DETERMINING PROTEINS USING BIURET TEST

Introduction
Proteins are one of the most amazing groups of molecules in the human body.
They are complex combinations of smaller chemical compounds called amino acids.
These are like the bricks or building blocks of a building. The main source of protein is
our diet. We eat proteins from animals and plants. Our bodies can synthesize some of
the amino acids, but many of them must be obtained from the diet.
This experiment help us in determining the presence of proteins in some food
samples using the "Biuret test", in which it is a chemical test used for detecting the
presence of peptide bonds.

Procedures
During the experiment we begin performing the Biuret test by preparing first the
materials and reagents needed. The materials needed are the Test tubes, Test tube
rack, Test Tube holder, test tube brush, Alcohol lamp, tripod, wire gauze, beaker,
graduated cylinder, evaporating dish, Funnel, filter paper, Dropper, stirring rod, and the
aspirator. The reagents needed are the 10% NaOH solution, Copper Sulfate solution
and acetic acid. The food samples we have used are the egg, milk, coconut cream,
distilled water, and the other 3 food samples.

The first one we have tested is the distilled water we put 3mL of distilled water at
the test tube and add 2mL of 10%NaOH and 8 drops of Copper Sulfate solution, we
have shaken it and observed it and recorded the results. The second food sample was
the freshly prepared egg. By using the aspirator we get the egg albumin solution and
added 5mL of distilled water to the egg white and shake it thoroughly. After that we put
3mL of albumin solution in a test tube and added 2mL of 10% NaOH solution followed
by 8 drops of copper sulfate solution. To the egg yolk we have just repeated the same
way of performing the test.
To the third sample which is the milk, we have put 20mL of it on the evaporating
dish and heated it with a gentle boil. After 3 minutes we have stopped heating it and we
let it cool. By using the stirring rod we have removed the milk skin and put it on the test
tube and performed the same test by adding it 2mL NaOH and 8 drops of Copper
Sulfate solution. To the remaining milk in evaporating dish, we added 10 drops of acetic
acid and stirred it, next we have filtered the mixture and tested the residue for the
presence of its proteins by putting it on the test tube and add the 2mL of 10% NaOH
solution and 8drops of copper sulfate. To the milk filtrate, we have place 20mL milk
filtrate and added 25mL distilled water on the beaker and add also 20mL of 50% acetic
acid and mixed it well. We have separated the solid by filtration and put together the
filtrate in the beaker. We have boiled the filtrate and after the solid appears, we let the
mixture cool and perform the second filtration. Lastly, we collected 3mL filtrate and
performed the Biuret test. The 4 th one is the Coconut Cream we have heated it at low
temperature and separated the oil from the residue formed after heating. We placed the
small amount of residue and added the 2mL of 10% NaOH solution and 8 drops of

Copper Sulfate solution. After testing the four food samples, we immediately used other
food samples that we have chosen which are the Dalandan Juice, Bread, and the
Coconut wine. We have performed the Biuret Test with it and do the same processes.
Results/Data

FOOD SAMPLES

RESULTS AND OBSERVATIONS

The 3mL distilled water together with the 2mL of 10% NaOH

Distilled water

solution and 8 drops Copper Sulfate solution resulted into a

light blue color.
The egg albumin solution added with the 2mL of 10% NaOh

Egg
Egg Albumin

and 8 drops of Copper Sulfate solution after shaking it

turned into a violet colored solution.
The egg yolk was mixed first with 5mL distilled water, and

Egg yolk

after that we get 3mL egg yolk solution mixed with 2mL of
10% NaOh and 8 drops of Copper Sulfate after shaking the

Milk
Milk Skin

mixture resulted into a dark-green color.

The milk skin with the 2mL of 10% NaOH and 8 drops
Copper sulfate solution before shaking the upper part of the
mixture is purple and the lower part is white but after shaking
the mixture the upper part leaves a white residues and the
lower part has a dark purple color.
The milk residue mixed with 10 drops of acetic acid, 2mL of

Milk Residue

10% NaOH and 8 drops Copper Sulfate Solution, after

shaking the upper part of mixture has a very light blue color
and the lower part is white.

Milk Filtrate

It coagulates. The mixture has light violet color in upper

potion and very dark on lower portion.

