Lab # 4

Date: February 9th, 2023

Topic: Biuret Test

Aim: To test for protein by means of a biuret test.

Material:

Test tube rack with four test tubes, and biuret reagents:

1. (i) 10% sodium hydroxide solution,

2. (ii) 0.5% copper (II) sulphate solution, both supplied in dropping bottles with teat

pipettes, and access to unknown solutions appropriately labeled.

Method:

1. Five test tubes were labeled A-E.

2. Then 2 cm3 of unknown solution was added to each test tube.

3. 2cm3 of 10% sodium hydroxide was then added to each sample and mixed by gentle

agitation.

4. Then 0.5% copper (II) sulphate solution was added, drop by drop, shaking the tubes well

between each addition until a defined color becomes visible. The process was repeated

for each test tube.

Data Presentation

Test Tubes

Color change A B C D E

Initial color Milky white Clear Clear White Clear

 Final color Lilac Orange Blue Light Purple Violet

Protein Yes Yes No Yes Yes

presence

TABLE SHOWING THE COLOR CHANGE IN UNKNOWN SOLUTIONS A-E WHILE

TESTING FOR PROTIEN’S USING THE BIURET TEST

Discussion:

The term "protein" refers to a class of big biomolecules that are constructed from chains of

amino acids joined together by peptide bonds. The chemical test used to determine whether

proteins are present in a sample is called a biuret test. In this experiment, a Biuret Test was

conducted to determine whether protein is present in any of the 5 assigned unknown solutions

A-E. The biuret reaction occurs when copper sulfate is introduced to a peptide structure with at

least two peptide bonds, resulting in the creation of a violet color. The Copper II ions (blue color)

when added to the substance can construct a bond with the peptide bonds of the tested solution.

The electronegativity of the unshared electron pairs in the nitrogen and oxygen of the peptide is

what causes them to establish a connection with the Copper II ion. The Cu2+ ion, carbonyl

oxygen, and amide nitrogen of the peptide bond work together to produce the test's colors. As a

result, proteins are composed of several peptide bonds that are joined together, so anytime the

test for peptides yields a positive result, protein is in fact present in the solution. There are no

proteins present in the solution if it becomes blue. Protein is present if the solution turns pink,

lilac, or purple; the more purple the solution is, the more protein is present.
The image below illustrates the formation of the peptide-copper complex:

Throughout the experiment 5 color changes were observed after the biuret test, test tube A

solution color initially was milky white, but after the biuret test it changed to a lilac color

indicating protein was present. Test Tube B started off clear before turning orange, suggesting

the presence of protein also. Test Tube C started off clean as well, but it quickly turned blue,

indicating that no proteins were present or simply a negative result. Test tube D initially had a

white tint, then turned to a pale purple color, signifying minute protein traces. The final test tube

E similarly started out clean, but after the biuret test, it turned violet.

Lastly, if Soy milk were to be used in the experiment then it would result in a positive color

(violet)indication for protein since soy milk has a high quantity of proteins. Whereas diluted

apple juice would come back with a negative test(blue) for protein since it contains little to no

proteins therefore there would not be any color change.

Source of Error:

A source of error would be if the proteins with abnormally high or low percentages of aromatic

acids will result in either high or low readings.

Another source of error would be if a high concentration of the Copper II ion solution was added

to solution A, which may have caused the solution to go from a positive to a negative test for

Protein.

Limitation:

A limitation of the biuret test would be its non-sensitive nature, this would require greater

amounts of protein so that the test can work.

Another limitation would be that the Biuret test could be interfered by ammonium sulfate, as it

could mess with the color change for protein in the solution, leading to a false test for protein.

Conclusion: It was determined that test tubes A, B, D, and E were positive for

protein whereas test tube C was negative for protein.

References:

Biuret test for protein. Brilliant Biology Student. (n.d.). Retrieved March 19,
2023, from
http://brilliantbiologystudent.weebly.com/biuret-test-for-protein.html

Aryal, S., Lawrencium, Emmanuel, M., katabalo, J. J., Steven, L., Shield,
chipengray, G., Raphael, E., Parameswaran, Amare, E., & Lot. (2023, January
18). Biuret test for protein- definition, principle, procedure, results, uses.
Microbe Notes. Retrieved March 19, 2023, from
https://microbenotes.com/biuret-test-for-protein/#:~:text=A%20Biuret%20test
%20is%20a,treated%20with%20alkaline

Admin. (2022, November 17). Biuret test - checking for peptide bonds with
Biuret reagent. BYJUS. Retrieved March 19, 2023, from
https://byjus.com/chemistry/biuret-test/
Lab 4 - Protein Mark Scheme
(Observation/Recording/Reporting)
Report Format & Procedure (5 marks)
- Aim is clearly stated [1]
-Correct spelling, grammar and punctuation [1]
- Procedure: clear, logical sequence of steps [1]
- Procedure: past tense/passive voice/3 rd person [2]
Observations & Results (7 marks)
- Acceptable scientific reporting & proper
documentation of results. [2]
- Accurately identify if protein is present in samples [2]
-Report includes complete relevant directly related [3]
discussion