Materials Included In Kit

Additional Materials Required

Prelab Preparation

Carbohydrate solutions, 1%: Prepare 1% carbohydrate solutions by adding 100 mL of distilled water to 1 g of each carbohydrate. Randomly assign the unknown letter codes A–E to these samples. Place one “unknown” label on each carbohydrate solution. Be sure to record the assignments in your notes before placing the corresponding “unknown” label on the bottle! Prepare these solutions within one week of use.

Safety Precautions

Iodine solution contains iodine and potassium iodide and is an eye and skin irritant. Benedict’s solution contains copper sulfate, sodium citrate 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 harmful if swallowed, in hydrochloric acid, a corrosive liquid. It causes severe skin and eye burns and may cause respiratory irritation. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Please view current Safety Data Sheets for additional safety, handling and disposal information. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory.

Disposal

Please consult your current Flinn Scientific Catalog and Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Carbohydrate solutions may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b. Leftover solutions remaining after the iodine and Seliwanoff classification tests should be collected in separate containers. Iodine test solutions may be reduced with sodium thiosulfate according to Flinn Suggested Disposal Method #12a. Leftover Seliwanoff test solutions may be neutralized with base according to Flinn Suggested Disposal Method #24b. Benedict’s and Barfoed’s test solutions may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b.

Lab Hints

• This experiment may be completed within a typical 2-hour lab period.
• The Preaboratory Assignment is designed to help students determine which unknown carbohydrate is revealed using each of the four classification tests. Students will need this information to follow the procedure and assign the identities of the unknown carbohydrates.
• To avoid cross-contamination of samples, consider dispensing smaller amounts of each solution for each lab bench to use separately. To avoid students sharing their results, provide different unknown assignments for each group.
• 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 students to time the reactions. The specified times 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.
• The Seliwanoff reagent contains 3 M HCl, which will hydrolyze the ketal linkage in sucrose upon prolonged heating. It may also cause the isomerization and/or interconversion of different monosaccharides.
• Lactose intolerance may be demonstrated by setting up four Erlenmeyer flasks with attached balloons. Place lactose (solution) and yeast in the first; glucose and yeast in the next; galactose and yeast in the third, and lactose, yeast and Lactaid in the last flask. Only the second and fourth flasks will show evidence of gas evolution due to fermentation of glucose by yeast.
• Compare and contrast the structures of starch and cellulose to illustrate how small structural changes can lead to large changes in the biological roles of carbohydrates. Both starch and cellulose consist of thousands of glucose units joined together. Glucose units are joined together via α-1,4-linkages in starch and via β-1,4-linkages in cellulose. The difference between them is the spatial orientation of the linkage joining the monosaccharide units together. This seemingly small structural change makes cellulose totally indigestible to humans! (Termites, on the other hand, thrive by digesting celulose.)

Further Extensions

As an extension, consider testing common foods for the presence of specific carbohydrates. Each pair of students could 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—such as crackers, cereals, raw fruits and vegetables—may also be tested by mashing them first in a mortar with a pestle and then adding water to obtain an “extract.” The results of testing different foods may be shared as part of a collaborative class discussion. All food-grade items that are brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the chemistry laboratory and do not remove any remaining food items from the lab after use.

Answers to Prelab Questions

Complete the following flow chart to show how the identities of the unknown carbohydrate samples can be revealed using a sequence of four classification tests.

Sample Data

Laboratory Report 

*Note: Carbohydrate identities were assigned as shown in the first and last columns. Samples were removed from further testing once their identities were revealed by a prior test in the sequence (those test boxes are shaded).

Answers to Questions

Laboratory Report

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. Fill in the following blanks to identify the reactants that undergo oxidation and reduction, respectively, when glucose reacts with Benedict’s solution. Name the oxidizing and reducing agents involved.

   Substance oxidized: ___Glucose___
   Oxidizing agent: ___Cu²⁺ ions___
   Substance reduced: ___Cu²⁺ ions___
   Reducing agent: ___Glucose___

4. Prior to the advent of more accurate enzyme methods to analyze the amount of glucose in urine (a test for diabetes), the presence of glucose was routinely detected using tablets containing the solid reagents needed for Benedict’s test. Describe a disadvantage of this method of testing for glucose.

