Chemical Hypotheses I & II Lab (Biomolecules) PDF

Generating and Testing Chemical Hypotheses: Biomolecules Have you had anything to eat in the last two-four hours? Most of us have been snacking on something, but do you REALLY know what you have been snacking on? To answer that question most would say, "breakfast" or "lunch" or maybe even "a candy...

Generating and Testing Chemical Hypotheses: Biomolecules Have you had anything to eat in the last two-four hours? Most of us have been snacking on something, but do you REALLY know what you have been snacking on? To answer that question most would say, "breakfast" or "lunch" or maybe even "a candy bar." Whatever you might have eaten, you just ate atoms, groups in molecules and compound that were used to create biomolecules. There are four types of biomolecules: carbohydrates, lipids, proteins, and nucleic acids. Basically, a carbohydrate is a carbon atom that has a water attached to it (carbo= carbon, hydrate = water). Thus, the base chemical structure for all carbohydrates is CH2O. There are three types of simple sugars (monosaccharides) that are important in human biology. These are glucose, fructose, and galactose. These simple sugars all have the same chemical formula, C6H12O6, but have different structural formulas. The next level of carbohydrates is the disaccharide. Disaccharides are formed when two monosaccharides are combined using a process called dehydration synthesis. This means that water is removed from the two compounds as they are bonded together. When a molecule of glucose is combined with a molecule of fructose, sucrose is produced. When a molecule of glucose and galactose are combined, lactose is formed. Finally, when two molecules of glucose are combined together, we produce the disaccharide maltose. Finally, within animals, the polysaccharide called glycogen can be found. Glycogen is the stored form of sugar within the human body and is many glucose molecules strung together. One biochemist refers to glycogen as a G-string. In plants there are two forms of polysaccharides that are important in human biology. The first is the class called starch (i.e., potato, wheat) and the second form is called cellulose (fiber). Digestion of these molecules use a chemical process called hydrolysis. Hydrolysis is when water is "lysed" (broken down) into H+ and OH- and it then inserted between monosaccharides to produce a smaller molecule. The next class of biomolecules is lipids. Like carbohydrates, lipids are composed o f carbon, hydrogen, and oxygen. However, there is not a set ratio between the atoms. All lipids are considered non-polar and hydrophobic; thus, they do not dissolve in water. There are four distinct groups within the class of lipids. They are: fatty acids/triglycerides, steroids, eicosanoids, and phospholipids. It is important to realize that not all lipids are fats. The term fat specifically deals with the class of triglycerides. Triglycerides are biomolecules that are made from a single glycerol molecule with three fatty acids attached. Triglycerides are the stored form of lipid (i.e., they are stored in adipose tissue) and are a great reserve for energy production within the human body. Triglycerides can be saturated, meaning that all carbon molecules have two hydrogens attached. In an unsaturated triglyceride, there is at least one carbon double bond. A trans fat is a triglyceride that has been modified to include a double carbon bond, but in the process the H is attached to the opposite side of the carbon molecule. Although that may not seem significant, we are beginning to understand the health consequences of trans fats. Trans fats disrupt the body’s ability to metabolize essential fatty acids like Omega 3s. The other types of lipids are used differently within the human body. Eicosanoids and steroids are used as messengers/hormones to help direct the functioning of the body. (We will study these types of lipids when we learn about immunity and hormones.) Phospholipids are the last group of lipids and are an integral portion of the cell membrane. Proteins are the next biomolecule class. Like carbohydrates and lipids, proteins contain carbon, hydrogen, and oxygen. However, proteins also contain the atom nitrogen. Proteins are important in the human body because the function as structural support and they catalyze the biochemical reactions within the human body. Proteins are made up of amino acids connected together with peptide bonds. There are twenty different amino acids that are used to produce the proteins in the human body. Eight of these amino acids are considered essential amino acids because they must be ingested within our diet. The other twelve can be synthesized through biochemical