Urea is heated to 180°C to create the compound biuret. When doing a chemical test, you’ll use the Biuret reagents, which are made up of 1% CuSO4 and are used in the Biuret test. The Biuret reagent’s Cu2 creates a complex with protein peptide bonds thanks to the presence of the Cu2. Peptide bonds can be identified with this test since it helps to determine the composition of the material.

Peptide bonds are formed when the carboxyl and amino groups of two acids are joined together. Most importantly, for students preparing to sit for their exams shortly, this experiment is critical.

Principle of the Biuret Test:

The Piotrowski test, sometimes known as the Piotrowski manoeuvre, was developed in 1857 by a Polish physiologist called Gustaw Piotrowski. For example, the modified Lowry test and the BCA test are both used. The approach used for this test, on the other hand, is based on a variety of theories.

It is produced when biuret reacts with diluted copper sulphate in the presence of alkaline, resulting in a purple-coloured product. Copper coordination complexes are formed during chelate complexes, which gives this colour its distinctive look.

A violet-coloured chelate complex is formed by the interaction of Cu (II) ions with oxygen from water and unshared electron pairs from peptide nitrogen. Cu (II) or cupric ions are formed by oxygen from water and unshared electron pairs from peptide nitrogen.

This chemical appears violet because it absorbs light at 540 nm. In the presence of protein, the colour of the substance changes from blue to violet. The colour of a protein grows increasingly vivid as the number of peptide bonds in the protein increases.

The technique used in this test may be used in any biological fluid to detect peptide bonds. This reaction involves the formation of H2N-CH2-, H2N-C-, and H2N-CS- or related groups, which can occur either directly or indirectly through the presence of a nitrogen or carbon atom.

The coordinate bonds of a single cupric ion are frequently related to six neighbouring peptide linkages, which is not unusual. The details of the protein Biuret test can only be learned when you have mastered the test principles. It has been discussed in further depth in the following paragraphs.

Manual for the Biuret Test

Requires the following materials:

• Biuret reagents
• Deionised water (as negative control)
• Dry test tubes
• Water bath
• Pipettes

Reagents for Biuret

Copper sulphate (CuSO4), sodium hydroxide (NaOH), and sodium-potassium tartrate make up the biuret reagent (also known as Rochelle salt). This reagent does not include Biuret ((H2N-CO-)2NH, despite its name. The Biuret protein test would be incomplete without it.

Method for Preparation of the Biuret Reagent

• By adding NaOH to a solution of CuSO4, you can create this alkaline compound. To make 1000mL of Biuret reagent, follow these instructions.
• Using 500 ml of distilled water, combine 1.5 grams of pentavalent copper sulphate with sodium potassium tartrate to create a solution (6gm).
• Copper ions are stabilised with sodium potassium tartrate, a chelating agent.
• Take a 2 molar hydroxide solution now (375 ml)
• Now combine the two solutions in a volumetric flask.
• Pour distilled water to get the total to 1000 ml.

Procedure:

This test is simple to carry out if you follow the guidelines below:

• To begin, gather three clean and dry test tubes.
• Add the test solution, albumin, and deionised water to the test tubes, about 1 or 2 mL each.
• Each test tube should include 1-2 mL of Biuret reagent.
• Wait for five minutes after shaking the solution vigorously.
• Finally, take note of the shift in hue.

Precaution

This test is frequently hampered by ammonium and magnesium ions in the environment. Excess alkali, on the other hand, maybe eliminated.

Observation

• There is no change in colour as the solution remains blue.
• The solution changes colour from blue to violet.
• The solution changes colour from blue to pink.

Positive Outcomes

• The colour shifts to a purple hue
• To put it another way, every protein and every peptide gives you something good.
• Only one amino acid, Histidine, yields a favourable outcome.

Negative Outcomes

There has been no shift in colour. Add a few drops of a 5% sodium hydroxide solution to each test tube to verify the sample is alkaline. The Biuret reagent, produced with 1% Copper II sulphate and 5% sodium hydroxide, should be shaken vigorously before use.
Notably, demonstrate your knowledge of how 5% sodium hydroxide alkalises the sample and 1% copper II sulphate produces a complex with peptide bonds in chemical processes.

Sensitivity Increase for the Biuret Test

Molybdenum blue is produced in the Folin-test Ciocalteu’s by reacting Mo(VI) with Cu, a powerful reducing agent. If you proceed in this manner, the amounts of main proteins between 0.005 and 2 mg/mL will be observed. Malachite green and Auramine O are examples of organic colours that Molybdenum blue may bond to. The signal is amplified even more as a result of this.

BCA (bicinchoninic acid) and copper ions combine to form a rich purple compound. It has a sensitivity range of 0.0005 to 2 mg/mL, making it suitable for detecting proteins. In honour of a reagent kit maker, this is also known as the “Pierce test.”

Advantages of Biuret Test

• This is the quickest and easiest approach to check if a sample contains protein. Compared to the Kjeldahl test, it’s also less costly.
• The colour is steady. Thus there are no fluctuations such as UV absorption, Folin-Lowry, and so on.
• Few substances interfere with the test except for protein.
• It only recognises N that is found in protein or peptide links as being present. Therefore, non-protein nitrogen cannot be detected.

Disadvantages of Biuret Test

• Folin Lowry test has a higher sensitivity, while this one has a lower sensitivity. Despite this, a protein concentration of 2-4 mg must be found.
• The reaction is influenced by the presence of a lot of ammonium salts and bile pigments.
• Different colours represent various proteins. Gelatine, as an illustration, lends a pinkish-purple hue. Aside from that, carbohydrates and lipids distort the solution’s colour.
• For protein detection, the proteins must be soluble.
• This isn’t a final, conclusive test. For well-known proteins like BSA, the colours must be harmonised.
• A little heat or 30% isopropyl alcohol will speed up the test. It cuts the reaction time in half, from 35 minutes to only 10 minutes.

Conclusion

• The Biuret test is a common tool for figuring out how much protein is in your urine.
• Using spectrophotometric analysis, the protein biuret test may also be used to determine the total protein concentration.