Introduction
In its 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 the common food categories listed on Nutrition Facts panels. 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 characteristic fashion, often a color change, if a particular molecule is present.
Each test will include a positive control and a negative control. A positive control is a test substance that should reliably produce a positive result. It shows what a positive reaction looks like. It contains the compound for which we are testing and all the appropriate chemical indicator(s). A negative control is a test substance that should reliably produce a negative result. It shows what a negative reaction looks like. It usually contains just distilled water (dH2O) and the appropriate indicator(s). To be valid, a negative control is placed through all the physical steps of a positive control 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.
Exercise 1: *Wear protective goggles and gloves during this activity.*
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 1 Results of Carbon Flame TestSubstance
Did it blacken?
Is it organic or inorganic?
Salt
Sugar
Gelatin
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, but in most biological situations containing water they exist in the ring structure (Figure 1). Two simple sugars bound 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 2). Lactose is also a disaccharide composed of galactose and glucose.
Figure 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)
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 are able to 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 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 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.
Figure 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)Benedict’s Test for Reducing Sugars
Materials Required
Test tube rack Distilled water (dH2O) Test tube holder
7 test tubes Transfer pipettes Red wax pencil (or Sharpie)
Starch solution Benedict’s reagent Boiling water bath (or heat block)
Unknown (#1- #4) Glucose solution Potato
Cow’s milk Sucrose solution
*Each lab group should pick only ONE of the 4 unknown solutions to use for each of the following tests. Different lab groups will use different unknown solutions and then relay the results of their unknown solution to the other lab groups.
Procedure:
*wear gloves and safety goggles when performing this activity*
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
6
Potato
7
Unknown # ____
Exercise 3: Testing for Carbohydrates - Iodine Test for Starch
Polysaccharides are very long chains of monosaccharides and 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 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 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 Required
dH2O Transfer pipettes sucrose solution Toothpicks
Starch solution Glucose solution
Iodine Unknown (#1 - #4) cracker
Potato paper 9 or 12-well spot plate (Figure 5)
Figure 5. Well plate (original photo)Procedure:
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
7
Cracker
8
Unknown # ____
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. Proteins serve diverse and vital roles in our bodies. 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 DNA polymerase, are proteins and 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.
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 causing a color change from its original color to purple or pink. 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 or pink 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.
Figure 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
5 Test tubes Toothpicks Albumin
Unknown (#1 - #4) Biuret reagent Milk
Sucrose solution Transfer pipettes dH2O
Procedure:
*wear gloves and safety goggles when performing this activity*
Test Tube
Substance Tested
Final color after Test
Conclusion:
Positive (+) or Negative (-)
1
Water
2
Albumin (egg white)
3
Sucrose Solution
4
Milk
5
Unknown # ___
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 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 7. Negative ethanol emulsion test (left) and positive ethanol emulsion test (right two tubes). (original photo)Materials Required
Test tube rack Transfer pipettes dH2O
12 test tubes Ethanol Half and half
Red wax pencil (or Sharpie) Vegetable oil Unknown (#1 - #4)
Sucrose solution Mortar & pestle Parafilm
Procedure:
Test tube
Substance Tested
Observations at end of test
Conclusion:
Positive (+) or Negative (-)
1
Distilled Water
2
Vegetable Oil
3
Sucrose Solution
4
Half and half
5
Potato chips
6
Unknown #____
Exercise 6: Grease Spot Test for Lipids (alternative lipid test)
The grease spot test is a simple test to observe the presence of lipids in a substance. In the grease spot test, a drop of the test substance is placed onto brown paper and allowed to dry. If the test substance is a solid substance, then the solid is crushed and rubbed onto the brown paper. The liquids must be given ample time to dry. Water placed onto brown paper will dry and the spot will disappear. Substances containing lipids dry but still appear wet, leaving a translucent spot that is easily visible when the brown paper is held up to the light (Figure 8). You might have observed this type of result if you’ve noticed grease spots on a paper bag when picking up greasy take-out foods.
Figure 8. Negative grease spot test (left) and positive grease spot test (right). (original photo)Materials Required
Sucrose solution Transfer pipettes dH2O
Brown paper squares Half and half Pencil
Vegetable oil Unknown
Procedure:
Table 6. Results of grease spot test for lipids
Brown paper
Substance Tested
Observation after drying
Conclusion:
Positive or Negative
1
Distilled Water
2
Vegetable Oil
3
Sucrose Solution
4
Half and half
5
Potato chips
6
Unknown #____
Exercise 7: Testing Unknown Substances
Table 7. Analysis of Unknown Substances
Unknown Solution #
Benedict’s Test
(+ or -)
Iodine Test
(+ or -)
Biuret Test
(+ or -)
Ethanol Emulsion Test
(+ or -)
Grease spot test (+ or -)
List Organic Molecule(s) Present
1
2
3
4
Questions for Review
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
Practical Challenge Questions
Biuret
Light purple color
Iodine
Yellowish color
Benedicts
Orange color
Ethanol Emulsion
Clear liquid
Flame test
blackened
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
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