Prelab for Exercise 4-1 Chemical Tests for Biomolecules. Name. 1. Three parts of this lab use chemical reagents. What are chemical reagents?

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1 77 Prelab for Name 1. Three parts of this lab use chemical reagents. What are chemical reagents? 2. Each part of this lab uses a control. What is a control and why is it important? 3. In this lab a Benedicts test is used to identify reducing sugars. a. What is a reducing sugar? b. What color change occurs in Benedict s solution when heated in the presence of reducing sugars? 4. What color change occurs in Lugol s iodine reagent in the presence of starch or glycogen? 5. Describe the test you will be using to identify the presence of triglycerides (a type of lipid). 6 a. Proteins are polymers composed of subunits called. b. Bonds that join amino acids together are called. c. What type of reagent can be used to identify proteins by detecting these bonds? d. What color indicates proteins are present when the Biuret reagent (a light blue solution) is mixed with protein?

2 78 7. What is the difference between a positive and negative control?

3 79

4 80 Exercise 4-1 All living organisms are composed of 4 major groups of organic compounds: carbohydrates, proteins, lipids and nucleic acids - along with water and various electrolytes (or salts). These chemicals are the building blocks of life. In this lab you are going to perform some exercises that can be used to identify some specific types of chemicals commonly found in foods. Since each of the major groups of compounds has unique chemical and physical properties, they can be identified them by a chemical reaction that is distinctive for these properties. While performing these exercises you will also be learning the basic concepts involved in the experimental method. One of the more important parts of this is the use of a control. The control is an extra experiment or set of experiments against which all other experimental samples are compared in order to determine exactly what is causing a change. You will use a control in the following exercises. Be aware of what the control is and what it tells you. Identification of Carbohydrates Carbohydrates consist of simple sugars (e.g. glucose, fructose and sucrose) and complex carbohydrates (large molecules called polymers made of hundreds of simple sugars). There are a large number of carbohydrates in living organisms, varying from small sugar molecules such as the simple sugar glucose, which provides all cells with the fuel needed to do cell work, to polymers such as cellulose (structural molecules of plants) and glycogen (a storage carbohydrate in animals). Because there are so many different types of carbohydrates, one chemical test cannot identify all of them. Different chemical reagents, or testing agents, are used to test for simple sugars and carbohydrate polymers. Activity 1 Identification of Reducing Sugars Materials Required: Benedict's solution 10-ml Graduated cylinder Test tube rack Test tubes China marker (grease pencil) Hot plate Beaker Test tube clamp Small piece of apple Small piece of banana Small piece of onion Dropper bottles of: 5% Glucose solution 5% Fructose solution 5% Sucrose solution Salad oil Milk Distilled water

5 81 A reducing sugar is a type of sugar that can cause a specific chemical reaction, called a reduction. A reduction is a very common type of chemical reaction where a substance "gains an electron". Reducing sugars can cause certain types of molecules to get reduced (gain an electron). Not all sugars have the chemical structure to be reducing sugars. A reduction is always coupled to a chemical reaction called an oxidation, where a substance loses an electron. Reducing sugars lose electrons when they cause a reduction of some other chemical. Oxidation-reduction, or Redox, reactions are very important in the energy transfer chemical reactions of living organisms. A Benedict s test can be used to identify reducing sugars. The Benedict s solution (a chemical reagent) contains a blue soluble form of copper ions (Cu++) that can undergo a reduction (that is the copper ions gain electrons) when heated in the presence of a reducing sugar. When the blue copper ions are reduced, they change from the soluble blue color to reddish color copper ions (Cu + ) that are insoluble. The color of the test solution changes from blue ---> green ---> orange ---> red-brown or rust color as more reduced copper ions are formed. You will use the Benedict's solution to test for the presence of reducing sugar in various substances. Procedure: 1. Set up a boiling water bath in a beaker on the hot plate. When the water boils reduce the heat setting to keep the water at a simmer. Leave the stirrer knob turned off. 2. Number your test tubes from 1 to 9. Label the tubes at the top so the numbers won't wash off in the boiling water bath. 3. Follow the directions in the chart below for filling your test tubes. To Tube Number Add 1 20 drops of 5% glucose 2 20 drops of 5% fructose 3 20 drops of salad oil 4 20 drops of 5% sucrose 5 20 drops of milk 6 small piece of banana 7 small piece of onion 8 small piece of apple 9 20 drops of distilled water 4. Add 3 ml of Benedict's solution to each of the 9 test tubes.

