By Mild, Petch, Prim, Eve, Ging 10-7 Purpose: To use various chemicals to test for the presence of different organic molecules, identify what are positive and negative controls and identify the contents of three unknown samples. Hypothesis:
Benedicts Test for sugars
Chemical Color Water Blue (no change) Monosaccharide and Disaccharide Varies with the concentration: Very low: Green Low: Yellow Moderate: Yellow-orange High: Orange Very high: Orange-red Starch (Polysaccharide) Blue (no change)
Iodine Test for Starch
Chemical Color Water Yellowish-brown (no change) Monosaccharide and Disaccharide Yellowish-brown (no change) Starch (Polysaccharide) Blue-Black
Biuret Test for Proteins
Chemical Color Water Blue (no change) Proteins or peptide Purple
Test for fat- If the solution contains fat there would be an oily spot on the paper after all the water have been evaporated from it. This is because fats and oil do not evaporate, unlike water.
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Materials:
Test tube Gloves Goggle Hot plate Stirrer Water Glucose solution Starch solution Gelation solution Diluted millk Onion Potato Biuret solution Iodine solution Benedic's solution
Procedure:
For Carbohydrates and proteins
1) Fill 300ml of water into the 500ml beaker with a magnetic stirrer. Place the beaker on the hot plate. (DO NOT TURN ON THE HOTPLATE UNTIL TOLD) 2) Ask an instructor or a lab technician to show you how to use a hot plate. Turn on the hot plate and the stirrer to the instructed levels. 3) Make labels (using the abbreviations in the Table 4) on provided marking tape to be placed on the outside of each test tube. This is done so you do not forget which test tube contains what sample. Use the chart below for abbreviation. 4) Apply the label on each test tube. And arrange the test tube on the rack according the Table 4. 5) Put on protective gloves and a goggle (for members who will be dealing with chemicals). 6) Adding approximately 1cm of each sample according to the label. Do NOT forget to arrange the test tube according to the chart. 6.1 In total you should have 3 tubes of each of the following samples: Water : negative controls Glucose solution (Monosaccharide): positive controls for Benedicts solution Starch solution (Polysaccharide): positive controls for Iodine solution Gelatin solution (Protein): positive controls for Biuret solution Diluted milk 3-4 pieces of diced onion + water 3-4 pieces of diced potato + water Unknown1 Unknown2 Unknown3 3
7) In ROW1: Add 5 drops of Biuret solution into each of the sample. 7.1 Gently swirl the test tube around so that the solution is evenly spread out in the test tube. 7.2 Observe and take note any the color change in the result section. 8) In ROW2: Add 5 drops of Iodine solution into each of the sample. 8.1 Gently swirl the test tube around so that the solution is evenly spread out in the test tube. 8.2 Observe and take note any the color change in the result section. . 9) In ROW3: Add 5 drops of Benedicts solution into each of the sample. 9.1 Gently swirl the test tube around so that the solution is evenly spread out in the test tube. 9.2 Use the beaker tong to transfer the test tube ONE AT A TIME into the boiling water in the beaker for around 3-5 minutes. 9.3 Observe and take note any the color change in the result section. . 10) Turn of the hot plate and stirrer. Be careful not to touch the hot plate as it will remain hot for a long time. . 11) Once you have finished the experiment, please help remove the marking tape on the test tubes. . 12) Continue to the next part of the experiment. For Oils (Lipids)
1) Collect two test tubes labeled A&B from your instructor. 2) Unscrew the cap of the tube 3) Add approximately the same amount of absolute ethanol into each tube. **CAUTION: Absolute ethanol in highly flammable, DO NOT POUR absolute ethanol NEAR THE HOT PLATE. Failure to follow this rule will result in 10% deduction from your lab report score** 4) Screw the cap back on tightly. 5) Shake each tube vigorously. Observe the change and record the findings in the results section. 6) Call your instructor to show you the brown-paper test and record the findings in the results section.
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Results:
Conclusion: From the experiment, all the positive and negative controls display the correct colors (there is no change for water, all the other controls display their right colors) From the hypothesis that Benedicts solution will react with sugar (monosaccharide and some disaccharide), Iodine will react with starch (polysaccharide), Biuret will react with protein, and lipids will leave to oily spot on the paper, the results are following the hypothesis. The Benedicts solution will change color from blue to green, yellow, orange, or red when it reacts with sugar molecules. However the Benedicts solution will not react with starch because starch is polysaccharide. The Iodines solution will change color from Yellow-brown to blue-black if the solution contains starch. Biurets solution will change color from blue to purple, because it can react with protein. However the Biurets solution is not to use for testing protein directly, so we have to water bath them to see the results. Solution containing high protein will change color to orange. The last thing is testing lipids. We drop the water and oil in a different sheet of papers and left it for a while. The water-drop evaporates from the paper, so some oil spot will be left on the paper. The results from the experiment show what organic compound is present in each sample, as listed bellow: Water: nothing (no change) Glucose Solution: Benedict = Orange - High Glucose Starch Solution: Iodine = Black - Starch Gelatin Solution: Biuret = Purple Proteins or peptide Diluted milk: Benedict = Orange-Yellow Moderate Glucose Biuret = Purple Proteins or peptide Onion + water: Benedict = Orange-Yellow Moderate Glucose Potato + water: Benedict = Milky Yellow Low Glucose Unknown 1: Biuret = Purple Proteins or peptide Unknown 2: Benedict = Milky Orange - High Glucose Unknown 3: nothing (no change) 5 Some errors might be that some weird color that has turned out in the experiment (as seen on the results table), even though we knew that it should not change color (or it might contains some substances that may react to the reagents). To improve this we should be more careful of our experiment and make sure to not mix the substances together. Question: 1.Does potato store glucose as starch? How do you know from your result? Yes, because the color of the potato with iodine is black, which means that potato have starch.
2.Does Onion store glucose as starch? How do you know from your result? No, because onion changes to yellow, this means that onion store glucose in another way that is not starch.
3.What sugar is most likely present in the diluted milk? Monosaccharide, disaccharide, protein (or peptides)
4.Even though, gelatin is made of protein, it changed color to purple after heating with Benedict's solution? Gelatin might have polysaccharide so it shows a different color that's not a result of monosaccharide and disaccharide.
5.A test tube contains starch, a digestive enzyme for starch, and water. The Biuret test is negative showing no change. However, after 30 minutes, the Benedict's test is positive. What substance is present? How do you know? The substance in the test tube might be a digestive enzyme for starch because it is the only substance that has sugar but not protein. Also, water doesn't have sugar so it cannot react with Benedict, and starch will not react to Benedict too.
6.How would you test an unknown solution for each of the followings: A. Sugar-use Benedict's test B. Fat-use absolute ethanol C. Starch-use Iodine D. Protein-use Biuret test