Materials Included In Kit

Additional Materials Required

Prelab Preparation

Starch Solution: Make a paste of 1 g of soluble starch with a small amount of water and add the paste to 100 mL of boiling water. Stir the mixture until it appears homogeneous and allow the colloidal starch solution to cool slowly to room temperature before using.

Carbohydrate Solutions: Prepare 1% carbohydrate solutions by adding 100 mL of distilled water to each carbohydrate sample bottle. Randomly assign the unknown letter codes A–E to these samples. Use the labels provided with this kit to make “unknown” labels A–E and conceal the identity of each solution. Place one “unknown” label on each carbohydrate solution. Be sure to record your assignments in your notes before placing the corresponding “unknown” label on the bottle!

Safety Precautions

Iodine solution contains iodine and potassium iodide and is an eye and skin irritant. Benedict’s solution contains copper sulfate and sodium carbonate; it is moderately toxic by ingestion and a skin and body tissue irritant. Barfoed’s solution contains copper acetate and acetic acid; it is moderately toxic by ingestion and a skin and body tissue irritant. The Seliwanoff reagent consists of resorcinol, which is toxic by ingestion, in hydrochloric acid. It is a corrosive liquid. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All carbohydrate samples can be flushed down the drain with excess water according to Flinn Suggested Disposal Method #26b. The waste solutions remaining after each classification test should be collected in separate containers. The waste iodine test solutions can be disposed of according to Flinn Suggested Disposal Method #12a. The waste Benedict’s and Barfoed’s test solutions can flushed down the drain with excess water according to Flinn Suggested Disposal Method #26b. Waste Seliwanoff test solutions can be disposed of according to Flinn Suggested Disposal Method #24b.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The experimental work for this lab can reasonably be completed in one 50-minute lab period.
• The pre-lab activity is included to allow students to determine which unknown carbohydrate is revealed using each of the four classification tests. Students need this information to follow the procedure and to assign the identities of the unknown carbohydrates. The prelab activity may be assigned as homework in preparation for the lab or may be used the basis of a cooperative class discussion prior to lab.
• To avoid cross-contamination of samples, consider dispensing beforehand smaller amounts of each solution for each lab table to use separately.
• Positive results for Benedict’s test range from green to yellow to orange in color, depending on the nature of the carbohydrate, the temperature of the bath, and the reaction time. In each case, however, a reddish precipitate will be evident. Negative test results do not contain a precipitate.
• Both the Barfoed and the Seliwanoff tests require the students to time the reactions. The specified time periods must be carefully followed in order to avoid getting false positive results for disaccharides, in the case of Barfoed’s test, and aldoses, in the case of the Seliwanoff test.
• Would you like to test common foods for the presence of specific carbohydrates? The volume of each test solution included in this kit is sufficient to allow each pair of students to take one food sample through the entire sequence of classification tests, if desired. Samples that might be tested include milk, fruit and vegetable juices (choose light-colored ones, such as apple juice), soda pop (again, colorless is better) and honey or syrup. All of these are more concentrated than the carbohydrate solutions tested in this activity. They should be diluted prior to testing—a good rule of thumb is 1 mL diluted to 50 mL with water. Solid foods (e.g., crackers, cereals, raw fruits and vegetables) may also be tested by mashing them first in a mortar with pestle and adding water to obtain an “extract.”

Answers to Prelab Questions

Sample Data

Carbohydrate identities were assigned as shown in the first and last columns. The samples were removed from further testing once their identities had been revealed by a prior test in the sequence (the test boxes are then shaded).

Answers to Questions

1. Maltose, a product of partial digestion of starches, is a disaccharide composed of two glucose units. It is a reducing sugar. In your mind, take maltose through the sequence of classification tests used in this experiment. Would you be able to distinguish maltose from lactose in an unknown sample?

   Maltose would give a negative result in the Iodine test (it is not a starch). As a reducing sugar, it should give a positive result with Benedict’s solution. Since it is a disaccharide and not a monosaccharide, however, it gives a negative Barfoed’s test. Finally, since maltose is composed only of glucose units joined together, it is an aldose and will give a negative test result with the Seliwanoff reagent. This is the same pattern of results that would be observed with lactose. It is not possible to distinguish maltose and lactose with the classification tests used in this experiment.

