3: Macromolecules

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Jun 28, 2025
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- 2: Scientific Method
- 4: Enzyme Lab

Brad Basehore, Michelle A. Bucks, & Christine M. Mummert
Harrisburg Area Community College

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Purpose:

This lab introduces students to testing for biological macromolecules using chemical indicators. Students will learn to identify carbohydrates, proteins, and lipids in various substances, including unknowns, while applying the scientific method.

Tasks:

Perform chemical tests for carbohydrates, proteins, and lipids using Benedict's, Iodine, Biuret, and Ethanol Emulsion tests.
Record and analyze color changes to identify macromolecules.
Compile results and deduce the identity of unknown substances based on macromolecule content.

Criteria for Success:

Correctly execute chemical tests and record observations in tables.
Accurately identify macromolecules present in each sample.
Use data to infer the identity of unknown substances.

Timeline

Introduction and Setup (10 minutes): Overview of macromolecules, lab objectives, and safety guidelines.
Carbon test (5 minutes): Instructor demonstration.
Benedict’s and Iodine Tests (20 minutes): Perform and record results.
Biuret and Lipid Test (20 minutes): Perform and record results.
Unknown Analysis (15 minutes): Test unknown and record observations.
Data Compilation and Discussion (15 minutes): Complete tables, analyze data, and infer unknown identities.

Introduction

In the simplest definition, organic compounds include all molecules that contain carbon. By this definition, simple molecules such as carbon monoxide (CO) and carbon dioxide (CO₂) would be defined as organic molecules, however, these simple molecules behave more like inorganic molecules than organic molecules. Therefore, other definitions of organic molecules state that organic molecules are molecules containing both hydrogen and carbon. For our studies, we define organic molecules using the latter definition.

The four main groups of biologically important organic compounds are carbohydrates, lipids, proteins and nucleic acids. These compounds are also known as biological macromolecules and all but the nucleic acids are commonly listed on Nutrition Facts panels on all the food you buy. These biologically important macromolecules play essential roles in cell and organismal structure, energy, and heredity. In addition to carbon and hydrogen, these biologically important organic compounds also contain the four other “building block” elements: oxygen (O), nitrogen (N), phosphorus (P) and sulfur (S).

In this lab, we will use chemical indicators and chemical tests to detect the presence of biological macromolecules. Chemical indicators are substances that react in a predictable fashion, often a color change, to show if a particular molecule is present.

Each test will include a positive control and a negative control. A positive control is a known test substance that will reliably produce a positive result. The purpose of a positive control is to show what a positive reaction looks like as a basis for comparison. A negative control is a test substance that will reliably produce a negative result meaning it shows what a negative reaction looks like for comparison to unknown substances. A common substance to use for the negative control is simply distilled water (dH₂O) and the appropriate indicator(s) because we know that the only chemical in distilled water is H2O. To be valid, controls go through all the physical steps of the experiment, such as heating, changing of pH, etc., if required.

Positive and negative controls differ from the control groups we studied in the Scientific Method lab. Remember, a control group is a test group of subjects that does not receive the treatment under investigation and is used as the baseline for comparison to an experimental group.

Safety Guidelines for Handling Chemicals and Samples:

Wear safety goggles and gloves at all times.
Handle Biuret reagent and ethanol carefully, as they are hazardous.
Dispose of chemical waste as directed by the instructor.
Wash hands thoroughly after completing the lab.

I. Flame test for Carbon (instructor demonstration)

Exercise 1:

As discussed, organic molecules contain carbon and hydrogen. Substances that contain carbon will burn and blacken. To test a substance for carbon, place the substance in a test tube and hold it over a flame for a few moments. If the substance blackens then it contains carbon and is an organic molecule.

Table $\PageIndex{1}$ : Results of Carbon Flame Test
Substance	Did it blacken?	Is it organic or inorganic?
Salt
Sugar
Gelatin

II. Carbohydrates

Carbohydrates include sugars and starches and are composed of monosaccharide building blocks. Glucose and fructose are examples of monosaccharides and are often called simple sugars. Simple sugars can exist in linear or ring structures (Figure $\PageIndex{1}$ ), but in most biological situations water will be present and the sugars will only exist in the ring structure (Figure $\PageIndex{1}$ ). Two simple sugars can bond together form a disaccharide. An example of a disaccharide is sucrose, commonly known as table sugar. Sucrose is formed by a glycosidic covalent bond linking glucose and fructose (Figure $\PageIndex{2}$ ). Lactose is also a disaccharide composed of galactose and glucose.

