converts copper (II) to copper (I) ion. Also, it can react with the Mo (VI) during the test to make molybdenum blue and this leads to the detection of protein concentration in the analyte. The range of the protein concentration in the sample is 0.005 and 2 mg/ml.
Ques: What are the advantages of performing a biuret test? (2 marks)
Ans: Biuret test is one of many tests conducted to detect the protein in the sample. As compared with other tests, it is considered as simple, rapid and less expensive. Moreover, the resultant pale purple colour is stable and it does not cause deviation. Also, the test is used for detecting various types of compounds in the sample apart from protein.
Ques: What are the disadvantages of the biuret test? (2 marks)
Ans: The demerits of testing the sample with biuret test is its sensitivity as it is less sensitive than the Folin Lowry test because about 2-4 mg of protein must be present in the sample to get detected. Also, sometimes the ions of ammonium and magnesium cause a hindrance in the result of the test. Thirdly, the colour is not specific which means that the test shows different colour for different protein.
Ques: When an analyte is present in a sample, the biuret will turn _____. (2 marks)
a) blue
b) purple
c) green
d) yellow
Ans: (b)
Explanation: The biuret test is used to identify whether the peptide bond is present in analyte or not. Also, the amount of protein in the analyst can be calculated with the help of a biuret test. When the solution is sufficiently alkaline, the copper (II) ion forms the pale purple coloured coordination complexes which reflects the presence of peptide bonds in the analyte.
Ques: What is the significance of biuret test? (3 marks)
Ans: The following are the two significances of using biuret test:
- In the pathology labs, the biuret test is used to detect the presence of protein in the urine sample of an individual.
- Biuret test for protein is also useful to total protein's quantitative determination, using spectrophotometric analysis.
- The Biuret test for protein is useful in the case of both cheese and meat as they get dissolved in potash lye or solutions of alkaline detergent
Ques: Give the brief about observation and result of biuret test? (2 marks)
Ans: After performing the test, observing if the colour of the solution changes from blue to violet implies that the analyst contains protein. Also, if the resultant solution is of pink colour that means the protein is present in it. But if the colour of solution remains unchanged then it implies that the analyst does not have protein in them.
Ques: Which bond makes the biuret reagent purple? (2 marks)
Ans: The biuret reagent turns to purple when added to the sample if it contains protein. The bond that makes the change in colour is peptide bond. It is an amide type of covalent chemical bond linking two consecutives alpha amino acids from carbon number one of one alpha amino acid and nitrogen number two of another along a protein chain.
Ques: What is lowry test? (2 marks)
Ans: The advanced variant of biuret test is lowry test. It is used to determine the concentration of protein present in the solution. The protein concentration is directly proportional to the intensity of colour. The principle of the lowry test is the reactivity of the peptide nitrogen with the copper (II) ions under alkaline conditions.
Ques: What is the use of CuSO4 in the Biuret test? (2 marks)
Ans: The biuret reagent is made by mixing three chemicals i.e., Sodium hydroxide (NaOH), hydrated copper (II) sulphate, potassium sodium tartrate. The use of copper (II) sulphate is the source of the copper (II) ion in the solution which reacts with the nitrogen ion present in the protein sample.
Check-Out:
Comments