Lab 2
Learning Objectives Compare and contrast organic and inorganic molecules Relate hydrogen bonding to macromolecules found in living things Compare and contrast the four major organic macromolecules: lipids, carbohydrates, nucleic acids and proteins Introduction There are over 100 known elements in the periodic table. Elements are pure substances made of only one type of atom. Interestingly, the number of naturally occurring elements is unclear. More than 90% of all matter is composed of combinations of just four of the approximately 88 naturally occurring elements: oxygen, carbon, hydrogen, and nitrogen. Living organisms, in general, require about 20 elements. The periodic table continues to develop as new elements are synthesized in laboratories. Figure 1: The periodic table of elements categorizes all of the known elements. Groups are listed vertically as 1 7. Periods are listed horizontally as 1-18. 35
Organic Molecules Although the term organic is frequently used to refer to foods or clothing, in biology and chemistry, the terms organic and inorganic are used to categorize molecules. Organic molecules contain carbon and are typically identified by the presence of carbon-hydrogen bonds. Figure 2: Glucose, fructose, and galactose are all examples of carbohydrates. The human body is made up of roughly 27% organic molecules and 73% inorganic molecules. Carbohydrates (sugars) and proteins are examples of organic molecules. Ammonia (NH 3 ), table salt (NaCl) and water (H 2 O) are all examples of inorganic molecules. Macromolecules There are many classes of organic compounds, based on the functional groups they contain. In living organisms, the most important organic Figure 3: Water is an inorganic molecule. Interestingly, life on Earth would not be possible without it! compounds belong to a classification of molecules called macromolecules. The term macromolecule simply means a large molecule. Recall that molecules are substances that contain two or more atoms bonded together. The same four types of macromolecules are used by all living 36
organisms for cellular metabolism and reproduction. These common biological macromolecules are lipids, nucleic acids, carbohydrates and protein. The properties they convey are of great importance to cell function. Lipids, or fats, have many functions within living organisms including energy storage, membrane structure, and aids in the formation of internal cellular components. All genetic material is composed of nucleic acids. Nucleic acids comprise the basis of DNA. Another nucleic acid, RNA, functions in the production of proteins and other cellular processes. Carbohydrates are commonly referred to as sugars. This class of organic compounds serves as one of the primary sources of metabolic energy. The carbohydrate monosaccharide subunit most commonly used for energy is glucose. Other useful carbohydrates include maltose, lactose, sucrose, and starch. Proteins are the most abundant macromolecule in living systems. Like lipids, proteins perform a variety of functions. For example, proteins are major components of tendons, ligaments and muscles in the human body. Figure 4: Omega-3 Fatty acid. Figure 5: Approximately 70% of a human adult body is composed of water. Macromolecules are formed by the covalent bonding between subunits. Recall that covalent bonds can produce molecules that are either polar or non-polar. Lipids, for the most part, are non-polar. The 20 amino acids found in proteins may be either non-polar or polar. When macromolecules are created, non-polar regions tend to be located close together. Polar regions can form a type of chemical bond called a hydrogen bond. Hydrogen bonds result from the interaction of the positive region of one polar molecule and the negative region of another polar molecule. Although relatively weak bonds, these are extremely important in the maintenance of chemical structures. In fact, the double strands found in DNA, as well water molecules, are held together by hydrogen bonds. Living things require a constant supply of energy. Throughout this manual, you will learn about the reactions that take place inside of organisms. The sum of these reactions is called metabolism, and is a general term used to describe the energy required to keep those reactions occurring. 37
Pre-Lab Questions 1. Nitrogen fixation is a natural process by which inert or unreactive forms of nitrogen are transformed into usable nitrogen. Why is this process important to life? 2. Given what you have learned about the hydrogen bonding shared between nucleic acids in DNA, which pair is more stable under increasing heat: adenine and thymine, or cytosine and guanine? Explain why. 3. Which of the following is not an organic molecule; Methane (CH 4 ), Fructose(C 6 H 12 O 6 ), Rosane (C 20 H 36 ), or Ammonia (NH 3 )? How do you know? Experiment 1: Testing for Proteins The protein molecules in many foods provide the amino acid building blocks required by our own cells to produce new proteins. To determine whether a sample contains protein, a reagent called Biuret solution is used. Biuret solution contains copper ions, similar to Benedict s solution (another common reagent). However, the chemical state of the copper ions in Biuret solution causes them to form a chemical complex with the peptide bonds between amino acids (when present), changing the color of the solution. Biuret solution is normally blue, but changes to pink when short peptides are present and to violet when long polypeptides are present. 38 Figure 6: Biuret solution only is located on the far left side of the image (blue). Note the transition from blue to violet as proteins are added to the solution, causing the solution to transition from blue to violet.