2. When disaccharides are heated in water in the presence of a strong acid, the linkage joining the two monosaccharide components is “broken” (a reaction called hydrolysis). Using this information, explain why sucrose might give a false positive result with the Seliwanoff reagent.

   The Seliwanoff reagent contains strong acid (3 M HCl). If a test solution of sucrose were heated with this reagent, the linkage joining the glucose and fructose units would be broken. The fructose would be released into solution and would give a positive test result with the reagent. This is one reason why the directions for running the Seliwanoff test call for heating the solution for a short period of time only (to avoid hydrolysis).

3. Prior to the advent of more accurate test-strip methods to analyze the amount of glucose in urine (a test for diabetes), the presence of glucose in urine was routinely detected using Clinitest™ tablets. These tablets contain all of the solid reagents needed for Benedict’s test. Do you think this is a good method of testing for glucose?

   Benedict’s test is not a specific test for glucose. All reducing sugars would give positive test results with Clinitest tablets. Since all monosaccharides and most disaccharides are reducing sugars, the presence of a wide variety of carbohydrates would give false positive readings. Clinitest tablets do provide a valuable general screening tool, however, because the presence of other carbohydrates in urine could be symptoms of other diseases.

4. In the Background section, it was stated that fructose is used as a “lower calorie and lower cost sweetener than table sugar.” Explain how and why this statement might be true.

   Since fructose is about 30% sweeter per gram than table sugar, a desired level of sweetness can be achieved using fewer grams of fructose compared to sucrose. Since the calorie content of all sugars is about the same (4 Calories per gram), food sweetened with fructose would supply fewer calories than food sweetened with table sugar. Fructose is a lower cost sweetener than table sugar only if the costs of the two sugars are similar (using fewer grams of fructose costs less than using more grams of table sugar). Of course, all of these observations would no longer be true if more fructose were added to make the foods even sweeter than they would be with added table sugar!

References

Mahlerbe, J. S. and Meyer, C. J. J. Chem. Ed. 1999, 76, pp 80–81.

Introduction

What is a carbohydrate? What are the roles of carbohydrates in energy, metabolism and cell structure? Explore the structure and properties of different types of carbohydrate molecules and learn how they can be identified and analyzed.

Concepts

• Carbohydrates
• Monosaccharide vs. disaccharide vs. polysaccharide
• Classification tests
• Reducing vs. nonreducing sugar

Background

Carbohydrates are the most abundant biological molecules. It is estimated that more than 50% of the total carbon content on the Earth is present in the form of carbohydrate compounds. The term carbohydrate dates back to the 1800s, when it was found that the molecular formulas of the simplest carbohydrates (called monosaccharides) could be expressed in the form (CH₂O)_n. Glucose and fructose, for example, both have the formula C₆H₁₂O₆ and can be expressed as (CH₂O)₆. Carbohydrates appeared to consist of “hydrates” of carbon. Carbohydrates are often referred to as sugars, a more common name that reflects the fact that many carbohydrates are naturally occurring sweeteners.

The biological properties of carbohydrates are usually divided into two categories. Many carbohydrates play an important role in meeting the energy requirements of cells. In this role, they function either as an immediate source of energy or as a means of storing chemical energy for future use. Other carbohydrate molecules serve a structural role within cells and organisms. Carbohydrate molecules are the primary components of the cell wall in both plants and bacteria.

The simplest carbohydrates, as noted above, are called monosaccharides. The two most common monosaccharides are glucose (also called dextrose) and fructose (“fruit sugar”). Monosaccharides are the fundamental units, or building blocks, that make up all other carbohydrate molecules. When two monosaccharide units are joined together, they form disaccharides. Examples of disaccharides include sucrose (cane or table sugar) and lactose (“milk sugar”). Polysaccharides are huge organic molecules— called polymers—composed of hundreds or thousands of monosaccharides joined together. The most familiar polysaccharides are starch and cellulose.

Glucose is the most abundant monosaccharide in the body. It is the chemical “fuel” that is carried in the bloodstream to tissues as an energy source for metabolism. All other carbohydrates which are absorbed by the body must first be converted to glucose prior to metabolism. Fructose is the most abundant carbohydrate in many fruits. Honey is a 1:1 mixture of glucose and fructose. Although glucose and fructose share the same molecular formula, the arrangements of the atoms in these two molecules are different, giving them different structures and biological properties. Fructose is the sweetest sugar—about 30% sweeter on a per gram basis than table sugar. For this reason, fructose is widely used as a lower calorie and lower cost sweetener than table sugar. Many soft drinks and fruit drinks are sweetened with “high fructose corn syrup.”

