Figure 2. Figure 1. Name: Bio AP Lab Organic Molecules

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1 Name: Bio AP Lab Organic Molecules BACKGROUND: A cell is a living chemistry laboratory in which most functions take the form of interactions between organic molecules. Most organic molecules found in living systems can be classified as carbohydrates, fats, proteins, or nucleotides. Each of these classes of molecules has specific properties that can be identified by simple chemical tests or assays. In this lab you will learn to identify three of the four major types of organic molecules: carbohydrates, fats and proteins. You will then test for the presence of these important compounds in food. Carbohydrate: The basic structural unit of carbohydrates is the monosaccharides (or simple sugar). One way to classify monosaccharides is by the presence of a terminal aldehyde group or an internal ketone group (figure 1). Both of these groups contain a carbonyl group, or a double bonded oxygen atom. The double bonded oxygen atom bonds with the Benedict s reagent forming a colored precipitate. When two monosaccharides are joined together through a condensation reaction, they form a disaccharide. If either the aldo or keto group are involved in the glycosidic link (the bond between the monosaccharides units), the disaccharide is no longer able to react with Benedict s reagent, as in sucrose. If however the aldo or keto group is not involved in the formation of the glycosidic link it remains free to react with the Benedict s reagent, as in maltose (figure 2). Sugars with free carbonyl groups (aldo or keto) whether monosaccharides or disaccharide are called reducing sugars. Figure 1 Figure 2 When a solution containing Benedict s reagent and a reducing sugar is heated, the copper (II) ions in the Benedict s reagent are reduced to copper (I) ions and the solution changes from blue to green to orange to red-orange to brick-red. A brick-red precipitate (solid), copper (II) oxide (Cu 2 O) may appear in the bottom of the tube. The more reducing sugar present in the mixture, the more precipitate will form in the bottom of the tube. The Benedict s reagent will therefore test for the presence of reducing sugars. Starch is a polysaccharide composed of many monosaccharides units bonded together. When glucose chains are sufficiently long they coil up like springs. This coil is supported by weak links between the glucose molecules. When the chains are longer than about 9 glucose molecules a triiodide ion (I 3 - ) fits inside the coil. Once the iodine is inside the coil, the resulting molecule absorbs light, which causes the color reaction between iodine and starch. The longer the glucose chains the more iodine molecules fit into the coils and the more intense the color. The iodine will change from amber to blue/black. Remember the links break down at high temperatures and the glucose chains uncoil, which is why the iodine test does not work with heated samples. 1

2 Lipids: Lipids are molecules that are soluble in non-polar solvents, but are insoluble in polar solvents such as water. Although lipids include fats, steroids, and phospholipids, this lab will focus on fats or triglycerides, the most common form of fat. They consist of three fatty acids attached to a glycerol molecule (figure 3). They are found predominantly in adipose tissue and store more energy per gram than does any other molecule. At room temperature some lipids are solid (generally those found in animals) and are referred to as fats, while others are liquid (generally those found in plants) and are referred to as oils. Since both solid and liquids fats are non-polar we will test for their presence by using Sudan IV, a non-polar dye that dissolves in nonpolar substances, but not in polar substances. Sudan IV dye stains lipids simply because the dye is soluble in the lipid. The dye imparts a pinkish to reddish color to the lipid. Sudan IV is not soluble in water. If fats or oils are present, these will appear as floating red droplets or as a floating red layer. Figure 3 Fats can also be detected by a simple paper or grease-spot test. Placing a drop of a fat on a piece of unglazed paper, such as brown wrapping paper, an oil based substance will make the paper look translucent when dried. Protein: Proteins are made up of one or more polypeptides, which are linear polymers of smaller molecules called amino acids. Polypeptides are formed when amino acids are joined together by peptide bonds between the amino group of one amino acid and the carboxyl group of a second amino acid (figure 4). The Biuret reagent reacts with peptide bonds so it reacts with proteins, but will not react with free amino acids because there are no peptide bonds present. Biuret reagent contains a solution of sodium or potassium hydroxide (NaOH or KOH) and a small amount of a copper sulfate solution. In this test for proteins there is a reaction between the copper ions and the amino groups in the peptide bonds. Biuret reagent is light blue, but in the presence of proteins (longchain polypeptides) it will turn violet and in the presence of short-chain polypeptides it will turn pink. Figure 4 LEARNING OBJECTIVES: To understand the chemical basis for each of the assays To identify food groups based upon the result of the assay test performed GENERAL SAFETY PRECAUTIONS: You must wear safety glasses or goggles and lab aprons. The reagents used in this lab contain copper and should be disposed of according to teacher direction. These reagents are stains and precautions must be taken to not get them on your skin or clothing. Remember when heating glassware to use appropriate equipment so as not to burn your skin. Do not work in the laboratory without your teacher s supervision. Talk to your teacher if you have any questions or concerns about the experiments. THE INVESTIGATIONS: This investigation consists of two major parts. In Part I you will learn how to test for the presence of reducing sugar, starch, lipid and protein by using control samples of water, starch, glucose, maltose, sucrose and egg albumin (protein). 2

