Organic Molecules of Life - Exercise 2 Objectives -Know the difference between a reducing sugar and a non-reducing sugar. -Distinguish Monosaccharides from Disaccharides and Polysaccharides -Understand the purpose of a control -Understand what gives you positive and negative reactions for the following assays and know what those assays are testing for what specific type of sugar (mono, di, or Polysaccharides): Benedict, Barfoed, Iodine, Biuret, Miscibility, and Sudan Red. KNOW THE COLORS OF TUBES WHEN THERE POSTIVE AND NEGATIVE FROM THE ASSAYS
There are four major molecules, also known as macromolecules, and these include carbohydrates, proteins, lipids and nucleic acids. Each of these molecules contain macromolecular varieties which can be chemically degraded by hydrolysis (broken down into smaller pieces). -Carbohydrates are polymers of monosaccharides (monomer). -Proteins are polymers of amino acids (monomer). -Nuclei are polymers of nucleic acids (monomer). -Lipids have the subcategories of neutral fats, and can be digested into glycerol and fatty acids.
Carbohydrates, proteins, and nucleic acids are polymers (and they are made from smaller monomers). Lipids are not polymers (they are not made from monomers) You could say there are subunits in lipids, such as, Glycerol & fatty acids. Assay = Test
Carbohydrates Complex Sugars Monosaccharides one sugar molecule Disaccharides two sugar molecules Oligosaccharides a few sugar molecules Polysaccharides many sugar molecules
What are Biological Molecules? Carbohydrates Sugar Starch Cellulose Fats Lipids Fatty acids Cell membranes Proteins Amino acids Collagen Fibrin Nucleic Acids DNA RNA
Carbohydrates Monosaccharide which is the basic unit of a carbohydrate, and can t be broken down anymore (smaller molecules) or it can also be called simple sugar. One significant group of Carbohydrates with 6 carbons is called hexoses. The very common hexoses are glucose, galactose, and fructose. Each of the three sugars have the exact same molecular formula, which is C6H12O6, but there arranged differently, so these are called isomers. These sugars also exist in different forms one is a ring and the other is a chain. Monoasccharides in the chain form will have one carbon atom that is doubled bonded to oxygen (C=O) which is known as a carbonyl group. Solutions of these sugars will contain both the chain and ring form in equilibrium, and each of the forms can freely convert in to the alternative form under favorable chemical conditions.
Reducing Sugars When talking about chemistry the main things to focus on are electrons, those are the things that make reactions happen. You will learn more about this in organic chemistry. An atom or molecules that receives one or more electrons form another substance is said to have been reduced. The thing that donates an electron is known as the reducing agent. A reducing sugar is any sugar that possess a carbonyl group (C=O). Reducing sugars are capable of donating an electron to another molecule. Reducing sugars can be monosaccharides or disaccharides. The chain form of any monosaccharide has the chemical group (C=O). In some, but not all, disaccharides one of the two rings may be open, and they will have the C=O group and thus be reducing sugars. Those that cannot open one of the rings will not be a reducing sugars, because it won t be able to donate its electrons.
All of these molecules are held by specific bonds, and these bonds can be used to chemically identify the molecules that posses them. The test that you performed tested for the chemical reactions of these bonds (simple chemical test) which are also known as assays, and these test could can be used to identify the presence of a particular type of molecules in a simple mixture. Reducing agent: Substance that loses electrons (oxidizes). Oxidation agent: The electron lets the substance get oxidize.
Carbohydrates Solutions of these sugars will contain both the chain and ring form in equilibrium, and each of the forms can freely convert in to the alternative form under favorable chemical conditions.
Monosaccharides Disaccharides Polysaccharides Glucose Galtose Fructose Ribose Monnose Sucrose Maltose Lactose Starch Cellulose Glycogen
What s a control, what s the purpose of having it?
Control When doing an assay (test) a control test must be done. The control tube will contain everything that the assays contain except the substance to be tested. In the assays, water is the solvent for all of the other substance and will be in the control, along with the test reagents. The control test is the negative standard against which the assays are compared.
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Page 22 Lab Book
Disaccharides: Lactose & Maltose Monosaccharides: Glucose, Fructose, Galactose Polysaccharide: Starch Sucrose Diasscahride: This always has rings, so it s a non-reducing sugar. You could put sucrose into any solution, and it s the same thing as throwing water, so nothing would change.
Benedict s Assay for Reducing Sugars The Benedict reagent is a clear blue solution of Copper hydroxide (Cu (OH)2) at an alkaline ph (7). When this is mixed with a solution containing a reducing sugar and heater the Cu ion is reduced to cuprous form, which will make the solution the solution would become turbid (cloudy). The color change in a positive assay can vary from greenish to bright red, depending open the strength of the reducing sugar (which determines how much precipitate will be formed). A positive Benedict s assay indicates the presence of a reducing sugar either mono or disaccharides.
