The Biuret test detects the presence of peptide bonds in proteins. Peptide bonds form when amino acids link together to make proteins. The test involves adding a protein sample to an alkaline solution of copper sulfate and sodium hydroxide. In an alkaline environment, the copper ions will form a coordination complex with the nitrogen atoms from any peptide bonds present. This complex is purple in color, so a color change from blue to purple indicates that proteins containing peptide bonds are present in the sample.

1. Biuret test for Proteins
Protein formed by peptide bond between amino
acid. Peptide bond is unique in protein The
Biuret Test is done to show the presence
of peptide bonds. These bonds will make
the blue Biuret reagent turn purple.

2. TEST USED TO DETECT
Protein
Kjeldahl method
Titration based –
measure nitrogen
then protein are
detected
Enhanced
Dumas method
High
temperature
required
Biureate test
WHEN High
concentration OF
protein present
Lowery
methods
Low concentration of
protein present
unable to detect
through Biurreate
Biurate reagent
+phosphomolyb
date and phosph
otungstate

3. Biureate test
Making of test
sample
alkaline
Different
purposes of
make alkaline
are next slide
To make the
protein
soluble

4. In alkaline
solution
dissociation
takes place
around pH 8,
and above pH 10
dissociation and
denaturation
proceed
For studying purification : from crude plant
extract (protein /enzyme )at 35 centrigrade pH 10
for 18 hours
For studying Protein carbohydrate linkage O
linkage /N linkage :
For studying structure-function relationships:
Alkali treatment has also proved useful in of
glycoproteins
In food processing, sodium hydroxide is used
for peeling of potatoes and peaches,
solubilizing plant proteins,
neutralizing casein preparations (sodium
caseinate),
removal of toxic constituents such as aflatoxin and
protease inhibitors in production of texturized foods
and vegetable protein

5. Process
same amount of NaOH are
added to suppliedsolution
(Protein) and mix gentelly.
Add a few drops of 1% CuSO4,
the mixture should be placed
static without shaking.
A PURPLE COLOR is a positive
result: protein is present.
Fig : Representing
color
change

6. Fig: Representing formation of co-ordination compound
which is Purple color

7. Explanation
The reagent used in the
Biuret Test is a solution
of copper sulfate (CuSO4)
and sodium hydroxide (NaO
H).
The NaOH is there to raise
the pH of the solution
to alkaline levels; the
crucial component is
the copper II ion (Cu2+)
from the CuSO4.
When peptide bonds are
present in
this alkaline solution,
the Cu2+ions will form a
coordination complex with
4 nitrogen atoms from
peptide bonds.
The complex of Cu2+ ions
and nitrogen atoms make the
color
of CuSO4 solution changes
from blue to violet.
This color change is
dependent on
the number of peptide
bonds in the solution, so the
more protein, the more
intense the change.