introduction
Carbohydrates, proteins, lipids, and nucleic acids are the basic organic molecules present in living organisms. These biological macromolecules contain carbon and may also contain hydrogen, oxygen, nitrogen, phosphorus, sulfur, and other trace elements. Carbohydrates, which are composed of monosaccharide units, function primarily in energy storage (Mika et al., 2024). The presence of macromolecules is determined in various ways. For example, the presence of reducing sugars is detected using Benedict’s reagent, which changes color from green to reddish due to the oxidation of copper ions (Benedict, 2002). Starch, a polysaccharide with long chains of glucose units, is identified by the iodine test. Iodine interacts with the helical structure of the polymer, so the presence of starch turns blue-black. Proteins, which are composed of long chains of amino acids, play important roles such as enzymatic activity, molecular transport, and structural support (Pesek et al., 2022). The biuret test detects proteins by forming a purple complex with copper ions that bind to peptide bonds (Lubran, 1978). Lipids such as fats, oils, and phospholipids are primarily involved in energy storage and membrane structure. The presence of lipids is confirmed using the grease spot test, in which lipids leave a translucent mark on paper (Grease Spot Test, n.d.).
In this experiment, the Benedict test, the iodine test, the biuret test, and the grease spot test were performed to qualitatively determine the presence of carbohydrates, starch, proteins, and lipids in various solutions, respectively (Mika et al., 2024).The hypothesis of this experiment is that if carbohydrates, starch, proteins, and lipids are present in the tested solution, the Benedict test will produce a color change indicating the presence of reducing sugars, the iodine test will produce a blue-black color confirming the presence of starch, the biuret test will produce a purple color indicating the presence of proteins, and the grease spot test will produce a translucent mark indicating the presence of lipids.
Materials and Methods
To detect carbohydrates, a Benedict test was performed (Mika et al., 2024). Eight test tubes were prepared, each containing 10 drops of different solutions: onion juice, potato juice, sucrose solution, glucose solution, distilled water, fructose solution, starch solution, and chyme. 1 ml of Benedict’s reagent was added to each test tube and the initial color was recorded. The test tubes were then placed in a boiling water bath for 3 minutes. After cooling, the final color of each solution was recorded and analyzed to determine the presence of carbohydrates.
For starch detection, an iodine test was performed with a procedure similar to that of the Benedict test. Instead of Benedict’s reagent, 4–6 drops of iodine solution were added to each test tube containing the same set of solutions (Mika et al., 2024). No boiling was performed in this test.
The presence of protein was assessed using the Biuret test. Six test tubes were prepared containing 2 ml each of egg white, honey, amino acids, distilled water, protein solution, and chyme solution. The initial color of each solution was recorded. 1 ml of 2.5% NaOH solution was added to each test tube, followed by 8-10 drops of Biuret reagent. The solutions were mixed gently and the color change was observed to determine the presence of protein (Mika et al., 2024).
Lipids were detected using a grease spot test. Six small squares were cut from a brown paper bag and each was labeled. A drop of each substance was placed on a separate square: maple syrup, chocolate syrup, canola oil, peanut butter, icing, and chyme. The squares were allowed to dry and the presence of translucent grease spots was observed to confirm the presence of lipids (Mika et al., 2024).
result
In Benedict’s test, only the onion juice changed color from blue to green, indicating the presence of reducing sugars – all other samples remained blue, indicating the absence of reducing sugars.
Table 1: Results of the Benedict test
| tube | Solution (10 drops) | Initial Color | Color after boiling |
| 1 | Onion juice | white | green |
| 2 | Potato juice | brown | Blue |
| 3 | Sucrose solution | Clear | Blue |
| 4 | Glucose solution | Clear | Blue |
| 5 | Distilled water | Clear | Blue |
| 6 | Fructose Solution | Clear | Blue |
| 7 | Starch Solution | Clear | Blue |
| 8 | Chyme | Milky yellow | Blue |
The iodine test showed that all solutions turned brown upon addition of iodine, indicating that all samples were free of starch.
Table 2: Iodine Test Results
| tube | Solution (10 drops) | Initial Color | Color after iodine |
| 1 | Onion juice | white | brown |
| 2 | Potato juice | brown | brown |
| 3 | Sucrose solution | Clear | brown |
| 4 | Glucose solution | Clear | brown |
| 5 | Distilled water | Clear | brown |
| 6 | Fructose Solution | Clear | brown |
| 7 | Starch Solution | Clear | brown |
| 8 | Chyme | Milky yellow | brown |
The Biuret test showed the presence of protein in egg white, protein solution and chyme, turning them purple in colour, all other samples remained blue, indicating the absence of protein.