Coconut Cream

The 3mL Coconut cream residue mixed with the 2mL of 10%
NaOH and 8 drops Copper sulfate solution resulted in color

(residue)

Other food
samples

violet solution with some white particles and bubbles on top.

The 3ml Dalandan Juice was added first with 2mL of 10%
NaOH solution resulted into a pink color and after adding the
8 drops of Copper Sulfate solution it resulted into a light

Dalandan Juice

Bread

violet color.
The crushed bread with 3mL distilled water, 2mL of 10%
NaOH and 8 drops Copper Sulfate Solution, before it was
shaken the upper part of mixture is sky blue, middle part is
light purple and lower part has bread residues with yellowish
color.
The 3mL Coconut wine added with 2mL of 10% NaOH and 8

Coconut Wine

drops Copper Sulfate Solution, the mixture resulted into

yellow green in the upper part and the lower part is creamy
light brown.

Discussion
Almost all of the exciting stuff such as metabolism, memory, hormone action, and
movement involves proteins. In this experiment, I have learnt method of measuring
protein concentration, biuret test.
The biuret reaction is a method that can be used to determine the amount of
soluble protein in a solution. The biuret reagent (copper sulfate in a strong base) reacts
with peptide bonds (which join amino acids to form proteins) and changes color when it
does so. The spectrophotometer has been used to measure the intensity of the color
produced. The more protein present the darker the color. 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.
Based on the results of the conducted experiment, the first sample which is the
distilled water the solution turned into blue therefore, theres no protein present. The egg
white which contains the albumin turned into a violet color, therefore it has proteins with
peptide bonds while the egg yolk mixture after shaking it resulted into a dark green
color. Analyzing the egg yolk is more complicated because it composes a mainly fats
such as phospholipids, cholesterol, and other components.

The milk skin produces a dark violet color casein is a protein that is found in milk
and used as a binding agent. To the milk residue, which is mixed with the acetic acid, it
resulted into a very light blue color,
Adding vinegar to milk can cause it to curdle. The suspended proteins in milk,
caseins, aggregate together forming lumps. Milk is Basic because it has Calcium and
Magnesium in it while vinegar is Acidic because it has acetic acid (organic acid). When
you mix the two a neutralization reaction can occur, hence the base and the acid
combine together and leave water behind, that is the reason why the mixture of milk
residue turned into light blue because the proteins are being drop out because of the
acetic acid.
The 3mL Coconut cream residue mixed with the 2mL of 10% NaOH and 8 drops
Copper sulfate solution resulted in color violet solution with some white particles and
bubbles on top, therefore it contains proteins.
The other 3 food samples tested by biuret test which are the dalandan juice,
bread and coconut wine also amazed us. The Dalandan juice when mixed first with
NaOH solution it produces pink color but when added by the Copper Sulfate it resulted
a very light violet color, therefore in my interpretation, Dalandan juice has peptides but
contains short chains of amino acid residues. The crushed bread also contains protein
because it has violet color of mixture while the coconut wine turned into a creamy light
brown in lower portion and dark yellow green in upper portion.
The Biuret test is based on the ability of Cu (II) ions to form a violetcolored chelate complex with peptide bonds (-CONH-

groups) in alkaline conditions.

Lone electron pairs from 4 nitrogen atoms in the peptide bond coordinate a copper (II)
ion to form the chelate complex. The chelate complex absorbs light at 540 nm so it
appears violet. Hence a color changes from blue to violet indicates that proteins are
present. The greater the concentration of peptide bonds, the greater the color intensity.
If the concentration of peptide bonds is low such as when short-chain peptides are
present - the color change is from blue to pink.

Conclusion
The Biuret Test is a general test to detect peptide bond or not. When protein that
includes peptide bond reacts with copper(II) sulfate (blue), the positive test is the
formation of a violet colored complex. Biuret test will result in the formation of violetpurple complex/ color change to indicate the presence of two or more dipeptide bonds
present in the protein. The darker the color, the more dipeptide bonds present and
hence the larger the protein present.
This test requires that more than large volumes of protein be available to bind in
order to give a positive result. Therefore, more than two peptide bonds must be
available to complex with the cupric ions.

References
http://edusanjalbiochemist.blogspot.com/2013/01/urinalysis-chemical-examination.html
http://www.ukessays.com/essays/biology/quantitative-tests-for-aminoacids-andproteins-biology-essay.php
http://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-experiment.html