Chemical Tests for Biomolecules: Key Concepts and Techniques

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Name Lab Partner Date U N I T 2 P R E L A B ■ ■ EXER CISE 2- 2 CHEMICAL TESTS FOR BIOMOLECULES 1. Each part of this lab uses a control. What is a control and why is it important? A control is an element in the experiment that remains unaffected by other variables. a control helps in minimizing effects of other factors other than those being tested 2. Why is it important to include the positive and negative control when performing a test for biomolecules? Positive controls are important as they scientists in understanding that the experiment was carried out well. Negative controls always test negative for biomolecules 3. Differentiate between monomers and polymers. Monomers are building blocks of polymers while polymers consist of repeating units of monomers. Monomers are single units which collectively form a polymer while polymers are repetitive units of monomers The molecular weight of a polymer is higher than that of a monomer 1

4. List the monomers for the following: a. Carbohydrates- monosaccharides b. Proteins- amino acids c. Lipids- Glycerol and fatty acids 2

5. In this lab a Benedict’s test is used to identify reducing sugars. a. What is a reducing sugar? A reducing sugar is any sugar that is capable of acting as a reducing agent b. What color change occurs in Benedict’s solution when heated in the presence of reducing sugars? blue to green to orange to red-brown 6. What color change occurs in Lugol’s iodine reagent in the presence of starch or glycogen? Deep blue color is observed 7. Describe the test you will be using to identify the presence of triglycerides (a type of lipid). Use Sudan dyes, which will be absorbed selectively by lipid molecules, and are visible when viewed through a microscope. 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. 8. a. Proteins are polymers composed of subunits called amino acids . b. Bonds that join amino acids together are called peptide bonds . c. What type of reagent can be used to identify proteins by detecting these bonds? Biuret Test d. What color indicates proteins are present when the Biuret reagent (a light blue solution) is mixed with protein? Turns from light blue to violet UNIT 2

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■ ■ E X E R C I S E 2- 2 CHEMICAL TESTS FOR BIOMOLECULES INTRODUCTION 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. PURPOSE The purpose of this lab is to perform some activities 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 by a chemical reaction that is distinctive for these properties. While performing these activities you will also be learning the basic concepts involved in the experi- mental method. One of the more important parts of this is the use of a control. In scientific testing, a control in the experiment allows the experimenter to keep a variable or variables constant so that the impact of another factor can be better understood. The control shows the experimenter what would have happened if there was no experimental condition from the variables. You will use positive and negative controls in the following activities. The positive control is an experiment that the experimenter knows will give a positive result, and the negative control removes the variable to give a negative result. If the controls do not give a 73

positive or negative result, then something has gone wrong in the experiment. For example, in Activity 1 (Identification of Reducing Sugars), the 20 drops of distilled water is the negative control because we expect that the result will be negative from this experiment. If this negative control yields a positive result, then something has gone wrong with the experiment. The positive control in this lab would be a sample such as glucose that is a carbohydrate and a simple sugar. This positive control should give you a positive result when added to Benedict’s solution. Be aware of what the control is and what it tells you. For each activity, identify the negative and positive controls and apply this information to Activity 5 (Identification of an Unknown). Your instructor may decide to split the class into two groups since you will only have time to do two of the four activities. Then, your group can share the data with other groups. 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. 74

■ ■ A ctivity 1: I den tific ation o f R educing S ugar s Materials • Benedict’s solution • Small piece of apple • 10 mL Graduated cylinder • Small piece of banana • Test tube rack • Small piece of onion • Test tubes • Dropper bottles of: • China marker (grease pencil) – 5% Glucose solution • Hot plate – 5% Fructose solution • Beaker – 5% Sucrose solution • Test tube clamp – Salad oil • Unknown sample – Milk – Distilled water 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.

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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-colored copper ions (Cu+) that are insoluble. The color of the test solution changes from blue  green  orange  red-brown or rust 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 10. 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. TUBE NUMBER SAMPLES 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 10 unknown sample 4. Add 3 mL of Benedict’s solution to each of the 10 test tubes. 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 2-1 on the Student Worksheet at the end of this lab. ■ ■ A ctivity 2: I den tific ation o f S tar ch Materials • Test tube rack • Dropper bottles of:

• China marker (grease pencil) – Lugol’s iodine reagent (I2/KI) • Small amounts of potato flakes (a pinch) – Starch suspension • Test tubes – Glycogen solution • Unknown sample – 5% Glucose solution – Distilled water – Salad oil – Albumin suspension The use of Lugol’s iodine reagent (I2/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 stor- age molecule of plants. Humans store glycogen (another carbohydrate) for short-term use, but our long-term fuel storage molecule is 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.

