Carbohydrates Adapted from Pellar OBJECTIVE: To learn about carbohydrates and their reactivities BACKGROUND: Carbohydrates are a major food source, with most dietary guidelines recommending that 45-65% of daily calories come from carbohydrates. Rice, potatoes, bread, pasta and candy are all high in carbohydrates, specifically starches and sugars. These compounds are just a few examples of carbohydrates. Other carbohydrates include fibers such as cellulose and pectins. In addition to serving as the primary source of energy for the body, sugars play a number of other key roles in biological processes, such as forming part of the backbone of DNA structure, affecting cell-to-cell communication, nerve and brain cell function, and some disease pathways. Carbohydrates are defined as polyhydroxy aldehydes or polyhydroxy ketones, or compounds that break down into these substances. They can be categorized according to the number of carbons in the structure and whether a ketone or an aldehyde group is present. Glucose, for example, is an aldohexose because it contains six carbons and an aldehyde functional group. Similarly, fructose would be classified as a ketohexose. glucose fructose A more general classification scheme exists where carbohydrates are broken down into the groups monosaccharides, disaccharides, and polysaccharides. Monosaccharides are often referred to as simple sugars. These compounds cannot be broken down into smaller sugars by acid hydrolysis. Glucose, fructose and ribose are examples of monosaccharides. Monosaccharides exist mostly as cyclic structures containing hemiacetal or hemiketal groups. These structures in solution are in equilibrium with the corresponding open-chain structures bearing aldehyde or ketone functional groups. The chemical linkage of two monosaccharides forms disaccharides. Sucrose, table sugar, is made when a glycosidic bond forms between glucose and fructose. Lactose and maltose are two other examples of disaccharides. Joining many monosaccharide units through glycosidic bonds forms polysaccharides. Starch, commonly called a complex carbohydrate on food labels, and cellulose are examples. Chemical reactivity of carbohydrates Oxidation: Carbohydrates that can undergo oxidation, such as glucose, are known as reducing sugars. All monosaccharides are reducing sugars sine they have either the aldehyde functional group or an alpha-hydroxy ketone group that can undergo oxidation. Many disaccharides are also reducing sugars. Sucrose, however cannot be oxidized as so is not a reducing sugar. Fehling’s reagent will be used to test the ability of carbohydrates to act as reducing sugars. A red precipitate signals a positive reaction. Barfoed’s reagent can also be used for oxidation. It is a much milder oxidizing agent than Fehling’s reagent and can be used to distinguish between monosaccharides and reducing disaccharides. Reducing disaccharides undergo a slow reaction or none at all with Barfoed’s reagent, while monosaccharides react quickly. Again, the presence of a red precipitate indicates a positive reaction. Dehydration: Carbohydrates can undergo dehydration reactions. To differentiate between aldoses and ketoses, Seliwanoff’s reagent will be used. When a ketose reacts with this reagent (resorcinol in 6M HCl), a cherry-red colored complex forms. Aldoses take much longer to react. Disaccharides and polysaccharides eventually hydrolyze to monosaccharides in the acidic environment of this test and will also form a red-colored solution slowly over time. Reaction with iodine: Starch reacts with a solution containing iodine to reveal a deep blue color. This color may vary according to the structure of the polysaccharide (one component of starch, amylose, gives a blue color while the other component, amylopectin, gives a violet color). Simpler carbohydrates will not change the color of the iodine solution. Hydrolysis: Polysaccharides and disaccharides can be broken down in the presence of acid. A carbohydrate such as sucrose that has a single glycosidic linkage bond becomes fully hydrolyzed in an acid solution. Large polysaccharides typically undergo a partial hydrolysis to produce many shortened starch molecules, known as dextrins. Enzymatic tests: Urinalysis for glucose used to rely primarily on the Barfoed’s test, which was non-specific for the type of sugar found in the urine. Modern test strips are more specific enzyme-based assays that react only in the presence of glucose. PROCEDURE: Perform each of the following tests on glucose, fructose, lactose, sucrose, starch and your unknown unless otherwise indicated. Fehling’s test: To each of 6 test tubes, add 6 drops of the substances to be tested. In a large test tube, mix 6 mL of Fehling’s solution A with 6 mL of Fehling’s solution B. Add 2 mL of this mixture to each tube and stir thoroughly. Place the test tubes in a boiling- water bath for 5 minutes and record your observations. The formation of a red precipitate indicates a positive reaction. Barfoed’s test: To each of 6 test tubes, add 1 mL of the solutions to be tested. Then add 3 mL of Barfoed’s reagent to each test tube and mix thoroughly. Place the test tubes in a boiling water bath for 5 minutes and record your observations. The formation of a red precipitate indicates a positive reaction. Seliwanoff’s test: (avoid contact with this solution since it contains hydrochloric acid) To each of 6 test tubes, add 10 drops of the solutions to be tested. A seventh test tube containing 10 drops of distilled water should also be prepared. Add 4 mL of Seliwanoff’s reagent to each test tube and mix. Place the test tubes in a boiling-water bath and note the time needed for any color change to occur. Discontinue heating after 10 minutes. A color change indicates a positive reaction. Record your observations. Iodine test: To each of 6 test tubes, add 1 mL of the solutions to be tested. A seventh test tube containing 1 mL of distilled water should also be prepared. Add 3 drops of iodine solution and record your observations. Hydrolysis: (test only sucrose and starch) To each of two tests tubes, add 5 mL of the solutions to be tested. Add 10 drops of 3 M HCl and mix thoroughly. Heat in a boiling water bath for 20 minutes. Allow the solution to cool and then add 3 M NaOH dropwise until the solution tests neutral on pH paper. Test the hydrolyzed sucrose solution using the Fehling’s test above. Perform the iodine test on the hydrolyzed starch solution. Urinalysis test strips: Each student should test a single solution. Create a table on the blackboard to ensure that all solutions are tested and everyone has all of the results. Place a single drop of the solution to be tested on a chemical test strip. Read the concentration of glucose using the scale on the side of the container. .