Introduction
Carbohydratesproteins, lipidsand nucleic acids are fundamental organic molecules present in living organisms. These biological macromolecules contain carbon and may also include hydrogen, oxygen, nitrogen, phosphorus, sulfur, and other minor elements. Carbohydrates, composed of monosaccharide units, function primarily in energy storage (Mika et al., 2024). The presence of macromolecules is determined using several methods. For example, the presence of reducing sugars is detected using Benedict's reagent, which changes color from green to reddish upon oxidation by cupric ions (Benedict, 2002). Starch, a polysaccharide with long chains of glucose units, is identified by the iodine test, which turns blue-black in the presence of starch due to the interaction of iodine with the helical structure of the polymer. Proteins, consisting of long chains of glucose units, are found in the presence of glucose and carbohydrates. amino acidsThey perform critical functions such as enzymatic activity, molecular transport, and structural support (Pesek et al., 2022). The Biuret test detects proteins by forming a violet complex with copper ions that bind peptide bonds (Lubran, 1978). Lipids, which include fats, oils, and phospholipids, are primarily involved in energy storage and membrane structure. The presence of lipids is confirmed by the fat spot test, where lipids leaves a translucent mark on the paper (Grease stain test, undated).
In this experiment, Benedict’s test, iodine test, Biuret test and fat 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 solutions, then Benedict’s test will produce a color change indicating the presence of reducing sugars, iodine test will result in a blue-black color confirming the presence of starch, Biuret test will produce a violet color showing the presence of proteins and fat spot test will reveal a translucent mark indicating the presence of lipids.
Materials and methods
To detect carbohydrates, Benedict's test was performed (Mika et al., 2024). Eight test tubes were prepared, each containing 10 drops of a different solution: onion juice, potato juice, sucrose solution, glucose solution, distilled water, fructose solution, starch solution, and chyme. To each test tube, 1 ml of Benedict's reagent was added and the initial color was recorded. Then, the test tubes were placed in a boiling water bath for 3 minutes. After cooling, the final color of each solution was noted and analyzed for the presence of carbohydrates.
For starch detection, the iodine test was performed following a procedure similar to that of the Benedict's test. Instead of Benedict's reagent, 4 to 6 drops of iodine solution were added to each test tube containing the same set of solutions (Mika et al., 2024). Boiling was not performed in this test.
The presence of proteins was assessed by the Biuret test. Six test tubes were prepared with 2 ml of each of the following solutions: egg albumin, honey, amino acidsdistilled water, protein solution and chyme. The initial colour of each solution was recorded. To each test tube was added 1 ml of 2.5% NaOH solution, followed by 8-10 drops of Biuret reagent. The solutions were gently mixed and any colour change was observed to determine the presence of proteins (Mika et al., 2024).
Lipids were detected using the fat spot test. Six small squares were cut from a brown paper bag and labeled accordingly. One drop of each of the following substances was placed in separate squares: maple syrup, chocolate syrup, canola oil, peanut butter, frosting, and chyme. The squares were allowed to dry and the presence of a translucent fat spot was observed to confirm the presence of lipids (Mika et al., 2024).
Results
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 that no reducing sugars were present.
Table 1: Benedict's test results
| 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 | United products | Milky yellow | Blue |
In the iodine test, all solutions turned brown after the addition of iodine, meaning the absence of starch in all samples.
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 | United products | Milky yellow | Brown |
The Biuret test showed the presence of proteins in the egg albumen, protein solution and chyme, as evidenced by the purple color change. All other samples remained blue, indicating no protein was present.
Table 3: Biuret test results
| Tube | Solution (2 ml) | Initial color | Color after Biuret |
| 1 | Egg albumin 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 | United products | Yellowish | Purple |
In the grease spot test, lipids were detected in canola oil, peanut butter, and frosting, as indicated by the formation of large, visible oil spots. No oil spots were observed in the other samples.
