Experiment 2

Objective:  Biuret Test for Proteins.

Introduction:

The Biuret test is used to detect proteins based on the ability of Cu (II) ions to form a violet-colored chelate complex with peptide bonds (-CONH-groups) under alkaline conditions.

Chemicals:

·       Hydrated Copper Sulphate: Provides the Cu (II) ions that form the chelate complex and give the reagent its characteristic blue color.

·       Potassium Hydroxide Solution: Provides the alkaline medium but does not participate in the reaction.

·       Potassium Sodium Tartrate (KNaC₄H₄O₆·4H₂O): Stabilizes the chelate complex.

Biuret Test Procedure:

1.  Add 2 cm³ of the liquid food sample* to a clean, dry test tube.

2.  Add 2 cm³ of Biuret Reagent.

Alternative method:

Add 1 cm³ of sodium hydroxide solution (40% or bench solution).Add 1% copper (II) sulphate solution dropwise to the food sample.

Fehling's Method (Optional):

Use freshly prepared Fehling’s A and B solutions.Add 1 cm³ of Fehling’s A (copper (II) solution) and 1 cm³ of Fehling’s B (sodium potassium tartrate and sodium hydroxide) to the food sample.

3.  Prepare controls:

Negative control: Repeat steps 1 and 2 with de-ionized water.Positive control: Repeat steps 1 and 2 with albumin (egg white).

4.  Shake well and allow the mixture to stand for 5 minutes.

5.  Observe any color change.

* To prepare liquid samples from solid foods: Crush the solid food, add a little de-ionized water, and decant the liquid. This liquid should be used for the food test. The amount of food and water depends on the number of tests conducted.

Observations:

Test

Observation

Inference

Positive

Solution turns from blue to violet (purple)

or pink

Proteins (purple) or Peptides (pink) are present

Negative

No change; the solution remains blue

No proteins present.

Questions:

1.    What is the composition of Biuret reagent?

2.    When is the chelate complex formed?

3.    What color is observed when the complex forms?