CgMr7C7pkkGeixhh1_Dstw9j_SEYt_n2Bt602xECXhah0W2qZYfn7kdv1QABZDtFukkVfXJr4DRNx_3geFWd6cNwD_uBeOHZz7tvGYgTLj3oycbNXBdujo1uWPv0C_LiDLbbFS5F — Figure $\PageIndex{1}$ . Glucose in ringed and linear forms. (Blausen.com staff (2014). "Medical gallery of Blausen Medical 2014". WikiJournal of Medicine 1 (2). DOI:10.15347/wjm/2014.010. ISSN 2002-4436., CC BY 3.0 <https://creativecommons.org/licenses/by/3.0>, via Wikimedia Commons)

wGsOtzvcfYA2Lw25cki9kIRbEheKgrEg4WfNMQfirSDkxRaJSCzpt9ByPaj7fDMux2_F5SEbzl-jhuniXltceSmc7UTkdaqvfGbZ7v7kwg9usWToGTOByd91AH58uI5IX2S8YvRA — Figure $\PageIndex{2}$ Sucrose ring structure. (CNX OpenStax, CC BY 4.0 <https://creativecommons.org/licenses/by/4.0>, via Wikimedia Commons)

Polysaccharides are long chains of many subunits of simple sugars covalently bound together. Polysaccharides are often referred to as complex carbohydrates due to their large structure. Starch and cellulose are polysaccharides found in plants. Plants store extra energy in the form of the polysaccharide starch. The complex carbohydrate, cellulose is an important structural material in many plants. Animals store some extra energy (for short-term storage) in the form of the polysaccharide glycogen.

Carbohydrates play important roles in organismal structure and as main sources of energy for cells. Simple sugars, such as glucose, enter directly into metabolic pathways (such as glycolysis) to provide ATP for cells. When larger polysaccharides, such as starch, are consumed by organisms the complex carbohydrates must be broken down by water and enzymes into simpler sugars before they can enter into metabolic pathways to yield ATP.

Exercise 2: Testing for Carbohydrates - Benedict’s Test for Reducing Sugars

All Monosaccharides are reducing sugars. The disaccharides maltose (glucose + glucose) and lactose (glucose + galactose) have a free aldehyde group and are also reducing sugars. Reducing sugars can reduce (add electrons to) other molecules. Reducing sugars have a free carbonyl group (a carbon atom double-bonded to an oxygen atom) that can react to donate electrons. The disaccharide sucrose lacks free carbonyl groups due to the glycosidic bond that links glucose and fructose to create the disaccharide (Figure $\PageIndex{2}$ ). Therefore, sucrose is not a reducing sugar. Benedict’s reagent is a solution of copper sulfate, sodium carbonate, and sodium citrate and is the indicator used to test for the presence of reducing sugars. In the absence of such sugars, Benedict’s reagent is a bright royal blue color, and clear (not cloudy). However, when heated in the presence of a reducing sugar, it accepts electrons from the reducing sugar and changes color. It also becomes cloudy as it forms a precipitate (an insoluble solid that emerges from a liquid solution) of cuprous oxide. Any color change is considered a positive reaction. However, the degree of color change depends on the amount of reducing sugar present (Figure $\PageIndex{3}$ ). A change from blue to yellow, or green, indicates a small amount of reducing sugar. A change from blue to red, or orange, indicates a large amount of reducing sugar. Note that heating the Benedict’s reagent for too long can cause false positive results.

CutZIXiwenpGHxYne22HUhS6tNx5ucC3mV-5YFH6337bR2zCWBgvqh2XuE4BMKrmX13DIk-fYM1Z4X5qCf_lYyrZshLDDjRDKWTc0Hy0ryUkY4Iiffuv11SThYuihqUNpzkr6O8J — Figure $\PageIndex{3}$ . Results of Benedict’s test for reducing sugars. Negative test remains blue (left tube) and positive results show color change (right three tubes). (original photo)

Suggested Student Roles and Group Size (for all activities)

For groups of 2–3 students:

Test Operator: Conducts chemical tests and handles reagents.
Data Recorder: Records observations and ensures tables are completed.
Materials Manager (optional): Prepares and cleans test tubes and supplies.

Benedict’s Test for Reducing Sugars

Materials and chemicals
Instruments	Materials
Test tube rack	Distilled water (dH₂O)
7 test tubes	Benedict’s reagent
Test tube holder	Starch solution
Waste beaker	Sucrose solution
Transfer pipettes	Milk
Sharpie	Glucose solution
Boiling water bath (or heat block)

Procedure:

*wear gloves and safety eyewear when performing this activity*

Use the Sharpie to number seven clean test tubes #1 through #7.