Materials (2) 250 ml Beakers 25 Drops Biuret Solution, H 2 NC(O)NHC(O)NH (1) Knox Gelatin Packet 5 ml 1% Glucose Solution, C 6 H 12 O 6 (1) 10 ml Graduated Cylinder (1) 100 ml Graduated Cylinder Permanent Marker 5 Pipettes 5 Test Tubes (Glass) Test Tube Rack 5 ml Unknown Solution *Egg White *Hot Water *Tap Water *You Must Provide Procedure 1. Predicted results based on a previously learned set of information is sometimes called an a priori prediction. Before you begin, take a moment to construct a priori predictions stating whether or not there are proteins present in each of the following solutions: Albumin (Egg White), Knox Gelatin, Glucose, and Water. Record these predictions in Table 1. 2. Then, use your knowledge of Biuret solution chemistry (refer to the experimental introduction) to predict the color of each of the four solutions when mixed with Biuret solution. You must predict the initial color, as well as the final color (the color after). Record these predictions in Table 1. 3. You may now begin your experiment by using the permanent marker to label five test tubes 1, 2, 3, 4 and 5. 4. Prepare your testing samples as follows: a. Mix one egg white with 25 ml water in a 250 ml beaker to create an albumin solution. Pipette 5 ml of this solution into Test Tube 1. b. Mix the packet of Knox gelatin with 50 ml hot water in a second 250 ml beaker. Stir until dissolved. Pipette 5 ml of this solution into Test Tube 2. 5. Pipette 5 ml of the 1% glucose solution into Test Tube 3. 6. Use the 10 ml graduated cylinder to measure and pour 5 ml of water into Test Tube 4. 7. Pipette 5 ml of the Unknown Solution into Test Tube 5. 39
8. Record the initial color of each sample in Table 2. 9. Add five drops of Biuret solution to each test tube. Swirl each tube to mix. 10. Record the final color in Table 2. Note: Protein is present in the sample if a light purple color is observed. Table 1: A Priori Predictions Sample Will There be Protein Present? Initial Color Final Color 1 - Albumin Solution 2 - Gelatin Solution 3 - Glucose 4 - Water 5 - Unknown Table 2: Testing for Proteins Results Sample Initial Color Initial Color Is Protein Present? 1 - Albumin Solution 2 - Gelatin Solution 3 - Glucose 4 - Water 5 - Unknown 40
Post-Lab Questions 1. How did your a priori predictions from Table 1 compare to your actual results in Table 2? If there were any inconsistencies, explain why this occurred. 2. Identify the positive and negative controls used in this experiment. Explain how each of these controls are used, and why they are necessary to validate the experimental results. 3. Identify two regions which proteins are vital components in the human body. Why are they important to these regions? 4. Diet and nutrition are closely linked to the study of biomolecules. Describe one method by which you could monitor your food intake to ensure the cells in your body have the materials necessary to function. 41
Experiment 2: Testing for Reducing Sugars Many of the foods we eat contain carbohydrates. Monosaccharides and short chains such as disaccharides taste sweet due to certain aspects of their chemical structure. A structural characteristic of some sugars can be identified using a chemical solution called Benedict s reagent. When heated, the copper ions in Benedict s solution react with the free end of any reducing sugars, such as glucose molecules. Copper ions are reduced by the sugars, producing an orange or red colored precipitate. Materials 5 ml Benedict s Solution 5 ml 1% Glucose Solution, C 6 H 12 O 6 10 ml Graduated Cylinder Permanent Marker 3 Pipettes Ruler Spatula 5 Test Tubes (Glass) *Fork *Hot Water Bath (stovetop or microwave and a deep, heat-safe bowl) *Knife *Onion *Potato *Stopwatch *Tap Water Thermometer 5 ml Unknown Solution *You Must Provide Note: Use great caution when handling a knife and/or cutting. Ask for assistance if you need help or are uncomfortable with knife work. Procedure 1. Label five test tubes as 1-5. 2. Prepare your testing samples as follows: a. Cut a raw potato into a 1.0 cm x 1.0 cm x 1.0 cm cube. Cut this cube into smaller pieces, and mash with a fork and approximately 5-10 drops of water. Place half of the mashed raw potato into Test Tube 1. Use the 10 ml graduated cylinder to measure and pour 5 ml of water into Test Tube 1. b. Cut a raw onion into a 1.0 cm x 1.0 cm x 1.0 cm cube. Cut this cube into smaller pieces, and finally mash with a clean or new fork. Place half of the mashed raw onion into Test Tube 2. Use the 10 ml graduated cylinder to measure and pour 5 ml of water into Test Tube 2. 3. Pipette 5 ml of the 1% glucose solution into Test Tube 3. 42