6 82 5. Agitate the test tubes gently. Do not shake the test tubes by placing your thumb over the top of the tube. Your instructor will demonstrate the acceptable method for mixing test tube contents. 6. Put the test tubes in the boiling water bath for 5 minutes. 7. Record your results and conclusions in Table #1 on the data sheet at the end of this lab.

7 83 Activity 2 Identification Of Starch Materials Required Test tube rack China marker (grease pencil) Small amounts of potato flakes (a pinch) Test tubes Dropper bottles of: Lugol s iodine reagent (I 2 /KI) Starch suspension Glycogen solution 5% Glucose solution Distilled water Salad oil Albumin suspension The use of Lugol s iodine reagent (I 2 /KI) is useful to detect the presence of starch or glycogen. Starch and glycogen are polymers composed of many glucose molecules. Starch is the most abundant storage molecule of plants. Humans store glycogen (another carbohydrate) for short-term use, but our long-term fuel storage molecule as triglyceride, a type of lipid. Lugol s reagent is normally brown, and changes from its brownish color to a purple or blue-black color in the presence of starch or glycogen. Procedure: 1. Number your test tubes from 1 to Follow the directions in the chart below for filling your test tubes. To Tube Number Add 1 Few potato flakes and 40 drops H 2 O 2 40 drops of starch suspension 3 40 drops of glycogen solution 4 40 drops of glucose solution 5 40 drops of distilled water 6 40 drops of salad oil 7 40 drops of albumin suspension 3. Add 2-3 drops of iodine to each of the 7 test tubes. 4. Agitate each tube gently. 5. Record your results and conclusions on Table #2 on the data sheet at the end of this lab. Activity 3 Identification Of Lipids Materials Required:

8 84 Small piece of French fry or a similar greasy food Small pieces of brown wrapping paper Dropper bottles of: Salad oil Distilled water Starch suspension There are many different types of lipids. Among these are triglycerides (fats and oils), the major fuel storage molecules of most animals. Many lipid tests involve the use of fat-soluble dyes, such as Sudan dyes, which will be absorbed selectively by lipid molecules, and are visible when viewed through a microscope. For those who lack experience with detecting Sudan-stained oil globules, there is a much simpler lipid test, one which each of us has done many times while consuming our favorite junk food treats. In this exercise this test will be done as an experiment, complete with control. Unglazed paper such as a grocery bag or a napkin is normally opaque (light scattering). When lipids are placed on unglazed paper they will leave a permanent translucent spot through which light can pass. Other substances may temporarily coat the paper, but only lipids will leave the paper permanently translucent. Procedure: 1. Mark your pieces of paper from 1 to Follow the directions on the chart below for adding substances to your papers. To Paper Number Add 1 1 drop oil 2 1 drop starch suspension 3 1 drop distilled water 4 smear French fried potato on surface 3. Allow the papers to dry and record your results in table #3. Activity 4 Identification of Protein Materials Required: China marker (grease pencil) Test tubes potato flakes soy flour Dropper bottles of: 0.5% CuSO 4 10% NaOH Starch suspension Distilled water Albumin (egg white) suspension Milk