Table 3: Biuret test results
| tube | Solution (2ml) | Initial Color | Color after Biuret |
| 1 | Egg white solution | Clear | Purple |
| 2 | Honey solution | yellow | Blue |
| 3 | Amino Acid Solution | yellow | Blue |
| 4 | Distilled water | Clear | Blue |
| 5 | Protein Solution | Clear | Purple |
| 6 | Chyme | Yellowish | Purple |
Grease stain testing showed lipids in canola oil, peanut butter, and icing, forming large visible grease stains, while no grease stains were observed in the other samples.
Table 4: Grease Spot Test Results
| tube | Solution (1 drop) | Grease Spot Reaction Explained |
| 1 | Maple syrup | The stain didn’t move and there was no oil. |
| 2 | Chocolate syrup | The stain didn’t move and there was no oil. |
| 3 | Canola oil | The spots have gotten bigger and oilier |
| 4 | Peanut butter | The spots have gotten bigger and oilier |
| 5 | Icing | The spots have gotten bigger and oilier |
| 6 | Chyme | The spots are stationary, liquid, and not oily. |
Discussion
The results support the hypothesis that the presence of carbohydrates, starches, proteins, and lipids results in color changes such as green, orange, brick red, purple, and oil stains upon addition of the respective reagents. Qualitative analysis of samples by evaluating the color changes can help identify the specific macromolecules in solution.
Benedict’s test showed that onion juice contained reducing sugars, which changed its color to green (Table 1). This color change confirmed the presence of reducing sugars, as they are capable of reducing copper ions to cuprous oxide, while the other samples did not change color, indicating that they did not contain reducing sugars (Benedict, 2002).
Iodine testing revealed the absence of starch in any of the samples, including the starch solution itself (Table 2). Normally, iodine forms a blue-black complex with starch, but our tests showed no color change, suggesting that the starch was degraded or the iodine solution was ineffective (Pesek et al., 2022). The starch solution has a limited shelf life and may degrade over time, which may have led to negative results. Additionally, issues with the iodine solution may have contributed to the lack of reaction.
The Biuret test indicates the presence of protein in egg white, protein solutions, and chyme by causing a purple color change (Table 3). The absence of a color change in amino acid solutions is due to the absence of peptide bonds. The Biuret reagent reacts specifically with peptide bonds, and since pure amino acids do not contain these bonds, no color change occurs (Lubran, 1978).
The Grease Spot Test detected lipids in canola oil, peanut butter, and icing, which left visible oily spots (Table 4). Maple syrup, chocolate syrup, and chyme did not have any oily spots, confirming that these samples did not contain lipids. The Grease Spot Test is effective at detecting lipids that leave an oily residue on paper (Grease Spot Test, n.d.).
To increase the reliability of your experiments, it is important to use freshly prepared solutions and incorporate both positive and negative controls with each test. For example, the iodine test on starch did not have a positive control, making it difficult to accurately interpret the results. Including a known positive sample makes it clear whether the problem is with the starch solution or the iodine reagent.
Further Experiments
To enhance the analysis of carbohydrates, starches, proteins, and lipids, quantitative methods such as colorimetry and Soxhlet extraction can be used to measure concentrations more accurately. Furthermore, the incorporation of advanced techniques such as specific enzyme activity assays and thin layer chromatography will provide a better understanding of the biochemical properties and types of these macromolecules.
References
- Benedict SR (2002). Reagents for the detection of reducing sugars. 1908. Journal of Biochemistry, 277(16), e5.
- Lubran MM (1978). Determination of total serum protein by the biuret method. Annual Report of Clinical Laboratory Science, 8(2), 106-110.
- Grease Spot Test. (nd) How to identify lipids. source: [https://cdn.agclassroom.org/media/uploads/LP841/Grease_spot_test_student_instruction s.pdf]
- Pesek, S., Lehene, M., Brânzanic, AMV, & Silaghi-Dumitrescu, R. (2022). On the origin of the blue color of iodine/iodide/starch supramolecular complexes. Molecular (Basel, Switzerland), 27(24), 8974.
https://doi.org/10.3390/molecules27248974
- Mika, TA, Klein, RJ, Brajan, AE, Connor, RL, Swimmer, LM, White, R.
E., Gosses, M. W., Carter, T. E., Andrews, A. M., Maier, J. L., and Sidiq, F. (Eds.). (2024). Anatomy and Physiology BIO 211 Laboratory Manual (3rd ed.). Owens Community College.
Amino Acids: Types, Functions, Sources, and Differences between Essential and Non-essential Amino Acids (thesciencenotes.com)
