Monosaccharides, disaccharides and polysaccharides are important biomolecules that store chemical potential energy. When this energy is released during controlled oxidation in catabolism, ATP and reducing power in the form of NADH and FADH2 accumulate in the cell. The stored energy can then be used in biosynthesis of proteins and other essential cellular building blocks.
Monosaccharides are classed as aldoses or ketoses, depending on the location of the carbonyl group in the molecule. They can be further described by the total number of carbon atoms in the chain, hence “aldopentose” or “ketohexose”. All simple sugars are capable of reducing reagents such as Tollen’s reagent, Fehling’s reagent or Benedict’s reagent since the cyclic hemiacetal or hemiketal functional group can be oxidized to a lactone. The oxidation reaction carries out a coupled reduction of the oxidizing agent.
Disaccharides are characterized by the identity of the linked simple sugars and by the nature of the glycosidic linkage joining them. When the anomeric carbon is converted to a ketal or acetal, it can no longer be oxidized. The reducing behavior of a disaccharide discloses whether either anomeric carbon remains as a hemiacetal or if both are converted to acetals.
Polysaccharides consist of sugar polymers and are characterized by the identity of the monomer and by the nature of the glycosidic linkage. Glucose storage homopolymers include amylose and amylopectin (the plant starches) and glycogen (the animal storage polysaccharide). The linear polymer amylose forms a blue-black colored complex with molecular iodine. Amylose forms a helix with 6 residues per turn. As amylose wraps around an I2 molecule, with the iodine molecules parallel to the long axis of the helix, interactions between the iodine electrons and the polar sugar hydroxyl groups change the molecular orbital energy for I2. As a result, the complex strongly absorbs in the visible provided at least 6 turns of the helix (36 glycosyl residues) wrap the iodine. Shorter chains do not produce the colored complex. Amylopectin gives a reddish-purple complex and glycogen a faint pink color.
In this experiment, we will prepare starch from a natural source, potatoes, and will investigate the behavior of the starch and some reference carbohydrates. We will classify the carbohydrates as reducing or non-reducing carbohydrates, will hydrolyze our starch using both acid and a naturally occurring enzyme in saliva, and will monitor the progress of the hydrolysis using the color-forming reaction with iodine.
Objectives: after this experiment, you will be able to
- Extract starch from a natural source
- Recognize the behavior of a reducing and of a non-reducing sugar
- Relate the color of a starch-iodine complex with the chain length of the polymer
- Potato, you’ll need to bring one from home, note the brand, type (Russet, Yukon, etc.), and where purchased
- Blender, cheesecloth, rubber bands, boiling chips, knife, scissors
- 6 M HNO3 and 6 M NaOH, litmus paper
- conc. H2SO4
α-naphthol solution (5% in 95% ethanol)
- iodine test solution (1% I<sub>2 in 10% KI solution)
- Benedict’s solution, Tollen’s reagent (AgNO3(aq) and conc. NH3(aq))
Phenylhydrazine solution, glacial acetic acid and sodium acetate (solid)
- Reference solutions of glucose, fructose, galactose, maltose, sucrose, and lactose
Part A: Isolation of Potato Starch
Wash and peel a potato then quickly cut it into small cubes. Homogenize the pieces for two minutes in a blender containing 200 mL of distilled water. (Note: turn on the blender with water only. Then carefully pour in the potato pieces.) Place several layers of cheesecloth over the top of a 600 mL beaker and secure with a rubber band around the top of the beaker. Pour the homogenized mixture onto the cheesecloth and allow the water and starch granules to filter through into the beaker. Resuspend the pulp in water and homogenize again for one minute then repeat the filtration. Discard the pulp retained by the cheesecloth.
Add water up to the 300 mL mark if needed, stir then allow the beaker to stand until the starch granules settle out. Decant the supernatant liquid. Add another 100 mL of distilled water to the starch grains, stir and let them settle. Once again, drain and discard the supernatant liquid. Finally, wash the starch grains onto a fluted filter paper using a stream of water from a wash bottle. Set the well-drained filter paper with product on top of a stack of folded paper towels to air-dry.
To use the starch in any of the following procedures, take ~0.5 g of starch and add enough cold water to make a thin paste that can be poured readily. Heat 100 mL distilled water to boiling then remove the heat. Pour the thin starch paste into the hot water and stir until the solution clears. Cool the solution and use portions for the following tests and procedures.
Part B: Acid Hydrolysis of Starch
Place 25 mL of starch solution in a 125 mL Erlenmeyer flask. Add 5 mL of 6 M HNO and boiling chips. Place a small funnel in the neck of the flask to act as a condenser. Boil the mixture gently for 15 minutes. Do not allow the liquid to boil away.
Cool the solution and neutralize it with 6 M NaOH (added slowly while stirring). Use this solution for the following tests and procedures.
Part C: Carbohydrate Qualitative Tests
Molisch test: Place one mL of a solution containing carbohydrate in a small test tube. Add two drops of α- naphthol solution and mix. Incline the test tube at about a 45° angle and let 1 mL of conc. sulfuric acid run down the side of the test tube and settle at the bottom. USE CAUTION WITH conc. H2SO4! AVOID SPILLS AND SKIN CONTACT. DISCARD WASTE IN A WASTE JAR FOR ACIDS, NOT IN THE SINK. Test with a water blank, the reference carbohydrate solutions, your original starch solution and your hydrolyzed starch solution.