4. Use the 10 ml graduated cylinder to measure and pour 5 ml of water into Test Tube 4. 5. Pipette 5 ml of the Unknown solution into Test Tube 5. 6. Record the initial color of each solution in Table 3. 7. Prepare a hot water bath using the following information: a. Heat water to a temperature between 85 and 100 C (not boiling) using a stovetop or microwave safe container. Be sure to confirm this temperature using the thermometer just prior to use in Step 9. The hot water bath must be of appropriate size and shape to fit five glass test tubes in a vertical orientation. 8. Pipette 10 drops of Benedict s Solution to each test tube. Swirl each tube gently to mix. 9. Place the five test tubes into the hot water bath and let sit for three minutes. Remove the tubes from water and place them in test tube rack to cool for five minutes. 10. Record the final color in Table 3. Note: A reducing sugar is present in the sample if a red, yellow or green precipitant forms. Wash your test tubes immediately after recording results to prevent permanent staining from the reaction products. Table 3: Testing for Reducing Sugars Results Sample Initial Color Final Color Reducing Sugar Present 1 - Potato 2 - Onion 3 - Glucose Solution 4 - Water 5 - Unknown 43
Post-Lab Questions 1. Write a statement to explain the molecular composition of the unknown solution based on the results obtained during testing with each reagent. 2. What can you conclude about the molecular make-up of potatoes and onions based on the test you performed? Why might these foods contain these substance(s)? 3. What results would you expect if you tested ribose, a monosaccharide, with Benedict s solution? Biuret solution? Experiment 3: What Household Substances are Acidic or Basic? In the following experiment, you will be using ph test strips to determine the ph of various household substances. ph stands for potential hydrogen and is broken into a scale of 1-14 to indicate the acidity or basicity of a solution. Generally speaking, more hydrogen ions in a solution correlates to lower ph values, and more acidic solutions. Conversely, fewer hydrogen ions correlates to higher ph values, and more basic solutions. 7 is located in the middle of this number scale, and represents neutral solutions. ph: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Acidic Neutral Basic Figure 7: Note that many strong acids and bases do not have a ph that is indicated on this scale. For example, lead battery acid has a ph that is below one. Refer to the color key provided in the module with your ph test strips to determine which color corresponds to each ph value. In this way, ph paper allows scientists to determine to what degree a substance is acidic or basic and can provide an approximate ph value. 44
Materials 5 ml 4.5% Acetic Acid (Vinegar), C 2 H 4 O 2 (3) 100 ml Beakers (3) 250 ml Beakers 10 ml Graduated Cylinder (10) ph Test Strips 5 ml Sodium Bicarbonate (Baking Soda) Solution, NaHCO 3 *4 Liquid, Household Solutions *Paper Towels *Water Source (Jug or Sink) *You Must Provide Procedure 1. Find four household substances to test (e.g., grape juice, lemon juice, dishwashing liquid, milk, tomato juice, shampoo, corn starch solution, etc.). You will use the vinegar (acidic) and sodium bicarbonate (basic) solution provided in your kit as standards. 2. Predict the ph of each substance before testing with a ph test strip. Record your predictions in Table 4. 3. Use the permanent marker to label each of the beakers with the name of one of the six solutions. It does not matter which size beaker is used for the different solutions. 4. Use the graduated cylinder to measure and pour 5 ml of vinegar into the beaker labeled Vinegar. 5. Thoroughly rinse the graduated cylinder with water to remove any remaining vinegar. Use paper towels to dry the graduated cylinder and repeat Step 4 with each of the five remaining solutions and beakers. 6. Measure the ph of each solution by dipping the pad of the ph strip into the solution for 5-10 seconds and comparing it with the ph test strip key (located in the lab module). Record your results in Table 4. Table 4: ph Values of Common Household Substances Substance ph Prediction ph Test Strip Color Acetic Acid (Vinegar) Sodium Bicarbonate Solution (Baking Soda) 45
Post-Lab Questions 1. What is the purpose of determining the ph of the acetic acid and the sodium bicarbonate solution before testing the other household substances? 2. Compare and contrast acids and bases in terms of their H + ion and OH - ion concentrations. 3. Name two acids and two bases you often use. 46