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PROCEDURE 1. Number your test tubes from 1 to 8. 2. Follow the directions in the chart below for filling your test tubes. TUBE NUMBER SAMPLES 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 8 unknown sample 3. Add 2–3 drops of iodine to each of the 8 test tubes. 4. Agitate each tube gently. 5. Record your results and conclusions on Table 2-2 on the Student Worksheet at the end of this lab. ■ ■ A ctivity 3: I den tific ation o f Lipids Materials • Small piece of French fry or a similar greasy food • Dropper bottles of: • Small pieces of brown wrapping paper – Salad oil

• Unknown sample – 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 exper ence with detecting Sudan-stained oil globules, there is a much simpler lipid test, one that each of us has done many times while consuming our favorite junk food treats. In this activity 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 5. 2. Follow the directions on the chart below for adding substances to your papers. PAPER NUMBER SAMPLES 1 1 drop oil 2 1 drop starch suspension 3 1 drop distilled water 4 smear French fried potato on surface 5 unknown sample 4. Allow the papers to dry and record your results in Table 2-3. ■ ■ A ctivity 4: I den tific ation o f P r o t ein Materials • China marker (grease pencil) • Dropper bottles of: • Test tubes – 0.5% CuSO 4 • Potato flakes – 10% NaOH • Soy flour – Starch suspension • Unknown sample – Distilled water – Albumin (egg white) suspension – Milk

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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 mem- brane 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) is 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 7 and use the chart below for filling your test tubes. TUBE NUMBER SAMPLES 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 7 unknown sample 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 7 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 2-4. 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. ■ ■ A ctivity 5: I den tific ation o f an Unkno wn

Now that you have some experience with using chemical tests to identify the different types of biologi- cal molecules, you will be asked to analyze the data from your unknown sample. For Activities 1-4, identify the negative control and select a positive control from your experimental results. Remember a negative control gives a negative result and a positive control gives a positive result that reacts with the test reagent. Record your data in the table in the Student Worksheet. 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. Place them in the used test tube bin to be put into the dishwasher. Make sure you do not leave bits and pieces of experimental materials in the sinks.

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Name Lab Partner Date S T U D E N T W O R K S H E E T ■ ■ E X E R C I S E 2- 2 CHEMICAL TESTS FOR BIOMOLECULES Data and Conclusions Table 2-1. Data from Identification of Reducing Sugars SUBSTANCE RESULTS (POSITIVE OR NEGATIVE) CONCLUSION (COLOR, TEXTURE, OTHER DETAILS) 1 Glucose positive Red-brown 2 Fructose positive Red-brown 79

3 Salad oil positive Red-brown 4 Sucrose negative No color change 5 Milk positive Red-brown 6 Banana positive Red-brown 7 Onion positive Red-brown 8 Apple negative No color change 9 Water negative No color change 10 Unknown positive Red-brown Table 2-2. Data from Identification of Starches SUBSTANCE RESULTS (POSITIVE OR NEGATIVE) CONCLUSION (COLOR, TEXTURE, OTHER DETAILS) 1 Potato flakes positive Purple 2 Starch positive Purple 3 Glycogen negative No color change 4 Glucose negative No color change 5 Water negative No color change 6 Salad oil negative No color change 7 Albumin negative No color change 8 Unknown positive Purple 80

Table 2-3. Data from Identification of Lipids SUBSTANCE RESULTS (POSITIVE OR NEGATIVE) CONCLUSION (COLOR, TEXTURE, OTHER DETAILS) 1. Oil positive Translucent spot 2 Starch positive Translucent spot 3 Water negative No spot 4. French fried potato positive Translucent spot 5. Unknown positive Translucent spot Table 2-4. Data from Identification of Proteins SUBSTANCE RESULTS (POSITIVE OR NEGATIVE) CONCLUSION (COLOR, TEXTURE, OTHER DETAILS) 1. Milk positive Violet 2. Starch negative No color change 3. Albumin positive Violet 4. Potato flakes positive Violet 5. Soy positive Violet 6. Water negative No color change 7. Unknown negative No color change Results from the Unknown ACTIVITY 1: IDENTIFICATION OF REDUCING SUGARS Positive control sample: glucose Test result (positive or negative): positive

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Negative control sample: distilled water Test result (positive or negative): negative Unknown sample test result (positive or negative): positive ACTIVITY 2: IDENTIFICATION OF STARCH Positive control sample: starch Test result (positive or negative): positive Negative control sample: distilled water Test result (positive or negative): negative Unknown sample test result (positive or negative): positive EXERCISE 2-2 CHEMICAL TESTS FOR

UNIT 2 ACTIVITY 3: IDENTIFICATION OF LIPIDS Positive control sample: oil Test result (positive or negative): positive Negative control sample: distilled water Test result (positive or negative): negative Unknown sample test result (positive or negative): negative ACTIVITY 4: IDENTIFICATION OF PROTEIN Positive control sample: albumin Test result (positive or negative): positive Negative control sample: water Test result (positive or negative): negative Unknown sample test result (positive or negative): negative What do you think is the identity of your unknown? Give an explanation for how you came to this conclusion. The identity of the unknown is water as it has tested negative of all elements tested. Analysis and Interpretations 1. In Activities 1–4, what were positive controls? What was the negative control? 81

Positive controls are all the items that contained the elements that were being tested. Negative controls lacked the elements tested 2. Name the reagent or testing material used to detect the presence of the following: a. reducing sugars: Benedicts Reagent b. proteins: Biuret solution c. fats: Ethanol d. starches: Lugol’s Iodine 82

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3. In each of the chemical tests you performed, how did you know when a positive reaction took place? Every positive reaction had a color change 4. Which test would you use to determine if a food has starch present? Use the Lugol’s iodine reagent 5. Which test would you use to determine if your hamburger contains protein? use the Biuret reagent to test for a color change

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