9 85 Proteins are an important group of large molecules found in all living cells. Special proteins called enzymes mediate the chemical reactions necessary to maintain life. Transport proteins in the cell membrane control the passage of various materials into and out of the cell. Proteins are polymers composed of amino acid subunits. The bonds that join amino acids together in a protein are called peptide bonds. Biuret reagent detects peptide bonds and can be used to identify proteins. Chemicals of the Biuret reagent are NaOH and CuSO 4 Biuret reagent is light blue, but in the presence of proteins it turns violet. Other types of molecules may cause other color changes, but only the violet color indicates proteins. Please note that sodium hydroxide (NaOH), a very caustic substance. Be very careful not to spill any sodium hydroxide. If you do get some on your hands, rinse them under cold water until they no longer feel soapy. Procedure: 1. Label your test tubes from 1 to 6 and use directions in the chart below for filling your test tubes. To Tube Number Add 1 60 drops of milk 2 60 drops of starch suspension 3 60 drops of albumin suspension 4 small pinch of potato flakes AND 60 drops of water 5 small pinch of soy flour AND 60 drops of water 6 60 drops of water 2. To each tube CAUTIOUSLY add 40 drops of 10% NaOH solution. If any NaOH is spilled, immediately flush the spilled area with cold water. NaOH can burn severely!!! 3. Now add 8 drops of 0.5% CuSO 4 (copper sulfate) to each of the 6 test tubes. 4. Agitate the tubes gently, then let the test tubes sit at room temperature for a few minutes. 5. Record your results in table #4. Be sure that you understand your results and conclusions before you leave the lab. Ask your instructor if you are unsure of any of your results.

10 86 Activity 5 Identification of an Unknown Now that you have some experience with some chemical tests to identify types of biological molecules you will be asked to identify an unknown. In the first three activities we used a negative control, a substance that will not react with the reagent being used. You must now use a positive control, a substance that definitely will react with the test reagent. Begin by designing a step-by-step procedure to use to identify your unknown. With each test, include both a positive and negative control. This will allow you to more easily decide if your unknown has given a positive or negative result. Write out the entire procedure you plan to use. Include data tables in which to take your data. Get your procedure checked before you continue with this experiment. Materials Required: Selection of Unknown solutions (some may contain more than one type of molecule) Test tubes Dropper bottles of: 0.5% CuSO 4 10% NaOH Lugol s iodine reagent (I 2 /KI) Distilled water Benedict's solution Data from Identification of an unknown. On a separate page: 1. Write out the procedure you will use to identify your unknown. Identify both the positive and negative controls in each test you make and data tables. Have your procedure checked by your instructor before you continue. 2. Perform the experiments and record your data 3. Write out your conclusion about the identity of the unknown with an explanation for making this identification. When you have completed all of the exercises clean up your laboratory supplies and return them to the areas designated by your instructor. Test tubes must be rinsed out, but you do not need to dry them. Do not leave bits and pieces of experimental materials in the sinks.

11 87 Data and Conclusions Name Table #1 - Data from Identification of Reducing Sugars Substance Reaction Conclusion 1. glucose 2. fructose 3. salad oil 4. sucrose 5. milk 6. banana 7. onion 8. apple 9. water Table #2 - Data from Identification of Identification of Starch Substance Reaction Conclusion 1. Potato flakes 2. Starch 3. Glycogen 4. Glucose 5. Water 6. Salad oil 7. Albumin Table #2 - Data from Identification of Identification of Lipids Substance Results Conclusion 1. Oil 2. Starch 3. Water 4. French Fried Potato Table #4 - Data from Identification of Identification of proteins Substance Results Conclusion 1. Milk 2. Starch 3. Albumin 4. Potato Flakes 5. Soy 6. Water

12 88 Analysis and Interpretations 1. In Activities 1-4, what was the control? 2. Name the reagent or testing material used to detect the presence of the following: a. reducing sugars: b. protein: c. fats: d. starch: 3. In each of the chemical tests you performed, how did you know when a positivereaction took place? 4. Which test would you use to determine if a food has starch present? 5. Which test would you use to determine if your hamburger contains protein?

13 89 6. Sometimes fructose is used as the sweetener in reduced calorie foods because fructose is much sweeter than glucose or sucrose. How would you determine that your food does not contain fructose?