Chemical Tests For Biologically Important Molecules Do not write on this document
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1 Chemical Tests For Biologically Important Molecules Do not write on this document Introduction The most common and important organic molecules found in living things fall into four classes: carbohydrates, lipids, proteins, and nucleic acids. Each class of molecules have different characteristics based on the combination of nitrogen, carbon, oxygen, and hydrogen used, as well as the actual structure and shape of the molecule. Each of these classes of organic molecules has a chemical test to indicate whether or not it is present in a solution. This is useful in identifying the types of molecules that make up a living organism or a product of a living organism. Purpose The purpose of this lab is to familiarize the student with a series of biochemical tests used to identify presence of specific types of organic molecules. Procedure 1: Benedict s Test for Reducing Sugars (Monosaccharides) Carbohydrates are made of monosaccharides, or simple sugars. There are three types of monosaccharides you will work with, each a major source of energy for organisms at the cellular level: glucose, fructose, and galactose. Benedict s test gives a color change of orange to brick red when reducing sugars (or monosaccharides) are present. Most complex sugars, such as disaccharides (made from two linked monosaccharides) and polysaccharides (made from three or more linked sugars) react poorly with Benedict s solution. Materials: Seven test tubes Test tube rack Hot plate 400mL beaker Test tube holder Benedict s solution Onion juice Potato juice Sucrose solution Glucose solution Water Starch solution Honey 1
2 Procedure 1. Fill up a beaker about halfway with water and place it on the hot plate. Heat the water to a low boil. 2. Number each of the test tubes Add 5 ml of the following materials to be tested to each test tube: Test tube 1: Water Rinse the graduated Test tube 2: Glucose solution cylinder and test Test tube 3: Sucrose solution tubes after Test tube 4: Starch solution measuring each Test tube 5: Onion solution solution to prevent Test tube 6: Potato solution Test tube 7: Honey contamination! 4. Make a prediction of how each material will react. Write a plus sign (+) for a positive test and a negative sign ( ) for a negative test. Table 1: Predicted Results of Benedict s Test A negative control does not contain the variable you are testing for. Negative controls should always test as negative. A positive control contains the variable and should always test as positive. Which of the solutions would be a negative control for Benedict s? Which of the solutions would be a positive control for Benedict s? 5. Add a dropper full of Benedict s solution to each test tube. Place each of the test tubes into the gently boiling beaker of water. 6. Allow 3 minutes for the reaction to complete. Observe any color changes in the test tubes. 7. Record your results below: Table 2: Actual Results of Benedict s Test Test Result Observed Color Which tested most strongly positive: glucose, sucrose, or starch? Was the presence of monosaccharides detected in either onion or potato? Explain Procedure 2: Iodine Test for Starches Starch is a polysaccharide, made of hundreds of repeating glucose molecules linked together. It is the main molecule of carbohydrate energy storage used by plants. A small segment of a starch molecule The coiled shape that the starch molecule takes allows it to react with iodine, forming a bluish black color. 2
3 Materials: Seven test tubes Test tube rack Iodine (I 2KI) solution Onion juice Potato juice Sucrose solution Glucose solution Water Starch solution Honey Procedure 1. Number each of the test tubes Add 5 ml of the following materials individually to each test tube: Test tube 1: Distilled water Test tube 2: Glucose Test tube 3: Sucrose solution Test tube 4: Starch solution. Test tube 5: Onion solution Test tube 6: Potato solution Test tube 7: Honey 3. Make a prediction of how each material will react. Write a plus sign (+) for a positive test and a negative sign ( ) for a negative test. Table 3: Predicted Results of Iodine Test Which of the solutions would be a negative control for iodine? Which of the solutions would be a positive control for iodine? 4. Add a dropper full of iodine solution to each test tube. Observe and record the results. Table 4: Actual Results of Iodine Test Test Result Observed Color Which is a polysaccharide: starch, glucose, or sucrose? What has a higher amount of starch: potato, or onion? Based on the iodine and Benedict s tests, compare how potatoes and onions store energy differently. Procedure 3: Sudan Test for Lipids Lipids are biological molecules made from three fatty acids bonded to a molecule of glycerol. Unlike carbohydrates, lipids are nonpolar and do not dissolve well in polar solvents such as water. Lipids only dissolve in nonpolar solvents. In the spaces provided in Figure 1, identify the saturated fatty acid chain, the monounsaturated fatty acid chain, and the polyunsaturated fatty acid chain in the space provided. 3