reactions within the body, thus they are referred to as nonessential. All amino acids have the same base structure: a carbon molecule with an amino group (nitrogen- based), carboxyl group (COOH), and a hydrogen molecule. They differ at the fourth attachment called the R group, or the rest of the molecule. Last, but not least, is the biomolecule class called nucleic acids. These biomolecules are made up of carbon, hydrogen, oxygen, nitrogen, and phosphorus. This class is a very important class because it includes the molecules deoxyribose nucleic acid (DNA), ribonucleic acid (RNA), and adenosine triphosphate (ATP). Both DNA and RNA are essential for the development of proteins within the human body. ATP is the energy molecule. There are two parts to this lab. In part A of this lab, we will be performing six biochemical reagent tests to indicate the presence of specific biomolecules (we will not be testing for nucleic acids). In part B, you will perform the same biochemical reagent tests on three unknowns to determine the chemical composition of the substance.  PART A: BIOCHEMICAL REAGENT TESTS FOR BIOMOLECULES Experiment One: Benedict ’s Reagent I Benedict’s reagent is blue. If heated in the presence of monosaccharides, the solution will turn to yellow or red, depending on the concentration of the test material present. Given what you know about Benedict’s reagent in the presence of monosaccharides, write a hypothesis with reasoning (include in your lab notebook): In the prediction column of the following chart, identify those solutions that you expect to give you a positive (+) Benedict’s result and those that will yield a negative (-) Benedict’s result. Experiment Protocol: Label six test tubes 1 through 6. To each of the test tubes add the Benedict’s reagent and water as indicated by the following chart. Add an equal scoop of designated substance to the test tubes. Agitate Heat in a boiling water bath for 5 minutes. Cool, agitate, and then record results Tube # Benedict’s Water Substance Prediction Result Reagent 1 3 mL 3 mL --- 2 3 mL 3 mL 1 scoop glucose 3 3 mL 3 mL 1 scoop fructose 4 3 mL 3 mL 1 scoop sucrose 5 3 mL 3 mL 1 scoop maltose 6 3 mL 3 mL 1 scoop starch Experiment Two: Benedict’s Reagent II As you have seen, Benedict’s reagent turns red or yellow when heated in the presence of monosaccharides. You have also seen how Benedict’s reagent responds to some disaccharides and polysaccharides. Unlike the bonds between glucose and glucose (maltose), the bonds between glucose and fructose (sucrose) do not readily break down in heated water. However, other chemicals, like acids, are powerful agents for causing corrosion and the breakdown of materials. Hydrochloric acid is an organic acid that has this property. Given what you just read regarding acids, write a hypothesis for a reaction using Benedict’s reagent, sugars, and hydrochloric acid with reasoning (include in your lab notebook): Experiment Protocol: Label the test tubes 1 through 7 As indicated by the following chart, add water, HCl, and the equal amounts of substance into the test tubes. DO NOT ADD THE BENEDICT’S REAGENT YET!!! Heat the test tubes in a hot water bath for 5 minutes. Cool. (You may run cold water on the outside of the test tubes to facilitate this.) Now add Benedict’s reagent to each of the seven test tubes. Return to the hot water bath for 5 minutes. Cool, agitate, and record results. Tube # Water HCl Substance Benedict’s Prediction Result Reagent 1 3 mL 5 drops 3 mL 2 3 mL --- 1 scoop maltose 3 mL 3 3 mL --- 1 scoop sucrose 3 mL 4 3 mL --- 1 scoop starch 3 mL 5 3 mL 5 drops 1 scoop maltose 3 mL 6 3 mL 5 drops 1 scoop sucrose 3 mL 7 3 mL 5 drops 1 scoop starch 3 mL Experiment Three: Potassium Iodide Reagent: Starch molecules will combine with the yellowish iodine in the reagent to produce a blue-black color. Write a hypothesis for the Potassium Iodide Test with reasoning (include in your lab notebook): Experiment Protocol: Label test tubes 1 through 4. Add to each of the three test tubes the water, potassium iodide, and 1 scoop of the substance as indicated below. Agitate on mixer Record your results Tube # Water Substance Potassium Prediction Result iodide 1 3 mL 3-4 drops 2 3 mL 1 scoop of glucose 3-4 drops 3 3 mL 1 scoop of starch 3-4 drops 4 3 mL 1 scoop of maltose 3-4 drops Experiment Four: Sudan IV Sudan IV is a reagent that tests specifically for lipids. The reagent is pinkish-brown. It acts as a stain, being taken up preferentially by any lipid that is present in a solution. Because lipids are less dense than water but do not dissolve, the float on the surface, if a lipid is present, a thin, translucent, stained ring will be seen at the surface of the water. Because of