Biuret test

for protein
Biuret is a compound produced by heating urea
at 180 ℃.

Biuret test is the name of a chemical test which utilises the

Biuret reagents which contains a 1% solution of Copper II
sulphate (CuSO₄).

It is the Cu₂⁺ in the Biuret reagent that forms a complex

with the peptide bonds found in proteins.

Hence, this test helps in determining peptide bonds in any

substance.
Biuret test principles
This test is also known as Piotrowski’s test
after the name of Gustaw Piotrowski, a
polish physiologist, who documented this
test in 1857.

However, the mechanism of this test

works through a series of principles.
● In the presence of alkaline, when Biuret is reacted with dilute
copper sulphate, a purple coloured substance is formed. The
reason behind this colour is the formation of a chelate complex
or copper coordination complex.

● Cu (II) or cupric ions create a chelate complex of violet

colour, using oxygen of water and the unshared
electron pairs of peptide nitrogen.

● Since this complex absorbs light in 540 nm, it

appears violet. In the presence of protein, it
changes its colour from blue to violet.
 ● Naturally, the colour intensifies as the number of peptide bonds
increases in protein.
● Depending on this tests principle, we can find the peptide bonds
in any biological fluid.
● This reaction takes place in a compound having at least two,
H₂N-CH₂-, H₂N-C, and H₂N-CS- or similar groups attached
directly or via a nitrogen or carbon atom.

● One cupric ion is generally attached to six nearby

peptide linkages through coordinate bonds.
 Biuret test materials
1% alanine and 5% Deionised water
Biuret reagents
albumin or egg white (as (as negative
positive control) control)

Water bath
Dry test tubes Pipettes
 Biuret reagents

Copper sulphate sodium hydroxide sodium potassium

(CuSO₄) (NaOH) tartrate (also known as
Rochelle salt).
Mercury Neptune Mars
Despitethe
Despite being red, Mars
name, this Neptune
reagent does isnot
the contain
fourth- Biuret Jupiter is a gas giant
((H₂N-CO-)2NH). It
is actually a cold place largest planet in the and the biggest planet
is afull
vital component
of iron oxide dust of Biuret protein assay.
Solar System in the Solar System
 Biuret reagent preparation
Take distilled water (500 ml) and
dissolve pentavalent copper sulphate
(1.5gm) and sodium potassium tartrate
(6gm).
2. Sodium potassium tartrate, a
chelating substance that stabilises
the copper ions.

4. Now take a volumetric flask 5. Finally, make it to 1000

3. Now take 2 molar
and mix two solutions. Pipettes
hydroxide (375 ml) ml by pouring distilled
water.
 Procedure:
1.First, take 3 dry and clean
test tubes.

2.Now add 1 or 2 mL of the test

solution, albumin and deionised
water in the test tubes.

3.Add Biuret reagent (1-2

mL) in each test tube.
Saturn is composed
Feedback &
mostly of hydrogen Activities
Goals
Jupiter is a gas giant
and helium and the biggest planet
4.Now shake the solution well
and let it stand for 5 minutes.
EvaluationDespite beingin the
red,Solar System
Mars is a cold place full
of iron oxide dust
5.Finally, observe how
the colour changes.
 Observation and Analysis of the Result for Each
Sample Solution
Observation Interpretation

No change in colour
(solution remains blue). Proteins are unavailable.

Solution turns violet Proteins are available.

from blue.

Solution turns pink from Peptides are available.

blue.
 to ensure that the test sample is
alkaline, add a few drops of 5%
sodium hydroxide solution to each
Introductio
test tube. Moreover, if you are using
Biuret reagent ensure that you shake
n
Jupiter is the biggest
that planet
well as it isSolar
in the a prepared
System with
reagentandof
the1%fourth-brightest
Copper II sulphate
object in the night sky
and 5% sodium hydroxide.
 Significance of Biuret test

800
● It is widely used
in determining
900
● Biuret test for protein
is also useful to total
proteins quantitative

K
the protein
Despite being
amountred,inMars
urine.
is
actually a cold
K
determination,
Saturn is
composed of
using
spectrophotometric
hydrogen and
planet analysis.
helium
 Thankyo
u!
ALMAZAN
AQUINO
ARGOSINO
AVERA

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