4 Figure 1: Classic Lipid Molecule (Do not write on this) Glycerol Three Fatty Acids Due to this property of lipids, an effective test for them is to add a nonpolar dye. This dye can be easily absorbed by nonpolar substances such as lipids, while polar substances are unable to dissolve it. A positive test will show a red layer on the surface of the test tube after a gentle tap. Materials: Five test tubes Sudan IV solution Vegetable oil Test tube rack Water Honey Lipid solution Procedure 1. Number each of the test tubes Combine 3 ml of vegetable oil with 3 ml of water in the first test tube and tap it. Is salad oil soluble in water? Based on this, is salad oil polar or nonpolar, hydrophobic or hydrophilic? 3. Add 3 ml of water, and 5 ml of each of the following to test tubes 2-5: a. Water b. Honey c. Vegetable oil d. Lipid Solution 4. Tap the test tubes gently, observe and predict the results of the Sudan IV test to follow. Table 5: Predicted Results of Sudan IV Test Water Honey Vegetable Oil Lipid Solution Which of these solutions would be the negative control for Sudan IV? Which of these solutions would be the positive control for Sudan IV? 5. Add a dropper full of Sudan IV into each of the test tubes. 6. Mix the contents of the test tube by lightly tapping it against your hand to mix it. 7. Record the results. 8. Table 6: Actual Results of Sudan IV Test Water Honey Vegetable Oil Lipid Solution 4
5 How does the dye react differently with the oil versus the water? Describe. Based on this test, would you consider honey to be a food high in lipids? Lipids supply about 9 calories per gram, while carbohydrates supply about 4 calories per gram. Given your results, what would you expect to have more calories, a tablespoon of honey, or a tablespoon of vegetable oil? Explain. Procedure 4: Biuret Test for Proteins Proteins, also known as polypeptides, are large organic molecules made of amino acids. Each amino acid molecule contains a amino group (-NH2), a carboxyl group (-COOH), and another group that can vary and is referred to as (-R). The bonds between carbon and nitrogen (called peptide bonds) react with Biuret reagent to produce a violet color. Biuret only reacts positively with peptides, not with individual amino acids. Materials Seven test tubes Egg white Honey Potato solution Amino Acid solution Water Protein solution Onion solution Procedure 1. Number the test tubes Add 5mL of the following materials individually to each test tube: Test tube 1: Water Test tube 2: Amino Acid Solution Test tube 3: Egg white Test tube 4: Honey Test tube 5: Onion solution Test tube 6: Protein solution Test tube 7: Potato solution 3. Predict the results of the Biuret test. Table 7: Predicted Results of Biuret Test Amino Acid Water Egg White Honey Onion Solution Protein Potato 5
6 Which of these solutions would be a negative control for Biuret? Which of these solutions would be a positive control for Biuret? 4. Add a dropper full of Biuret reagent to each tube and mix gently. 5. Record the results of the Biuret test. Table 8: Actual Results of Biuret Test Result Water Amino Acid Solution Egg White Honey Onion Protein Potato Color Based on your results, what had a higher amount of protein, egg white or honey? In general, would you expect to find higher amounts of protein in plant-based foods or animal-based foods? Explain. Procedure 5: Testing Unknowns Your instructor will provide each of you with one unknown food from three groups A, B, C and D. The letter gives possible unknowns that you might have in each group: Determine the unknown food sample and record below. Table 9: Possible Food Group A Group B Group C Group D glucose corn starch glucose protein solution milk sugar honey salt solution honey enriched flour table sugar potato starch water soy flour egg white gelatin table sugar Table 10: Unknown Results (Use + or -) Possible Unknowns in My Unknown Groups Component A B C D reducing sugar starch fat protein The Unknown food is: Application: Using the food that has been brought from home, determine what kind of biological molecules it contains. Make two tables, one showing your predictions before you test and a second table showing results. Write a brief summary of your findings. *Note: you will need enough of one solid food substance to run four tests per person. (About four pea-size amounts of one food made into a slurry will do). 6
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