Benedict s Assay Macromolecule Monomer Assay Name Assay Indicates Positive Result looks like: Carbohydrate Monosaccharide (simple sugar) Benedict s Assay Presence of a reducing sugar (could be a monosaccharide or disaccharide) A cloudy (turbid) color change from blue to greenish or bight red color
Barfoed s Assay for Monosaccharides Monosaccharides can be distinguish from reducing disaccharides by using Barfoed s reagent, which is similar to Benedict s, but set to an acidic ph (about 4.5). In an acidic environment the reducing disaccharides cannot reduce the cupric ion, but the monosaccharide can. All rings in disaccharide are closed; therefore, cant be reducing sugar. A positive Barfoed s assay indicates the presence of a monosaccharides and only monosaccharides, and is reenergize by the production of a slight turbidity and a slight color change from blue to red/violet or green. If you see particles in the bottom of the test tube, it s a positive result.
Barfoed s Assay Macromolecule Monomer Assay Name Assay Indicates Positive Result looks like: Carbohydrate Monosaccharide (simple sugar) Barfoed s Assay Presence of a monosaccharide A slightly cloudy (turbid) color change from blue to green or red/violet (looks for redish particles at bottom of tube)
Summary of Benedcit s & Barfoed s Assay Note: Those that cannot open one of the rings is not a reducing sugar. Benedcit s assay: Used to test for monosaccharides and disaccharides. It has a ph of about 7, but when the solution gains (reduces) an electron from copper ion the solution becomes a reducing sugar (positive result). In the Benedcit s assay five carbohydrates were reduced - three were monosaccharides, and the other two were disaccharides. Barfoed s Assay was used for testing monosaccharides too. Monosaccharides can be distinguish from reducing disaccharides by using this test. But Barfoed s test had a ph of 4.5, which is a more acidic environment. A positive result indicates presence of monosaccharides and only monosaccharides. In an acidic environment, disaccharides can t gain an electron from the copper ion, but monosaccharides can gain an electron from copper ion. Those that cannot open one of the rings will not be a reducing sugars, because it won t be able to donate its electrons.
Starch: Iodine Assay Starch is a polysaccharide composed of hundreds of units of glucose covalently linked together. The structure of starch is a such that the molecule is coiled. Iodine [I2KI] will interact specifically with these coiled molecules to form a blue to black colored complex. Note: Iodine will not react with other carbohydrates, thus retaining its original color of yellow or amber. If it is still the color of yellow or amber but diluted somewhat, the assay is negative, but if it is any other color, such as, brown to reddish brown to blue to black, then its positive. A positive iodine assay indicates the presence of starch, so if the color turns dark blue, or black, this is a positive result.
The Iodine Assay - Starch Macromolecule Monomer Assay Name Assay Indicates Positive Result looks like: Carbohydrate Monosaccharide The Iodine Assay Presence of Starch A color change from yellow to reddish brown or bluish/black
Proteins (peptide bonds) - Biuret Assay Proteins are composed of atoms or elements C,H,O, N, & S. Proteins are very large polymers. The monomer unit of proteins is an amino Acid. Although each different protein has a unique sequence of amino acids, the covalent bonds between adjacent amino acids are essentially the same. A covalent bond between two amino acids is called a peptide bond. The presence of peptide bones can be determine by using Biuret test. In a strongly alkaline environment (ph 8-14), copper sulfate reacts with the peptide bones, causing the blue color of the reagent to change to a violet color. A violet color indicates a positive Biuret assay and also indicates the presence of peptide bonds and of course of proteins.
Polypeptides Polypeptides Are polymers of amino acids A peptide bond is a covalent bond between 2 amino acids. A protein Consists of one or more polypeptides
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Page 24 Lab Book
Biuret Assay- Protein Macromolecule Monomer Assay Name Assay Indicates Positive Result looks like: Protein Amino acids Biuret Test Presence of peptide bonds Color change from blue to violet
Lipid - Miscibility All lipids are insoluble in water. Families of lipids, including fats and oils, phosopholipids, steroids, and carotenoids. We will look at triglycerides in lab. Fats and oils are polymers. The subunit of fats/oils are glycerol and three fatty acids. Miscibility Assay: If a fat or oil mixes with water in the test tube, two distinct layers of liquid will form because oil and water do not mix, this would indicate a negative result (two layers of liquid), but if two the substances mix, then it s a positive result. Basically what this means is that the substance assayed does not mix with water (negative miscibility). Friendship and money oil and water. -Michael Corleone (Godfather III)
Lipids: Fatty Acids Charged polar end Long hydrocarbon chains Very hydrophobic Oil and water don t mix Used for energy storage Very high energy Long-term storage Lipid tail end
What s so important about lipids, what barrier do they create in a cell? Hint it has multiple heads & tails.
Phospholipid bilayer - Membrane Structure Fluid Mosaic Model: fluid membrane with various proteins embedded in or attached to a double layer (bilayer) of phospholipids. Hydrophilic head WATER WATER Hydrophobic tail
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Page 25 Lab Book
Miscibility Assay - Lipid Macromolecule Subunit Assay Name Assay Indicates Positive Result looks like: Lipid Glycerol + fatty acid Miscibility Assay Presence of an insoluble (hydrophobic) substance Here we look for a negative result, namely no mixing. If an insoluble substance is present, there will be two visible layers.
