Identification of Unknown Macromolecule

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The purpose of this lab was to use the scientific method to identify an unknown solution, based on the reactions (i.e. color changes) of known solutions with indicator solutions. These known solutions contained different types of macromolecules. Each type of macromolecule reacted with at least one indicator solution in a unique way, which allowed us to identify the macromolecule based upon the presence or absence of a color change.

The hypothesis is that protein will have a positive reaction with Biuret reagent, changing the color to violet.

Starch will have a positive reaction when treated with the iodine solution, changing the color to blackish-blue.

Lipids will show a positive reaction when coming in contact with Sudan III, changing the color to red-orange.

Vitamin C will diminish the color of iodine when it is positive for presence of ascorbic acid.

General Background

Macromolecules can be divided into four classes; carbohydrates, lipids, nucleic acids and proteins. These molecules are all considered to be organic molecules, because they consist of carbon, hydrogen, oxygen, sulfur, phosphate and nitrogen elements. Macromolecules have different building blocks to form carbohydrates, proteins, lipids and nucleic acids. These building blocks are amino acids, monosaccharaides, nucleotides, fatty acids and glycerol.

The water will be used as a control, to see if there are any false positive reactions during our experiment. The water will also show us what the most natural response is for each of the indicators. Water will also serve as a great way to compare color changes, since everything that looks the same as the water will be negative.

Proteins are one of the components of macromolecules and are very important in different biological processes. They are catalysts and are capable of transporting and storing molecules throughout the body. Proteins are build up out of several amino acids bound together by peptide bonds. The function of a protein depends primarily on its 3D structure.

Common foods that contain proteins are milk, eggs, meat and cheese.

Lipids are another form of macromolecules. These molecules are non-polar molecules and non-soluble in water. Lipids consist of two parts, a glycerol and a fatty acid tail. Lipids have large stores of energy that are released when the molecule is oxidized. Lipids come in different types, such as waxes, oils and certain vitamins.

Common foods that contain lipids are any oils such as olive oil or grape seed oil and butter.

Starch is a carbohydrate that is not found in humans, but in plants. It consists of multiple Glucose molecules bound to each other by Glycosidic bonds.

Common foods that contain carbohydrates are potatoes and bread.

Vitamin C is also known as ascorbic acid and is an antioxidant. This vitamin is soluble in water and plays an important role in growth and repair of the bodies tissues.

The most common foods that contain Vitamin C are citrus fruit.

Focused Background

Iodine test is the most common test for detecting the presence of starch. If the substance that is being tested has starch present, then there will be a change in the color to a blackish-bluish liquid.

The best method for testing proteins will be the Biuret test. This test can also determine whether there are peptide bonds in the protein. A positive result for proteins will show a color change from a blue to a light purple. If there are peptide bonds present, the color will be a much darker purple.

If Biuret can’t be used, Fehling’s Solutions A and B and Sodium hydroxide and copper (II) sulphate solutions can be used.

The test that best shows the presence of lipids is the Sudan III test. Sudan III is a

fat-soluble compound that will cause the color of the solution with lipids present to change to red. An easier test to preform is using a brown paper bag, lipids are fats, thus on a brown paper bag it will leave “greasy”, translucent stains. Another test for testing the presence of lipids is an Emulsion test

The main test for Vitamin C is adding drop for drop the Vitamin C solution to Indophenol solution. If the dark blue color of the Indophenol solution becomes colorless, then the suspected solution does indeed contain Vitamin C. In our experiment, we will use the Iodine solution to test for the presence of Vitamin C, because it follows the same principles that Indophenol does.



Testing with Iodine solution:

Clean all your test tubes thoroughly with ethanol and then rinse them with distilled water.

Label each test tube as water, protein, starch, lipid, vitamin C and unknown.

Add 3ml of each of the macromolecules to the correct test tube, except Vitamin C.

Add 2-5 drops of the Iodine solution to each of the test tubes, except Vitamin C.

