Laboratory Report: Biomolecule Detection

Objective

1. Identify and apply reagents that detect carbohydrates, lipids, and proteins.
2. Describe the role of negative and positive controls.
3. Develop an experiment to determine the biomolecule composition of unknown solutions.
4. Apply appropriate controls during analysis and interpretation of experimental data.
5. Understand the importance of accurate measurement techniques.

Introduction

In this laboratory exercise, we aim to detect biomolecules from three major classes - proteins, lipids, and carbohydrates using chemical reagents. These biomolecules play crucial roles in living organisms. Proteins catalyze chemical reactions, transport substances, and provide structural support.

Lipids serve as energy reserves, while carbohydrates, like starch, provide a source of glucose for cellular work. We will use negative and positive controls to validate our experiments and interpret the results accurately.

Biomolecule Classes

• Carbohydrates: Polysaccharides like starch are composed of glucose molecules linked by covalent bonds. We will use Lugol's iodine (IKI) to test for the presence of starch.
• Monosaccharides/Disaccharides (sugars): Benedict's solution will detect sugars containing free aldehyde or ketone groups.
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  It changes color from blue to green, yellow, orange, or brick red based on sugar concentration.

• Proteins: Amino acids form proteins, and peptide bonds link them. Biuret reagent will detect proteins by changing color from blue to lavender in their presence.
• Lipids: Lipids include fats (triglycerides), phospholipids, waxes, and steroids. We will use Sudan III to detect the presence of fat.

Part I: Detection of Biomolecules in Known Solutions

We will start by testing six known solutions (Table 1) for the presence of proteins, fats, glucose/fructose, and starch. Predictions will be made before experimentation and compared to observed results.

Carbohydrates - Starch Detection

Starch, a polysaccharide, will be detected using Lugol's iodine (IKI). The change in color from amber to dark blue indicates the presence of starch due to the interaction of IKI with the starch polymer.

Protocol for Starch Detection

1. Obtain 5 glass test tubes labeled 1ST - 5ST.
2. Measure 5 ml of each solution indicated in the table below with a graduated cylinder and transfer to the labeled test tube.
3. Add 3 drops of Lugol's iodine to each test tube (1ST - 5ST).
4. Cover the tube with parafilm and gently mix. Observe and record your data in Table 2.
5. Analyze the data to determine which solutions contain starch.
6. Identify and retain positive and negative controls for Lugol's reagent.

Monosaccharides/Disaccharides - Sugar Detection

Benedict's solution will be used to detect sugars containing free aldehyde or ketone groups. The color change from blue to green, yellow, orange, or brick red indicates the presence of sugars.

Protocol for Sugar Detection

1. Obtain 5 glass test tubes labeled 1SU - 5SU.
2. Measure 5 ml of each solution indicated in the table below with a graduated cylinder and transfer to the labeled test tube.
3. Add 5 ml of Benedict's solution to each test tube (1SU - 5SU) and incubate in an 80°C water bath for 5 minutes.
4. Observe the results and record your data in Table 3.
5. Analyze the data to determine which solutions contain sugar.
6. Identify and retain positive and negative controls for Benedict's reagent.

Proteins - Protein Detection

Biuret reagent will be used to detect proteins by changing color from blue to lavender in their presence, due to the interaction between copper ions in the reagent and peptide bonds in the protein.

Protocol for Protein Detection

1. Obtain 5 glass test tubes labeled 1PR - 5PR.
2. Measure 5 ml of each solution indicated in the table below with a graduated cylinder and transfer to the labeled test tube.
3. Add 3 ml of Biuret's solution to each test tube (1PR - 5PR).
4. Cover the tubes with parafilm and gently mix. Observe and record your data in Table 4.
5. Analyze the data to determine which solutions contain protein.
6. Identify and retain positive and negative controls for Biuret reagent.

Lipids - Lipid Detection

Sudan III is used to detect the presence of lipids. It binds to hydrocarbon chains found in lipids. If a solution contains lipids, Sudan III dye will be retained on the filter paper.

Protocol for Lipid Detection

1. Obtain 6 glass test tubes labeled 1F - 6F.
2. Measure 2 ml of each solution (except vegetable oil) indicated in the table below with a graduated cylinder and transfer to the labeled test tube. Measure 2 ml of vegetable oil with a transfer pipette.
3. Add 5 ml of ethanol (95%) to each solution to dissolve any fats before exposure to Sudan III. Cover the test tubes with parafilm and vigorously shake. Let the tubes incubate at room temperature for 2 minutes.
4. Divide a piece of filter paper into 6 equal sections and label them 1F - 6F.
5. Place one drop of each solution onto the corresponding section of the filter paper using a different transfer pipette for each solution, avoiding cross-contamination.
6. Transfer the filter paper to a hot plate and allow it to dry.
7. Once the filter paper is dry, add a second drop of each solution onto the filter paper, directly on top of the first drop to concentrate the solutions on the filter paper. Use the hot plate to dry it again.
8. Repeat the process one last time by adding a third drop of each solution onto the filter paper and then using the hot plate to dry it.
9. Submerge the filter paper in the Sudan III solution and incubate it for one minute.
10. After the incubation in Sudan III, rinse the filter paper in distilled water for 1 minute.
11. Take your filter paper back to your bench and record your observations in Table 5.
12. Analyze the data and determine which solution(s) contain fat. Also, identify and retain positive and negative controls for the Sudan III reagent. Make sure to keep your filter paper for the next exercise.

Part II: Determination of Biomolecule Composition in Unknown Solutions

In this part, we will test several unknown solutions and compare the experimental data to the negative and positive controls established in Part I to determine the biomolecule composition of these unknown solutions.

Conclusion

In this laboratory experiment, we successfully detected the presence of biomolecules in known solutions using various chemical reagents. We used Lugol's iodine to identify starch, Benedict's solution to detect sugars, Biuret reagent to detect proteins, and Sudan III to identify lipids. By comparing our predictions to experimental observations, we determined which solutions contained specific biomolecules. These experiments helped us understand the importance of negative and positive controls in validating our results and ensured the accuracy of our biomolecule detection techniques.