Biuret test procedure for protein

The Biuret Test is a chemical test used to detect the presence of proteins in a sample.

It relies on the reaction between copper ions and peptide bonds in the protein, producing a color change that indicates the presence of proteins.

Here’s a step-by-step Biuret test procedure for protein.

Label three test tubes , “Test”, Positive control, negative control.
Add 1 to 2 ml of sample in a test tube. Add 1-2 mL of albumin solution in positive control tube, and in the test tube labeled as ‘negative’, dispense 1-2 mL of distilled water.
Now Add about 1-2 mL of Biuret reagent to the all three tubes.

Shake well and place them at room temperature.
After five minutes observe the color.
If sample contain protein, blue color change into purple color. like positive control.
In the case of negative result. sample remain blue in color like negative control.

How to prepare biuret reagent

The reagent primarily contains copper(II) sulfate (CuSO₄), sodium hydroxide (NaOH), and sometimes potassium sodium tartrate (Rochelle salt) to stabilize the solution.

Follow these steps for biuret reagent preparation.

Measure 1.5 grams of Copper(II) sulfate pentahydrate (CuSO₄ · 5H₂O). Dissolve it in about 90 mL of distilled water in a 100 mL volumetric flask (or adjust quantities proportionally if preparing a larger volume).
Measure 6 grams of Sodium hydroxide (NaOH). Slowly add the NaOH to the copper sulfate solution while stirring continuously. The solution will turn a light blue color.
Add distilled water to the solution to bring the final volume up to 100 mL (or the intended volume).
Store the Biuret reagent in a well-labeled glass bottle, away from light and heat.

Safety Precautions

Wear protective gloves, safety goggles, and a lab coat while handling chemicals.
Sodium hydroxide is highly caustic; handle with care to avoid contact with skin or eyes.
Work in a well-ventilated area or under a fume hood.
Dispose of chemical waste according to local regulations.