Add 3ml of the Iodine solution to the test tube marked as Vitamin C.

Drop wise, add Vitamin C to the test tube until the color changes.

Mix the contents well.

Note any color changes.

Testing with Biuret reagent:

Clean all your test tubes thoroughly with ethanol and then rinse them with distilled water.

Label each test tube as water, protein, starch, lipid, vitamin C and unknown.

Add 3ml of each of the macromolecules to the correct test tube.

Add 2-5 drops of the Biuret reagent to each of the test tubes.

Mix the contents well.

Note any color changes.

Testing with Sudan III reagent:

Clean all your test tubes thoroughly with ethanol and then rinse them with distilled water.

Label each test tube as water, protein, starch, lipid, vitamin C and unknown.

Add 3ml of each of the macromolecules to the correct test tube.

Add 2-5 drops of the Sudan III reagent to each test tube.

Mix the contents well.

Note any color changes.


Apparatus used:

Test tubes for containing the solutions.

Test tube rack to keep all test tubes organized.

Pipet will make the transfer of different liquids easier and the measuring there of.

A piece of white paper will make it easier to determine whether a color change has taken place.

Safety goggles to protect your eyes from any harmful reagents.

A white lab coat to protect your skin from any harmful reagents.

Chemical Compounds:

Distilled Water – used for rinsing test tubes and making the control solutions

Ethanol – to clean the test tubes

Ascorbic Acid (Vitamin C)




Unknown substance

Iodine solution – used for testing starches

Sudan III solution – used for testing lipids

Biuret & Copper sulfate solution – used for testing proteins


Table 1






Vitamin C





Dark blue, almost black

Yellow drop


Purple black

Sudan III

Light red

Light red

Light red


Light red

Light red

Biuret &

Copper sulfate

Light blue

Dark purple

Light blue

White with blue drop


Light blue


Water was used as the representative to show the natural change that will take place when the different solutions were added.

The protein had no different effect than that of the water when Iodine and the Sudan III was added, thus we can assume it to be negative. When it reacted with the Biuret solution, it yielded a different reaction than water did, it changed to a dark purple, resulting in a positive reaction, the hypothesis was correct.

Starch showed no different changes than water with the Sudan III and Biuret test, thus we assume it to be negative. While with the Iodine test, it changed color to a dark black-blue color, as indicated by the hypothesis. This color change indicates a positive reaction to iodine, meaning that there is starch present in our compound.

The suspected lipids solution showed a changed in color when Sudan III was added to it. This solution changed to a red color, thus there were lipids present. The other two tests, Iodine and Biuret tests didn’t yield any changes that differed from the results obtained from water. This test proved the hypothesis was correct.

When testing for Vitamin C, the iodine solution proved that there was Vitamin C present because it turned the dark blue Iodine solution colorless. The other two tests, Sudan II and Biuret test show no difference from the water control subjects. The results from this test was as expected from the hypothesis.

The Unknown substance only showed a positive response when tested with the Iodine test, leading me to believe that it is some kind of carbohydrate compound and possibly starch. The Sudan III test was negative and so was the Biuret test.

Interpretation of results


The protein solution reacted with the biuret reagent to show a change in color to a dark purple. This means that there was indeed peptide bonds present in the solution. The Biuret reagent has both KOH (potassium hydroxide) and CuSOâ‚„ (copper sulfate). The solution’s pH is raised to an alkaline by the KOH, which is important for CuSOâ‚„ to react. Cu⁺² react with nitrogen atoms present in peptide bonds to form a complex. The CuSOâ‚„ solution is usually a blue solution, but when the Cu⁺² react with the nitrogen atoms to form the complex, the blue changes to violet, sometimes dark purple color.


The starch reacted with the iodine solution to bring forth a blackish-blue change in color. This color change is attributed to the iodine and iodide ions reacting with each other and bond, forming a triodide ion. The iodine, which is a ligand, fits into the space created by the hundreds and thousands of simple sugar molecules such as Glucose that makes up the structure of starch. The color change to black is associated with the absence of light, since iodine absorbs all the light wavelengths.