Lipid - Sudan Red Assay Sudan Red Assay: The Sudan stain will mix with fats and oils and stain them but will not stain water and water soluble compounds. A positive Sudan red assay indicates the presence of a lipid such as fat or oil and vice versa. So if the substance stains red, the assay is positive.
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Page 25 Lab Book
Sudan (Red) Assay - Lipid Macromolecule Subunit Assay Name Assay Indicates Positive Result looks like: Lipid Glycerol + fatty acid Sudan (Red) Assay Presence of a lipid Lipids (but water) will stain
Questions 1. You are given two unlabeled containers and told that one of them contains a solution of pure glucose and the other contains a solution of pure maltose, a disaccharide. How can you identify the content of the bottle by performing chemical assays? 2. What is a monosaccharide? Why all monosaccharides and most disaccharides are reducing sugars? 3. In solution, chain and ring forms of glucose molecules exist in equilibrium. Explain this statement? 4. Both fructose and glucose are reducing sugars. When these two simple sugars become chemically bonded, a disaccharide, sucrose, is formed. Why sucrose is not a reducing disaccharide?
Questions 1. You are given two unlabeled containers and told that one of them contains a solution of pure glucose and the other contains a solution of pure maltose, a disaccharide. How can you identify the content of the bottle by performing chemical assays? You can perform the Bendicts and Barfoed s assay to see what reactions are positive. Maltose will only be positive in the Bendcit s assay due to the ph (7) environment. And, if you perform the Barfoed s assay and get a negative result, then you know you got Benedict s assay for maltose. For glucose you get a positive reaction for the Bendict s assay and a negative result for Barfoed s assay. 2. What is a monosaccharide? Why all monosaccharides and most disaccharides are reducing sugars? A monosaccharide is a simple sugar that contain a carbonyl group. They can accept (gain) the Cu ion under acceptable ph levels, for instance, ph 7. 3. In solution, chain and ring forms of glucose molecules exist in equilibrium. Explain this statement? Solutions of these sugars will contain both the chain and ring form in equilibrium, and each of the forms can freely convert in to the alternative form under favorable chemical conditions. 4. Both fructose and glucose are reducing sugars. When these two simple sugars become chemically bonded, a disaccharide, sucrose, is formed. Why sucrose is not a reducing disaccharide? Sucrose always has rings, so it s a non-reducing sugar. You could put sucrose into any solution, and it s the same thing as throwing water, so nothing would change.
Questions 5. Write three names of monosaccharides. 6. Write two names of reducing disaccharides. 7. What is the difference between reducing disaccharide and non-reducing disaccharides? 8. What are three names of polysaccharides.
Questions 5. Write three names of monosaccharides. Glucose, Fructose, & Galactose 6. Write two names of reducing disaccharides. Lactose & Maltose 7. What is the difference between reducing disaccharide and non-reducing disaccharides? The environment, in our case, the ph was the factor. Under alkaline conditions a disaccharide could gain an electron from the copper ion, but in an acidic environment (ph 4.5) the disaccharide can t accept the electron from the copper ion. 8. What are three names of polysaccharides. Starch, Glycogen, & Cellulose
a. Sucrose b. Maltose c. Starch d. Glucose e. Egg albumin Questions 9. This substance gives a negative Benedict s assay, a negative Barfoed s assay, a positive iodine assay and a negative Biuret assay. 10. This substance gives a positive Benedict s assay, a positive Barfoed s assay, a negative iodine assay, and a negative Biuret assay. 11. This substance gives a positive Benedict s assay, a negative Barfoed s assay, a negative iodine assay, and a negative Biuret assay. 12. This substance is positive in Biuret assay and negative in all other assays.
a. Sucrose b. Maltose c. Starch d. Glucose e. Egg albumin Questions 9. This substance gives a negative Benedict s assay, a negative Barfoed s assay, a positive iodine assay and a negative Biuret assay. C - Starch 10. This substance gives a positive Benedict s assay, a positive Barfoed s assay, a negative iodine assay, and a negative Biuret assay. D - Glucose 11. This substance gives a positive Benedict s assay, a negative Barfoed s assay, a negative iodine assay, and a negative Biuret assay. B - Maltose 12. This substance is positive in Biuret assay and negative in all other assays. E - Egg albumin
Questions a. Sucrose b. Maltose c. Starch d. Glucose e. Egg albumin 13. All test (Bendict s, Barfoed s, iodine, and Biuret) on the substance came negative. ============================================================ 14. Benedict s assay is for Positive reaction for Benedict s assay produces color. 15. Barfoed s assay distinguishes between and. 16. A protein solution can be identified by performing the assay.
Questions a. Sucrose b. Maltose c. Starch d. Glucose e. Egg albumin 13. All test (Bendict s, Barfoed s, iodine, and Biuret) on the substance came negative. A - Sucrose ============================================================ 14. Benedict s assay is for reducing sugars Positive reaction for Benedict s assay produces red color. 15. Barfoed s assay distinguishes between monosaccharide and reducing sugar. 16. A protein solution can be identified by performing the Biuret assay.