Table 4: Grease stain test results
| Tube | Solution (1 drop) | Description of the grease stain reaction |
| 1 | Maple syrup | The stains didn't move, there's no grease. |
| 2 | Chocolate syrup | The stains didn't move, there's no grease. |
| 3 | Canola oil | The stain became larger and oilier. |
| 4 | Peanut butter | The stain became larger and oilier. |
| 5 | Icing | The stain became larger and oilier. |
| 6 | United products | The stain didn't move, liquid, no grease. |
Discussion
The results support the hypothesis that the presence of carbohydrates, starch, proteins and lipids causes specific colour changes (green, orange, brick red, purple and the appearance of a greasy spot) when their respective reagents are added. Qualitative analysis of the samples assessing the colour change helps to determine the specific macromolecule present in the solution.
Benedict's test indicated the presence of reducing sugars in the onion juice, which changed color to green (Table 1). This color change confirms the presence of reducing sugars, since they can reduce cupric ions to cuprous oxide. In contrast, the other samples did not change color, indicating the absence of reducing sugars (Benedict, 2002).
The iodine test did not reveal the presence of starch in any of the samples, including the starch solution itself (Table 2). Normally, iodine forms a blue-black complex with starch, but the lack of color change in our tests suggests that either the starch was degraded or the iodine solution was ineffective (Pesek et al., 2022). Starch solutions have a limited shelf life and can degrade over time, which could explain the negative results. Additionally, problems with the iodine solution could have contributed to the lack of reaction.
The Biuret test showed the presence of proteins in egg albumen, protein solution and chyme, as evidenced by the purple color change (Table 3). The absence of color change in the amino acid solution can be explained by the lack of peptide bonds; the Biuret reagent reacts specifically with peptide bonds and individual amino acids do not contain these bonds, resulting in no color change (Lubran, 1978).
In the grease spot test, lipids were detected in canola oil, peanut butter, and frosting, which produced visible oil spots (Table 4). The absence of grease spots in maple syrup, chocolate syrup, and chyme confirmed the lack of lipids in these samples. The grease spot test is effective in detecting lipids due to their ability to create an oily residue on paper (Grease Spot Test, n.d.).
To improve experimental reliability, it is important to use freshly prepared solutions and to incorporate positive and negative controls in each test. For example, the iodine test for starch lacked a positive control, making accurate interpretation of the results difficult. Including a known positive sample would have clarified whether the problem was with the starch solution or the iodine reagent.
Additional experiments
To improve the analysis of carbohydrates, starch, proteins and lipids, quantitative methods such as colorimetric assays and Soxhlet extraction could be used to measure concentrations more accurately. In addition, the incorporation of specific enzyme activity assays and advanced techniques such as thin layer chromatography would allow for an understanding of the biochemical properties and types of these macromolecules.
References
- Benedict SR (2002). A reagent for the detection of reducing sugars. 1908. Journal of Biological Chemistry, 277(16), e5.
- Lubran MM (1978). Measurement of total serum proteins by the Biuret method. Annals of clinical and laboratory sciences, 8(2), 106–110.
- Grease stain test. (undated). Lipid identification methods. Retrieved from (https://cdn.agclassroom.org/media/uploads/LP841/Grease_spot_test_student_instruction s.pdf)
- Pesek, S., Lehene, M., Brânzanic, A.M.V. & Silaghi-Dumitrescu, R. (2022). On the origin of the blue color in the supramolecular iodine/iodide/starch complex. Molecules (Basel, Switzerland), 27(24), 8974.
https://doi.org/10.3390/molecules27248974
- Mika, TA, Klein, RJ, Bullerjahn, AE, Connor, RL, Nadador, LM, White, R.
E., Gosses, M.W., Carter, T.E., Andrews, A.M., Maier, J.L., & Sidiq, F. (Eds.). (2024). Anatomy and Physiology Laboratory Manual BIO 211 (3rd ed.). Owens Community College.