The lipid solution reacted with the Sudan III reagent to change its color to an orange-red solution. Lipids are triglycerides, and have an oily appearance. Sudan III is a dye that is lipid soluble; it is also non-polar and will react with hydrocarbon chains in the lipids to form hydrophobic interactions.

Vitamin C:

Vitamin C reacted with the iodine solution to diminish the blackish-blue color of iodine to form an almost colorless, but milky solution. This is accounted for because when these two solutions react with each other, they are no longer what they started out as. During this reaction, these two are mixed together, the ascorbic acid losses its electrons to iodine, meaning that this reaction is a redox reaction. Ascorbic acid will be the one being oxidized while iodine is reduced. Ascorbic acid forms dehydroascorbic acid and iodine forms iodide ions.


The unknown solution reacted with the iodine indicator to form a blackish-blue solution. The only known solution which reacted with iodine to form a solution of that color was starch. Although these colors don’t match up perfectly, they are the closest match.

The difference in color between the starch solution and the Unknown solution could be explained by a difference in concentration between the two solutions. This difference in concentration could have resulted if the solutions weren’t both well shaken, since starch can settle out of solution.

Broader implications of results

Biuret test:

This test is used for detecting the presence of proteins in a solution. When there are proteins present, the mixture of our suspected solution and the Biuret reagent will undergo a color change to violet. This indicates the presence of peptide bonds in the protein solution. I did achieve this by proving that our suspected protein solution did indeed contain peptide bonds present in proteins when it changed color to violet.

Iodine test:

Iodine can be used to detect the presence of both starch and vitamin C.

When testing for starch, a positive test will be indicated by a change in color from colorless starch to a blackish-blue solution after the addition of iodine. Our suspected starch solution did test positive for starch when its color changed to blackish-blue.

When testing for the vitamin C, small amounts of vitamin C was added to the iodine solution, this determines whether or not a redox reaction will take place to transfer vitamin C electrons to the iodine. This was proved to be correct when the addition of vitamin C to the iodine eliminated the blackish-blue color of the indicator.

Sudan III test:

Sudan III is a lipid soluble reagent that is used to stain triglycerides. This is used to detect the presence of lipids. Both lipids and Sudan III are non-polar and react with each other to have a hydrophobic interaction, and a color change is present when the suspected solution contains lipids. The change is a red-orange color. Our test showed this when our suspected solution exhibited this change in color to red.


I can conclude that all the expected results did indeed happen. The protein solution did change color to violet-purple when tested with the Biuret test, resulting in a positive reaction. This was because the Cu⁺² reacted with the nitrogen atoms. The starch solution reacted with iodine to bring forth a blackish-blue change in the color, testing positive. This is attributed to the formation of a triodide ion. The lipids did indeed react with the Sudan III to show the positive reaction of color change to red, because of the formation of hydrophobic portion in the solution. The vitamin C showed the positive results when added to iodine when it eliminated the dark color associated with iodine. This result is because of the redox reaction that takes place between these two compounds. All of my results prove that my hypothesis were indeed correct. The unknown solution only showed a positive reaction with the starch, thus it can be concluded that it’s starch or maybe a form of a carbohydrate close to the composition of starch.


J.M Berg, J.L Tymoczko, L Stryer. Biochemistry, 5th ed. New York: W.H. Freeman; 2002

Dr Ananya Mandal. What are Lipids?. (accessed 29 September 2014).

R.B Smith, E.C Loucheed, E.W Franklin, I. McMillan. The Starch Test for Determining Stage of Maturation in Apples. Canadian Journal of Plant Science 1979; 59(3): 725-735.

SEP Staff. Testing for Lipids, Proteins and Carbohydrates. (accessed 29 September 2014)

Mack, S. How does Biuret ReagentCause a Color Change with Proteins?.,html (accessed 2 October 2014).

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