Use a pipette to transfer 1 mL of the test substances/solutions listed in Table 2 to the corresponding labeled test tubes.
1. Your pipettes may have marks on the side known as graduations. You can use these to measure 1mL
2. If your pipettes do not have graduations, 20 drops equals approximately 1mL. It is important to hold the pipette vertically when dispensing drops because holding it at an angle or horizontally will affect the volume of the drop dispensed.

Add 0.5 ml of Benedict’s reagent to each tube. Swirl to mix. Record the color of the solution in each tube in Table 2 below before heating the tubes.

Place the tubes in a gently boiling water bath (or heat block) for 2 minutes. Using a test tube holder, carefully remove the test tubes from the water bath and place in the test tube rack to cool. Hold a white sheet of paper behind the tubes to more easily see the colors. Record any color changes, or changes in appearance, in Table 2.

Use color change information to determine the conclusion for each tube (whether or not each solution contains reducing sugars). A positive result indicates that the solution contains the macromolecule being tested for (reducing sugars). A negative result means that the solution does not contain reducing sugars.

Discard the contents of test tubes in the location indicated by your instructor.
1. Use test tube brushes to wash tubes with soapy water. Rinse thoroughly and shake out excess water. Return test tubes to the proper location.
2. Some test tubes are disposable. If this is the case, recycle them in the proper receptacle after you empty them.

Table $\PageIndex{3}$ . Results of Benedict’s Test for Reducing Sugars
Test Tube	Substance Tested	Color before heating	Final color after Test	Conclusion: Positive (+) or Negative (-)
1	Distilled water
2	Glucose solution
3	Milk
4	Starch solution
5	Sucrose solution

Exercise 3: Testing for Carbohydrates - Iodine Test for Starch

Polysaccharides are long chains of monosaccharides that do not react with Benedict’s reagent. Starch, cellulose and glycogen are examples of polysaccharides. Because these complex carbohydrates are not reducing sugars, and therefore do not chemically react with Benedict’s reagent, a different indicator is required to test for the presence of these complex polysaccharides.

Starch is the storage polysaccharide of plants and is highly digestible when consumed by animals. Iodine (aka Lugol’s Iodine) (I₂KI), an amber-colored clear liquid, is the indicator used to detect the presence of starch. The starch molecules interact with iodine to produce a dark blue-black color (Figure $\PageIndex{4}$ ). Glycogen, the storage polysaccharide in animals, reacts to a lesser extent with Lugol’s to produce a red-brown or reddish-purple color.

Figure $\PageIndex{4}$ : Iodine Test for Starch. Negative (left) and positive (right two wells) results of iodine tests for starch (original photo)

Iodine Test for Starch: Materials

Equipment	Materials
Toothpicks	Distilled water (dH₂O)
Transfer pipettes	Iodine
Scrap paper	Starch solution
9 or 12-well spot plate (Figure 5)	Sucrose solution
	Glucose solution
	Potato
	Unknown (#1 - #4)

Figure $\PageIndex{5}$ : Well plate (original photo)

Procedure:

Use the red wax pencil or Sharpie to label the wells of the well plate with #1-8 (if not already labeled).
Use a transfer pipette to transfer 1ml of the test substances listed in Table 3 to the appropriately numbered well.
1. For the solid substances, cut a small piece of the substance and place it into the appropriately numbered well.
Add 2-4 drops of Iodine to each well (or onto the solid substance). Mix using a toothpick (for solutions). Use a new toothpick for each well.
Place the well plate over white paper so any color changes are easily visible. Record the color of each reaction in Table 3.
Discard the contents of the well plate as instructed. Rinse the plate thoroughly with soap and water.

Table $\PageIndex{3}$ . Results of Iodine Test for Starch
Well	Substance Tested	Final color after Test	Conclusion: Positive or Negative
1	Distilled Water
2	Starch Solution
3	Glucose Solution
4	Sucrose Solution
5	Paper
6	Potato

III. Proteins

Proteins are essential for organisms to survive and are a highly abundant macromolecule in the body. The monomer building blocks of proteins are amino acids. Amino acids are linked through covalent peptide bonds to form polypeptides, also known as proteins. The final function of a protein is entirely dependent on its structure and the sequence of amino acids from which it is made.

Some proteins are important structural proteins in cells, such as tubulin. Other proteins play vital structural and protective roles in organisms, such as keratin. Actin and myosin are proteins that work together in muscle cells to provide movement. Most enzymes, such as digestive enzymes, are proteins that are essential to speed up biological reactions in cells. Ribulose-1,5- bisphosphate carboxylase (commonly known as Rubisco), catalyzes carbon fixation during photosynthesis and is thought to be the most abundant enzyme on earth. Additionally, antibodies are proteins produced by the immune system to protect us from invading pathogens. All of this is to say, proteins serve diverse and vital roles in our bodies.

