Experiment 2: Qualitative analysis of protein content using Biuret Test

Objective:
1. To detect the protein in the given solution.
2. To demonstrate the presence of the peptide bond.

Principle:
Biuret test is a general test for compounds having a peptide bond. Biuret is a compound formed
by heating urea to 180° C. When biuret is treated with dilute copper sulfate in alkaline condition,
a purple colored compound is formed. This is the basis of biuret test widely used for identification
of proteins and amino acids. This test is given by compounds containing two or more peptide
bond (CO-NH group). Since all proteins and peptides possessing at least two peptide linkage ie.
tripeptide gives positive biuret test. The principle of biuret test is conveniently used to detect the
presence of proteins in biological fluids. Alkaline CuSO4 reacts with compounds containing two
or more peptide bonds to give a violet colored product which is due to formation of co-ordination
complex of cupric ions with un-shared electron pairs of peptide nitrogen and O2 of water.

The Biuret reagent is a solution composed of sodium hydroxide (NaOH) or potassium hydroxide
(KOH), hydrated copper (II) sulfate, and potassium sodium tartrate. Presence of magnesium and
ammonium ions interfere in biuret test. This can be overcome by using excess alkali.

Requirements:
1. 1 % alanine, 5 % egg white (albumin)
2. Biuret reagent:
a. Copper sulfate (CuSO40)
b. Sodium hydroxide (NaOH)
c. Sodium potassium tartarate (commonly known as Rochelle salt)
3. Water bath
4. Dry test tubes
5. Pipettes

Preparation Biuret reagent

1. Biuret reagent is prepared by adding NaOH in CuSO4 solution, making it alkaline.
2. To prepare 1000ml of Biuret reagent
a. 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
b. **Sodium potassium tartarate is a chelating agent and it stabilize the copper ion
c. Take 375 ml of 2 molar Sodium hydroxide
d. Mix both the solution in volumetric flask and make it final volume to 1000 ml by
adding distilled water.
Procedure:
1. Take 1 ml of test solutions in dry test tubes and in another tube take 1 ml distilled water as
control.
2. Add 1 ml of biuret reagent to all test tubes, mix well.
3. Look for the development of blue colors

Chemical Reaction(s):

Result:
Biuret test positive: Color changes to purple.

Fig 1: Negative & Positive Biuret Test

------------------*--*--*---------------------
Experiment 3: Qualitative detection of protein using Millon’s test.

Objective:
To detect amino acid containing phenol group (hydroxyl group attached to benzene ring) i.e.
Tyrosine

Principle:
Millon’s test is an analytical test used for the detection of the amino acid tyrosine, which is the
only amino acid containing the phenol group. Millon’s test is a specific test for tyrosine, but it is
not a specific test for protein as it also detects the phenolic group present in other compounds
as well. Therefore, while performing Millon’s test, it is essential that other tests like the Biuret
test and Ninhydrin test also be performed. As many proteins consist of tyrosine, the test is useful
in the detection of such proteins.

Millon’s test is based on the principle of nitrification of the phenol group in tyrosine, which then
forms complexes with heavy metals like mercury. The reagent used for the test is called Millon’s
reagent, and it consists of mercuric nitrate and mercurous nitrate that is dissolved in
concentrated nitric acid. In the test, the phenol group on the tyrosine molecule is nitrated by the
nitric acid present in the reagent. The nitrated tyrosine then combines with the mercury ions in
the solution to form a red-colored precipitate or solution. In some proteins containing tyrosine,
the initial reaction between mercuric nitrate results in a white or yellow colored precipitate. After
the addition of nitric acid and heating, however, the residue turns red in color. Both of these
results are considered positive results and indicate the presence of tyrosine in the solution.
Requirements:
1. Test solution: 1 % arginine, 1 % tyrosine, phenol solution
2. Millon’s reagent (Acidified mercuric sulphate)
3. 1 % sodium nitrite
4. Test tubes & stand
5. Pipettes
6. Water bath

Procedure:
1. Take 1ml test solution in dry test tube.
2. Similarly, take 1ml distilled water in another test tube as control.
3. Add 1ml of Millon’s reagent and mix well.
4. Boil gently for 1 minute.
5. Cool under tap water.
6. Now add 5 drops of 1 % sodium nitrite.
7. Heat the solution slightly.
8. Look for the development of brick red precipitate.
Chemical Reaction(s):

Fig 1: Negative & Positive Millon’s Test

Result:

Fig 1: Negative & Positive Millon’s Test

Positive Millon’s test: Brick red color (Tyrosine and phenol solution)
**all phenol give positive Millon’s test