Sucrose, or table sugar, is a disaccharide composed of a glucose unit joined chemically to a fructose unit. Harvested from sugar cane or sugar beets, sucrose is also called cane sugar. It is a widely used refined sugar in a typical Western diet. Lactose is a disaccharide composed of a glucose unit joined chemically with a different monosaccharide, called galactose. Lactose constitutes about 4–5% of cow’s milk and about 7–8% of human breast milk. A specific enzyme called lactase is required for the digestion of lactose (to break the linkage joining the glucose and galactose units). People who lack this enzyme are said to be lactose intolerant— they cannot digest milk or milk products.

The polysaccharide starch is composed of thousands of glucose units joined together to form a large and complex molecule. Found in storage vacuoles in plants, starch is the principal way in which plants store chemical energy for fuel.

Classification Tests

With so many different carbohydrates found in nature, and their diverse biological functions, how can scientists identify and analyze carbohydrates in the laboratory? Although carbohydrate molecules share many common structural features, the sometimes subtle structural differences among them give rise to unique chemical properties as well. These chemical differences are the basis of a variety of classification tests that have been developed to identify carbohydrates.

Reaction with iodine serves as a classification test to identify the polysaccharide starch. Because of its large and complex structure, starch has a unique ability to bind iodine molecules to form intense blue-colored complexes. Other carbohydrates do not form complexes with iodine.

Benedict’s test is a classification test that is used to identify reducing sugars, which include ALL monosaccharides and most disaccharides (excluding sucrose). In contrast, all polysaccharides are nonreducing sugars. Benedict’s test depends on the ability of certain types of carbohydrate molecules to undergo oxidation upon treatment with CuSO₄ in basic solution (pH >12). Oxidation and reduction reactions always occur in pairs. When a carbohydrate molecule is oxidized, another reagent, for example, Cu²⁺ ion in the case of Benedict’s solution, must be reduced. This is the origin of the term reducing sugar. Thus, a positive Benedict’s test result is marked by the disappearance of the bright blue color due to Cu²⁺ ion and the appearance of a reddish precipitate consisting of reduced copper(I) oxide (Cu₂O). A few disaccharides are unique in that, in contrast to most other disaccharides, they do not reduce Benedict’s solution. The most common nonreducing disaccharide is sucrose. Because of the way in which the two monosaccharide components are joined together in sucrose, it is not readily oxidized and hence does not reduce Benedict’s solution.

Benedict’s solution is a strong oxidizing agent that gives positive test results with all reducing sugars. Not all reducing sugars react at the same rate, however, with different oxidizing agents—disaccharides are considerably less reactive than monosaccharides. Reducing sugars can be classified as mono- versus disaccharides by reacting them with a weaker oxidizing agent, such as copper acetate in acetic acid. This classification test is called Barfoed’s test. A positive Barfoed’s test result is similar to that observed with Benedict’s solution. In the case of Barfoed’s test, however, an unknown reducing sugar is allowed to react for a only a specified, short period of time. Monosaccharides give positive test results within 2–3 minutes, whereas disaccharides give negative results under the same conditions.

The Seliwanoff test is used to distinguish between different types of monosaccharides, specifically, aldoses and ketoses. Glucose and fructose share the same molecular formula, C₆H₁₂O₆. The only difference between them lies in the nature of the organic functional group they contain. Glucose is an aldose—it contains an aldehyde functional group

Fructose is classified as a ketose—it contains a ketone functional group This difference is sufficient to give the compounds different properties in at least one chemical reaction, the ability to lose water upon treatment with strong acid. Ketoses readily lose water upon heating with 3 M HCl for 2–3 minutes. The resulting products react with another compound, called resorcinol, in the Seliwanoff test to form red-colored complexes. Aldoses do not react under the same conditions. A color change from colorless to red in the Seliwanoff test serves as a positive result to identify ketoses, such as fructose.