Exercise 4: Testing for Proteins

Biuret reagent, a light aquamarine-colored liquid, is used to detect the presence of proteins. Copper ions in the Biuret reagent react with peptide bonds of proteins, causing a color change from its original color to a purplish hue. Proteins with short peptide chains turn pink; those with longer chains turn purple (Figure 6). Other types of molecules can cause color changes, but only the purple colors indicate the presence of peptide bonds. Note that a positive Biuret reaction only occurs at an elevated pH; therefore, Biuret reagent contains a strong base (NaOH) turning it a turquoise color. Some protocols include adding additional NaOH to test tubes at the time of protein testing. Caution should be used with Biuret because of this addition of strong base.

Figure $\PageIndex{6}$ : Biuret test for protein. Negative test remains light blue (left tube). Positive result shows change in color to violet (right two tubes). (original photo)

Biuret Test for Proteins

Materials Required

Equipment	Materials
Toothpicks	Albumin Biuret reagent
Transfer pipettes	Milk
5 Test tubes	Sucrose solution
	dH₂O

Procedure:

*wear gloves and safety goggles when performing this activity*

Use the Sharpie to number 4 clean test tubes # 1 through # 4.
Use a pipette to transfer 1 mL of the test substances/solutions listed in Table 4 to the corresponding labeled test tubes.
Add 0.5 ml of Biruet reagent to each test tube. Swirl to mix. Record the color of the liquid in each well in Table 4. Hold a white sheet of paper behind the test tubes to more easily see the colors of the solutions.
Discard the contents of the test tubes in the location indicated by your instructor.
1. This will be the same disposal method you used in the Benedict's test.

Table $\PageIndex{4}$ :. Results of Biuret Test for Protein
Test Tube	Substance Tested	Final color after Test	Conclusion: Positive (+) or Negative (-)
1	Water
2	Albumin (egg white)
3	Sucrose Solution
4	Milk

IV. Lipids

Lipids are a diverse group of nonpolar, hydrophobic, energy-dense organic molecules. Lipids such as triglycerides, phospholipids and sterols play many important biological roles. All membranes in a cell are composed of phospholipids. Many hormones important in sexual development are derivatives of sterol molecules. The most abundant type of lipid in the human diet and human body is triglycerides. Triglycerides consist of three fatty acids bound to one glycerol molecule. If the fatty acids contain only single bonds between the carbon atoms then the fatty acid (and the triglyceride) is “saturated” with hydrogens and referred to as a saturated fatty acid. If the fatty acids contain one or more double bonds between the carbon atoms then the fatty acid (and triglyceride) is referred to as an unsaturated fatty acid. Saturated triglycerides are solid at room temperature and are commonly called fats. Unsaturated triglycerides are liquid at room temperature and are commonly called oils.

Exercise 5: Ethanol Emulsion Test for Lipids

Lipids are nonpolar molecules and cannot dissolve in polar solvents such as water. However, lipids can dissolve in nonpolar solvents such as ethanol. The presence of lipids can be tested using an ethanol emulsion test. An emulsion is formed when two substances that do not dissolve into one another are mixed together. A common example of an emulsion is oil and vinegar salad dressing. When undisturbed, the oil and vinegar separate out into two distinct layers. When you shake it up, the oil and vinegar combine and the oil forms tiny droplets floating in the vinegar.

Ethanol is an amphipathic molecule; it has both polar and nonpolar ends. Because of the nonpolar component of the molecule, ethanol can dissolve lipids; however, because of it’s polar component, ethanol can also mix with water. The ethanol emulsion test works because of the amphipathic nature of ethanol. When lipids are present in a sample, they dissolve in the first step when mixed with ethanol, and the mixture remains clear. However, in the second step of the test when added to water, the lipids are forced out of solution and appear as tiny fat droplets, which reflect light and appear whitish (Figure $\PageIndex{7}$ ). The ethanol emulsion test allows fats in solid materials (such as potato chips) to be extracted in ethanol and then form an emulsion when added to water.