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

5. 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, these observations might no longer be valid if more fructose were added to make the foods even sweeter than they would be with added table sugar!

6. Glucose is classified as an aldose because it contains an aldehyde functional group in its open-chain form. In aqueous solution and in cells, however, the aldehyde group undergoes internal cyclization with the C₅–OH group to form the cyclic structure shown below and in Figure 1. The open-chain and cyclic structures are in equilibrium, with > 97% of the molecules typically in cyclic form. Use Le Chatelier’s principle to explain why all glucose molecules will react with Cu²⁺ ions, even though Cu²⁺ reacts only with the aldehyde functional groups.
   {14048_Answers_Reaction_1}
   As Cu²⁺ ions react with and remove the aldehyde from solution, the equilibrium shifts to the left, according to Le Chatelier’s principle, to generate more of this reactant and restore the equilibrium ratio. This process continues until eventually all of the glucose molecules have reacted.

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 carbohydrates and learn how they can be identified.

Concepts

• Carbohydrates
• Monosaccharides
• Disaccharides
• Polysaccharides
• Classification tests
• Reducing vs. nonreducing sugars

Background

Carbohydrates are the most abundant biological compounds. The term carbohydrate dates back to the 1800s, when it was found that the formulas of the simplest carbohydrates could be expressed in the form (CH₂O)_n. Both glucose and fructose, for example, have the formula C₆H₁₂O₆ and can be written as (CH₂O)₆. Carbohydrates appeared to consist of “hydrates” of carbon (C•H₂O)_n. Carbohydrates are also referred to as sugars, a 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 a primary role in meeting the energy requirements of cells, either as an immediate source of energy or as a means of storing energy for future use. Other carbohydrates serve a structural role within cells—carbohydrates are the primary components of the cell wall in both plants and bacteria.

The simplest carbohydrates are called monosaccharides. The two most common monosaccharides are glucose (also called dextrose) and fructose. Monosaccharides are the fundamental units, or building blocks, that make up all other carbohydrates. When two monosaccharide units are joined together, they form disaccharides. Examples of disaccharides include sucrose and lactose. Polysaccharides are huge organic molecules—called polymers—composed of hundreds or thousands of monosaccharides joined together. The most familiar polysaccharides are starch and cellulose. See Figures 1–3 for the structures of key carbohydrates used in this activity.

Glucose (“blood sugar”) is the most abundant monosaccharide in the human body. It is the chemical fuel carried in the bloodstream to tissues as an energy source for metabolism. Other carbohydrates that are absorbed by the human body must be converted to glucose prior to metabolism. Fructose is the most abundant carbohydrate in fruits. Honey is a 1:1 mixture of glucose and fructose. Although glucose and fructose have the same molecular formula, their structures are different and they have different properties. Fructose is the sweetest sugar—about 30% sweeter per gram than table sugar—and is widely used as a low cost sweetener. 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 and a fructose unit joined together. Harvested from sugar cane or sugar beets, and also called cane sugar, sucrose is the most common refined sugar in a typical Western diet. Lactose is a disaccharide composed of glucose and a second monosaccharide called galactose. Lactose (“milk sugar”) constitutes about 4–5% of cow’s milk and 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. Found in storage vacuoles in plants, starch is the principal way in which plants store chemical energy.

Chemical Tests for Classifying Carbohydrates
The structural and chemical differences among carbohydrates are the basis for a series of classification tests that have been developed to identify carbohydrates in the lab.