3 INTRODUCTION: For EACH of the following assays, explain the chemical functioning of the specific indicator. Include in your discussion the chemical basis for the reaction, what the indicator is testing for, and what indicates a positive/negative test result. 1. Benedict s test 2. Lugol s test 3. Sudan IV test 4. Biuret test Part I A. Carbohydrates Benedict s Test For Reducing Sugars If Benedict s reagent is heated with a reducing sugar, the color of the reagent changes from blue to green to yellow to reddish-orange, depending on the amount of reducing sugar present. Orange and red indicate the highest proportion of these sugars. Set up a row of 6 test tubes. Use a wax pencil to number them in a series from 1 to 6. To each test tube add 2 ml of the solution whose number in Data Table 1 corresponds to the number on the tube. Add one dropper full (approximately 2 ml) of Benedict s reagent to each test tube. Mix the reagent and the solution by agitating the solution in each tube from side to side. Record the original color of each solution in data table 1.Heat the test tubes in a boiling water bath for three minutes. Record the final color of each solution. Determine which test tubes tested positive for the presence of a reducing sugar. NOTE: Benedict s test will show a positive reaction for starch ONLY if the starch has been broken down into maltose or glucose units by excessive heating. DATA TABLE 1 Benedict s Test 6 Egg albumin 1) Why did you test water with Benedict s reagent? 2) Did maltose react with Benedict s reagent? Explain why or why not. 3) Did sucrose react with Benedict s reagent? Explain why or why not. 4) Explain the limitations of the Benedict s test in determining whether or not sugar is present in certain food products. 5) Why do all monosaccharides, but only some disaccharides react with Benedict s reagent? Lugol s Test For Starch Lugol s reagent will change from a brownish or yellowish color to a blue-black color when starch is present. solution whose number in Data Table 2 corresponds to the number on the tube. Add several drops of the Lugol s reagent to each tube, mix and IMMEDIATELY record the final color of the solution. DO NOT HEAT the test tubes in the Lugol s test. Determine which test tubes tested positive for the presence of starch. 3

4 DATA TABLE 2 Lugol s Test Substance Original 1) A student wanted to determine how a potato and an onion store their carbohydrates. He tested each substance with Benedicts solution and Lugol s iodine solution and recorded his results in the data table below: Substance Benedicts Test Lugol s Iodine test B. Lipids Potato Blue Blue/black Onion Red/Orange Amber a) From the results of the Benedict s and Lugol s tests, would you conclude that a potato stores its carbohydrates as sugars or as starch? Defend your answer. b) How does an onion store its carbohydrate? Defend your answer. Grease Spot Test For Fats The familiar grease spot is the basis of a very simple test for fats. On a piece of unglazed paper, such as brown wrapping paper, place one drop of oil and one drop of water. Allow the drops to dry. Describe the difference between the oil spot and the water spot after a period of drying. Sudan IV Test For Fats Sudan is soluble in fats but not in water. If fats or oils are present, these will appear as floating red droplets or as a floating red layer. solution whose number in data table 3 corresponds to the number on the tube. To each test tube add 2 ml of Sudan IV. Gently shake each test tube and record your observation in the data table. Determine which test tubes tested positive for the presence of fat. DATA TABLE 3 Sudan IV Test 1) The leaves of many plants are coated with a waxy substance that causes them to shed water. How would you expect this substance to react in the Sudan IV test? Explain. 4

5 C. Proteins Biuret Test For Proteins The blue reagent is a light blue color, but turns violet in the presence of proteins, and changes to pink when combined with short-chain polypeptides. solution whose number in Data Table 4 corresponds to the number on the tube. Add one dropper full (approximately 2 ml) of Biuret reagent to each test tube. After an incubation period of 2 minutes, record your results. Determine which test tubes tested positive for the presence of protein or polypeptides. DATA TABLE 4- Biuret Test 1) What does this test tell you about the biochemical composition of starch or glucose? 2) Why has water been included as one of the test substances? 3) Ninhydrin is a strong oxidizing agent that removes the amino groups (NH 2 ) of amino acids. The reaction liberates ammonia, carbon dioxide, the corresponding aldehyde, and a reduced form of Ninhydrin. The ammonia then reacts with the reduced Ninhydrin to form a purple precipitate. The Ninhydrin reagent reacts with the amino group of free amino acids, but does not react with polypeptides. This Ninhydrin will turn purple (violet) in the presence of the free amino groups of amino acids. In the case of the amino acid proline it turns yellow because the amino group in this amino acid is part of a ring structure. A protein was mixed with a certain peptidase that breaks specific peptide bonds in the protein. The fragments were separated and each fragment was tested with Ninhydrin. The results obtained are shown below: What conclusion can be made concerning the composition of each fragment? Fragment Biuret test Ninhydrin test Conclusion A Violet Clear B Blue Purple C Blue Yellow D Violet Clear 5