Figure $\PageIndex{7}$ : Negative ethanol emulsion test (left) and positive ethanol emulsion test (right two tubes). (original photo)

Equipment	Materials
12 Test tubes	Ethanol
Test tube rack	Milk
Transfer pipettes	Sucrose solution
Mortar & pestle	dH₂O
Parafilm	Vegetable oil

Procedure:

Use the Sharpie to number 6 clean test tubes #1 through #6. Then, repeat, labeling a second set of test tubes #1 through #6.
Use a pipette to transfer 1 mL of the test substances/solutions listed in Table $\PageIndex{5}$ to the corresponding labeled test tubes. *For solid substances, crush the test substance into small pieces, using a mortar and pestle, and add it to the test tube to approximately the same height on the test tube as the liquid substances.
Use a clean pipette to transfer 2 mL of ethanol into each tube. Use a gloved finger or parafilm to cap the tubes and shake well to mix.
Allow the contents to settle for about 30 seconds.
Use a clean pipette to remove the top half of the solution and transfer it to a clean labeled test tube.
Add 2mL of dH₂O to each tube, and observe the results. The appearance of a milky whitish layer indicates the presence of lipids in the sample. Record your observations in Table 5 below.
Discard contents of test tubes in the location indicated by your instructor.
1. Use test tube brushes to wash tubes with soapy water. Rinse thoroughly and shake out excess water. Return test tubes to the proper location.
2. Some test tubes are disposable. If this is the case, recycle them in the proper receptacle.

Table $\PageIndex{5}$ : **Results of ethanol emulsion test for lipids**
Test tube	Substance Tested	Observations at the end of test	Conclusion: Positive (+) or Negative (-)
1	Distilled Water
2	Vegetable Oil
3	Sucrose Solution
4	Milk
5	Potato chips

Exercise 6: Testing Unknown Substances

Repeat exercises 1-5 for the four unknown substances. In Table 6 compile the data from all of the tests of your unknown.
Determine what compounds (reducing sugar, starch, lipid, protein) are present in your unknown.
Based on the macromolecules in the unknown, try to determine the identity of your unknown substances. The unknowns are all foods/beverages that are commonly consumed at breakfast time.

Table 6. Analysis of Unknown Substances

Table $\PageIndex{6}$ :
Unknown Solution #	Benedict’s Test (+ or -)	Iodine Test (+ or -)	Biuret Test (+ or -)	Ethanol Emulsion Test (+ or -)	List Organic Molecule(s) Present
1
2
3
4

Questions for Review

What simple test can you perform to tell whether a substance is organic or not?

What is the purpose of a positive control? Give examples of positive controls used in this lab. Be specific.

What is the purpose of a negative control? Why did we use water as a negative control in many experiments?

Most monosaccharides and disaccharides are reducing sugars. Explain why the disaccharide, sucrose, is not able to function as a reducing sugar?

Complete the table below:

Macromolecule	Indicator/Test used for detection	Positive results appear as..
Organic molecule
Reducing Sugars (most simple carbohydrates)
Starch
Ethanol Emulsion Test
Greasy Spot test
Proteins

What major characteristic(s) do ALL lipids have in common? Explain.

Practical Challenge Questions

You are given an unknown sample and get the following results:

Test	Color
Buret	Light purple color
Iodine	Yellowish color
Benedicts	Orange color
Ethanol Emulsion	Clear liquid
Flame test	blackened

Based on these results, what can you infer about which organic molecule(s) are in the unknown sample?

Speculate on the identity of this test substance. (what do you think the test substance could be).

Are these test results examples of quantitative or qualitative data? Explain.

Qualitative tests can seem simplistic and don't give you very much information. For example, we do not have any idea how much sugar is in an unknown substance. There are times when qualitative tests, such as these, can be powerful tools. The rapid COVID and flu tests that you may have gotten at a doctor’s office are qualitative tests using a process known as Polymerase Chain Reaction (PCR). Why do you think a qualitative test may be appropriate for this purpose?

True or False? When an Unknown Solution changes to a violet/black color during the iodine test, this constitutes a positive control. Explain your answer.

Diabetes is a chronic disorder that results in an increased level of glucose in the bloodstream. It is caused by inadequate insulin, a hormone produced by the pancreas that allows cells to use and store glucose. One symptom of diabetes is excess glucose in the urine (glycosuria). Which test performed in the lab today could be used to assay a person’s urine to indicate diabetes? Describe the results of that test that would indicate the person has diabetes.

References

Belwood, Jacqueline; Rogers, Brandy; and Christian, Jason, Foundations of Biology Lab Manual (Georgia Highlands College). “Lab 2: Organic Molecules,” (2019). Biological Sciences Open Textbooks. 18. CC-BY https://oer.galileo.usg.edu/biology-textbooks/18

Natale, E. G., Laura Blinderman, &. Patrick. (2021, March 19). Book: Unfolding the Mystery of Life - Biology Lab Manual for Non-Science Majors (Genovesi, Blinderman & Natale). “Exercise 5: Biomolecules.” CC-BY Retrieved April 5, 2021, from https://bio.libretexts.org/@go/page/24114