the color of Sudan IV, the solution will be pink, orange, or light brown without lipid; therefore, the concern is whether there is a darker surface ring. Write a hypothesis for the Sudan IV test with reasoning (include in your lab notebook): Experimental Protocol: Label four test tubes, 1 through 4. Add to each of the four test tubes the water and the amount of substance indicated below. Mix the contents of each test tube using a stir rod or agitate on the mixer. Let sit for a few minutes. Add 5 drops of Sudan IV to the test tube, letting the drops run down the side of the tube. Record your results. (If you are questioning the result, gently drop 2 or 3 drops down the inside of the test tube and see if the top stains with the Sudan IV.) Tube # Water Substance Sudan IV Prediction Result 1 3 mL --- 5 drops 2 3 mL 2-3 drops corn oil 5 drops 3 3 mL 1 scoop cholesterol 5 drops 4 3 mL 1 scoop of starch 5 drops Experiment Five: Ninhydrin Reagent Ninhydrin is a specific test for the presence of free amino acids. On each end of the free amino acid are active groups with which the reagent reacts. When heated with the free amino acids, the colorless ninhydrin turns a shade between lavender or deep purple depending on the concentration of amino acids. You will complete this experiment in the fume hood. DO NOT use the ninhydrin outside of the hood. Dispose of your ninhydrin waste in the waste container in the hood. Given the information above, write a hypothesis regarding a Ninhydrin test with reasoning (include in your lab notebook): Experimental Protocol: Label four test tubes with the numbers 1 through 4. Add to each of the four test tubes the water, ninhydrin, and the amount of the substance indicated by the following chart. Agitate on a mixer. Heat in a hot water bath for 3 minutes (or until you see a distinct color change). Remove from the water bath and cool. Record the results. Tube # Water Substance Ninhydrin Prediction Result 1 3 mL 10 drops 2 3 mL I scoop glycine 10 drops 3 3 mL I scoop egg albumin 10 drops 4 3 mL 2-3 drops corn oil 10 drops Experiment Six: Biuret Reagent The biuret reagent is a combination of two chemicals that you will add to a solution. The two chemicals are copper sulfate and a 15% sodium hydroxide solution. This is caustic and extremely hazardous. In the presence of two or more peptide bonds, the reagent turns a lavender color. Sometimes this is a subtle difference, so inspect carefully. Write a hypothesis for the biuret reagent test with reasoning (include in your lab notebook): Experimental Protocol: Label four test tubes 1 through 4. Add to each of the test tubes the indicated water, substance and COPPER SULFATE. Then, add the indicated amount of sodium hydroxide. Gently agitate by flicking the test tube. Record your results. Tube # Water Substance Sodium Copper (Cupric) Prediction Result Hydroxide Sulfate 1 3 mL 3 mL 3-4 drops 2 3 mL I scoop glycine 3 mL 3-4 drops 3 3 mL I scoop egg albumin 3 mL 3-4 drops 4 3 mL 2-3 drops corn oil 3 mL 3-4 drops PART B: DETERMING THE CHEMICAL COMPOSITION OF AN UNKNOWN SUBSTANCE Benedict’s Reagent One: Label 3 test tubes 1-3. Then add 3 ml of Benedict’s Reagent, 3 ml of water, and 1 scoop of unknown to the test tube. Heat in a hot water bath for 5 minutes Record results Benedict’s Reagent Two: Label three test tubes 1-3. Then add 3 ml of water, 5 drops of HCl and 1 scoop of unknown. Heat in a hot water bath for 5 minutes Remove and cool test tubes. Add 3 ml of Benedict’s Reagent to each test tube. Heat in hot water bath for 5 minutes Record results Potassium Iodide Reagent: Label three test tubes 1-3. Add 3 ml of water and 1 scoop of unknown to each tube. Add 3-4 drops of Potassium Iodide to each test tube. Record results Sudan IV Reagent Label three test tubes 1-3. Add 3 ml of water and 1 scoop of unknown to test tube. Mix well. Let the test tubes rest for 5 minutes. Add 5 drops of Sudan IV reagent to the test tubes. DO NOT RE-MIX. Record results Ninhydrin Reagent Label three test tubes 1-3. Add 3 ml of water and 1 scoop of unknown. Mix well. Add 10 drops of Ninhydrin reagent to each test tube. Heat in a hot water bath for 5 minutes. Record your result. Biuret Reagent: Label three test tubes 1-3. Add 3 ml of water and 1 scoop of unknown to test tubes. Add 3-4 drops copper sulfate to each test tube. Add 3 ml of sodium hydroxide to each test tube. Agitate if needed. Record your result. Reagent Unknown # Unknown # Unknown # Benedict’s One Benedict’s Two Potassium Iodide Sudan IV Ninhydrin Biuret’s Unknown Possibilities: glucose, sucrose, starch, albumin, glycine, cholesterol Each unknown may contain one, two, or three substances from the above list. Please note: no unknown will contain both sucrose and starch.