Experiment Overview

Five carbohydrate samples, labeled A–E, are provided. The identities of these carbohydrate samples, starch, glucose, fructose, lactose, and sucrose, have been scrambled. The carbohydrate “code” can be unscrambled and the sample identities determined by performing four classification tests in sequence. A “blank” sample—distilled water, which always gives negative test results—will be included in each test in order to tell the difference between a positive and negative test result. As each classification test is performed in sequence (see the Prelab Activity), the identity of one of the unknown samples will become known. This sample is then removed from the number of samples that must be carried over to the next classification test in the sequence. As a result, although the first test, the Iodine test, must be carried out on six samples (five carbohydrate unknowns plus the blank), the final test should only need to be run on three samples (the remaining two carbohydrate unknowns and the distilled water blank).

Materials

Prelab Questions

Complete the following Flow Chart Diagram to show how the identities of the unknown carbohydrate samples will be revealed using a sequence of four classification tests.

Safety Precautions

Iodine solution contains iodine and potassium iodide and is an eye and skin irritant. Benedict’s solution contains copper sulfate and sodium carbonate; it is moderately toxic by ingestion and a skin and body tissue irritant. Barfoed’s solution contains copper acetate and acetic acid; it is moderately toxic by ingestion and a skin and body tissue irritant. The Seliwanoff reagent consists of resorcinol, which is toxic by ingestion, in hydrochloric acid. It is a corrosive liquid. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Preparation

Prepare a boiling water bath for use in Parts B–D. Fill a 250-mL beaker half full with water. Add a boiling stone and heat the water to a gentle boil using a hot plate or a Bunsen burner.

Part A. Iodine Test

1. Obtain and label 6 test tubes for the five unknown carbohydrate solutions A–E plus a blank (distilled water).
2. Add 1 mL of each sample to be tested to the corresponding labeled test tube.
3. Add 2–3 drops of iodine solution to each test tube. Record the color of each solution in the data table. Note whether each result is positive or negative.
4. In the data table, record the identity of the unknown that has been revealed using this classification test. This unknown can be removed from further testing in Parts B–D.
5. Dispose of the test tube contents in the appropriate iodine waste container, as directed by the instructor. Wash the test tubes and rinse them with distilled water.

Part B. Benedict’s Test

6. Label 5 test tubes as needed with the letters of the samples remaining to be tested and identified, including the blank.
7. Place 1 mL of each sample to be tested in the corresponding labeled test tube.
8. Add 2 mL of Benedict’s solution to each test tube and place the test tubes in the boiling water bath.
9. After 2–3 minutes, remove the test tubes from the bath using a test tube holder. Record the color and appearance of each sample in the data table. Note whether each result is positive or negative for the presence of a reducing sugar.
10. In the data table, record the identity of the unknown that has been revealed using this classification test. Remember, this unknown can be removed from further testing in Parts C and D.
11. Dispose of the test tube contents down the drain with plenty of water. Wash the test tubes and rinse them with distilled water.

Part C. Barfoed’s Test

12. Label 4 test tubes as needed with the letters of the samples remaining to be tested and identified, including the blank.
13. Place 1 mL of each sample to be tested in the corresponding labeled test tube.
14. Add 3 mL of Barfoed’s Reagent to each test tube and place the test tubes in the boiling water bath.
15. After exactly 2 minutes, remove the test tubes from the bath using a test tube holder. Record the color and appearance of each sample in the data table. Note whether each result is positive or negative for the presence of a monosaccharide.
16. In the data table, record the identity of the unknown that has been revealed using this classification test. Remember, this unknown can be removed from further testing in Part D.
17. Dispose of the test tube contents down the drain with plenty of water. Wash the test tubes and rinse them with distilled water.

Part D. Seliwanoff Test

18. Label 3 test tubes as needed with the letters of the samples remaining to be tested and identified, including the blank.
19. Place 2 mL of the Seliwanoff reagent in each test tube.
20. Add 2 drops of each sample to be tested in the corresponding labeled test tube and place the test tubes in the boiling water bath.
21. After 5–6 minutes, remove the test tubes from the bath using a test tube holder. Record the color of each sample in the data table. Note whether each result is positive or negative for the presence of a ketose.
22. In the data table, record the identity of the unknown that has been revealed using this classification test.
23. The final unknown can be identified from the list after the first four have been eliminated. Record the identity of the final unknown in the data table.
24. Dispose of the test tube contents in the appropriate waste container, as directed by the instructor. Wash the test tubes and rinse them with distilled water.

Student Worksheet PDF

13371_Student1.pdf