• Reaction with iodine is used as a classification test to identify the polysaccharide starch. Starch binds iodine molecules to form dark blue–colored complexes. Other carbohydrates do not react with iodine.
• Benedict’s test is used to identify reducing sugars, which include all monosaccharides and most disaccharides (excluding sucrose). In contrast, all polysaccharides are nonreducing sugars. Benedict’s solution contains copper(II) sulfate dissolved in strong base. Cu²⁺ ions will oxidize carbohydrate molecules, a process that results in Cu²⁺ ions being reduced to copper(I). This is the origin of the term reducing sugar. A positive Benedict’s test is marked by the disappearance of the blue color due to Cu²⁺ ions and the appearance of a red precipitate for copper(I) oxide, Cu₂O. Not all disaccharides will reduce Benedict’s solution. The most common nonreducing disaccharide is sucrose. Benedict’s solution 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 using Barfoed’s solution, a weaker oxidizing agent consisting of copper(II) acetate in acetic acid. A positive Barfoed’s test gives similar observations as Benedict’s solution. Monosaccharides give positive Barfoed’s test results within 2–3 minutes, while disaccharides do not react under the same conditions.
• The Seliwanoff test is used to distinguish between different types of monosaccharides, specifically, aldoses and ketoses. Glucose and fructose have the same formula, C₆H₁₂O₆. They differ in the nature of one organic functional group they contain. Glucose is an aldose—it contains an aldehyde functional group in a cyclic hemiacetal structure. Fructose is a ketose—it contains a ketone functional group in a cyclic hemiketal structure. Ketoses react with hydrochloric acid and resorcinol, which are components in the Seliwanoff reagent, to form red products. 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

The purpose of this experiment is to investigate the structures and properties of carbohydrates and apply this knowledge to identify unknowns. Five carbohydrate samples, labeled A–E, are provided. The identities of these compounds, starch, glucose, fructose, lactose and sucrose, have been scrambled. The carbohydrate “code” can be unscrambled 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 a negative test result. As each classification test is performed in sequence (see the Prelaboratory Assignment), the identity of one of the unknowns should 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. Thus, the first test (iodine) must be carried out on six samples (five carbohydrate unknowns plus the blank), but the final test should only need to be run on three samples (the last two carbohydrate unknowns and a distilled water blank).

Materials

Prelab Questions

Complete the following flow chart to show how the identities of the unknown carbohydrate samples can 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, sodium citrate 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 harmful if swallowed, in hydrochloric acid, a corrosive liquid. It causes severe skin and eye burns and may cause respiratory irritation. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Before beginning the first test, prepare a boiling water bath. 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 at a medium setting.

Iodine Test

1. Obtain and label 6 test tubes for the five unknown carbohydrates A–E plus a blank (distilled water).
2. Add 1 mL of each sample to be tested to the correspondingly labeled test tube.
3. Add 2–3 drops of iodine solution to each test tube. Record the color of each solution and note whether each result is positive or negative.
4. Record the identity of the unknown that has been revealed using this classification test. This unknown can be removed from further testing.
5. Pour the contents of the test tubes into an appropriately labeled container for reduction of any leftover iodine, as directed by the instructor. Wash the test tubes and rinse with distilled water.

Benedict’s Test

6. Label 5 test tubes as needed with the letters of the samples remaining to be tested, including a blank.
7. Place 1 mL of each sample to be tested in the correspondingly 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 clamp. Record the color and appearance of each sample and note whether each result is positive or negative for the presence of a reducing sugar.
10. Record the identity of the unknown that has been revealed using this classification test. This unknown can be removed from further testing.
11. Rinse the test tube contents down the drain with plenty of water. Wash the test tubes and rinse with distilled water.

Barfoed’s Test

12. Label 4 test tubes as needed with the letters of the samples remaining to be tested, including a blank.
13. Place 1 mL of each sample to be tested in the correspondingly labeled test tube.
14. Add 3 mL of Barfoed’s solution 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 clamp. Record the color and appearance of each sample and note whether each result is positive or negative for the presence of a monosaccharide.
16. Record the identity of the unknown that has been revealed using this classification test and remove this sample from further testing.
17.  Rinse the test tube contents down the drain with plenty of water. Wash the test tubes and rinse with distilled water.

Seliwanoff Test

18. Label 3 test tubes as needed with the letters of the samples remaining to be tested, including a 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 correspondingly 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 clamp. Record the color of each sample and note whether each result is positive or negative for the presence of a ketose.
22. 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 its identity.
24. Pour the contents of the test tubes into an appropriately labeled container for neutralization of the acidic solution, as directed by the instructor. Wash the test tubes and rinse with distilled water.

Student Worksheet PDF

14048_Student1.pdf