Iodine test: Place one mL of test solution in a small test tube. Add one or two drops of iodine solution. Observe the color that develops in the solution. Test with a water blank, the reference carbohydrate solutions, your original starch solution and your hydrolyzed starch solution.
Benedict’s test: Place 5 mL of Benedict’s reagent in a test tube and add 10 drops of the carbohydrate test solution. Boil the solution for two minutes then cool. Record the results. Test with a water blank, the reference carbohydrate solutions, your original starch solution and your hydrolyzed starch solution.
Tollen’s test: Place 1 mL of AgNO3 solution in a clean and well-rinsed test tube. Add ammonia to the silver nitrate solution. Initially a brown precipitate of silver hydroxide will form; continue stirring in ammonia until the initial precipitate dissolves. Add 1 mL of test solution and place in a warm (but not boiling) water bath. Warning: aqueous silver ion can be reduced just by room light (the basis for back-and-white photography!). Test with a water blank, the reference carbohydrate solutions, your original starch solution and your hydrolyzed starch solution.
OMIT the derivative formation – Fall 2013
Formation of osazone derivatives: phenylhydrazine (or 2, 4-dinitrophenylhydrazine: 2,4-DNP) reacts with a carbohydrate’s carbonyl group and with hydroxyl groups adjacent to the carbonyl group to form a crystalline osazone derivative. Thus, the common monosaccharides each react with 3 equivalents of phenylhydrazine and attach the =N-NH-C6H5 group at carbons 1 and 2. Glucose, fructose and mannose all give the same osazone derivative since all differences at carbons 1and 2 are lost during the reaction while these have the same configuration at carbons 3, 4, 5 and 6. The color, appearance, time of formation of crystals, relative solubility in hot solvent and melting point of the derivatives can help to distinguish other sugars and to identify the original compound.
Note: phenylhydrazine is suspected to be a carcinogen. Do not spill the solution on your bare skin. The osazone products and the reaction solution can stain skin. Gloves are advisable when handling these test solutions. DISCARD ALL WASTE AND COMPLETED TESTS IN THE LABLED WASTE JAR! Acidify 5 mL of sugar solution with 10 drops of glacial acetic acid, add 3 drops of phenylhydrazine solution and a small scoop of sodium acetate. Warm in a water bath for 5 minutes with occasional shaking, filter, and warm the filtrate in the water bath for an additional 20 minutes. Cool slowly. Describe the appearance of any precipitate and whether it formed in hot or cool solution. Test with a water blank, the reference carbohydrate solutions, your original starch solution and your hydrolyzed starch solution. Note : 2, 4-dinitrophenylhydrazine (2, 4-DNP) forms a similar derivative of carbohydrates and may be substituted.
Part D: Hydrolysis of Starch with Salivary α-Amylase
Dilute a sample of your own saliva by collecting 0.5 mL in a clean test tube and adding 5 mL of distilled water. Pipet 1 mL samples of the unhydrolyzed starch solution into each of 5 test tubes. Add 0.5 mL of distilled water to one test tube (this is your control) and 0.5 mL of diluted saliva to each of the others. After 5 minutes, add 1 drop of iodine solution to the first (blank) and second test tubes. At five-minute intervals, add iodine solution to the remaining test tubes until the solution stops producing colored complex. If this takes less than 10 minutes or more than 20, repeat with either more dilute or more concentrated saliva solution as appropriate. The catalytic activity and concentration of α-amylase varies for each person so it may be necessary to adjust the enzyme concentration. It may be necessary to warm your test tubes up in a warm (no higher than 37 ºC) water bath. Conduct this test with one reference disaccharide and one monosaccharide of your choice.
Note: test solution with different concentration of saliva.
What are the experimental differences between a reducing sugar and a non-reducing sugar? How do the differences in behavior relate to the molecular structure? What evidence do you have for the successful hydrolysis of potato starch by a) acid and b) salivary α-amylase?
- Provide a citation for the isolation of potato starch.
- Sketch the structure of starch (draw at least 3 monomer units), label the linkage, and identify the monomer unit.
- Acid hydrolysis of starch will produce what? Provide a citation.
- What is the Molisch test? The results indicate what? Provide a citation.
- If the Molisch test is applied to the reference solutions, what are the results?
- What is the iodine test? The results indicate what? Provide a citation.
- If the iodine test is applied to the reference solutions, what are the results?
- What is the Benedict’s test? The results indicate what? Provide a citation.
- If the Benedict’s test is applied to the reference solutions, what are the results?
- What is the Tollen’s test? The results indicate what? Provide a citation.
- If the Tollen’s test is applied to the reference solutions, what are the results?
- Hydrolysis of starch with salivary α-amylase produce what product(s)? Provide a citation.
- Suppose that a fresh solution of sucrose were acidified then heated. After heating, the solution is neutralized and subjected to the same set of tests as the starch hydrolysate. Predict the results for each test on this sucrose hydrolysate.
- What was the purpose of neutralizing the starch hydrolysate before performing any tests?
- Cellulose is also a glucose homopolymer. Could we hydrolyze cellulose in a manner similar to our treatment of starch? Would we see the same results in the carbohydrate qualitative tests using a solution of cellulose that had been heated in acid?
On a new page, prepare data table(s) for the results section as part of the pre-lab.