Experiment 11: Carbohydrates (adapted with permission of Dr.

Ann Willbrand, USC-Aiken) Introduction Reducing sugars are those carbohydrates that can be oxidized by Benedict's reagent. The name reducing sugar comes from the reaction in which the aldehyde group of the carbohydrate is oxidized while Cu(II) ions in the reagent are reduced to Cu(I). A positive test with Benedict's reagent is readily discerned when the solution changes from a blue color to brick red as the reduced copper precipitates in the form of Cu2O.
CHO H HO H H OH H H HO O C OH OH H

+
OH OH CH2OH

Cu2+ blue

+
H H OH OH CH2OH

Cu2O red

In aqueous solution, most carbohydrates exist primarily in the closed hemiacetal form (A); however, the hemiacetal is always in equilibrium with the open aldehyde (B). Only a small amount of the open form is required for the sugar to give a positive test. As the open form reacts with Benedict's reagent and is oxidized to the carboxylic acid (C), more of the hemiacetal will open to replenish the supply of aldehyde. This process will continue until all of the sugar has reacted with the reagent.
CH2OH O OH OH H OH OH CH2OH OH OH H O OH OH CH2OH OH OH OH O

Cu

2+

+ Cu2O

A OH

C OH

Although the ketohexose fructose does not appear to be a reducing sugar, it readily isomerizes to glucose under the reaction conditions used for the test, so it also gives a positive test as a reducing sugar.
CH2OH O HO H H H OH OH CH2OH HO H H H OH OH H OH OH CH2OH O H O OH

H HO H H

OH H OH OH CH2OH

OH H OH OH CH2OH

Cu2+

HO H H

Fructose

Glucose

Unlike the hemiacetal group, the acetal group that joins two sugars together in a disaccharide is stable in water and aqueous base. Cleavage of an acetal only occurs by treatment with hot aqueous acid. Depending upon how the acetal in a disaccharide is formed, the disaccharide may or may not be a reducing sugar. If the hemiacetal groups from both monosaccharides are involved in the formation of the acetal linkage, then the disaccharide cannot be a reducing sugar. Sucrose is an example and is classified as a nonreducing sugar. If the hemiacetal group from only one of the monosaccharides is involved in the acetal linkage, then the other hemiacetal can still react with the reagent. Thus, a disaccharide such as maltose will give a positive test and is considered to be a reducing sugar.
CH2OH O OH OH OH O OH CH2OH O OH OH OH H OH fructose CH2OH O

H CH2OH

H , H2O

OH CH2OH

sucrose

glucoseOH

Cu2+

No reaction
CH2OH CH2OH O OH OH OH O CH2OH OH O OH OH OH

CH2OH O OH OH OH O

O OH H OH OH

Cu2+

maltose Polysaccharides containing a few sugar units will give positive results, since the concentration of hemiacetal groups is still relatively high. Large polymers of glucose, such as starch, are not reducing sugars, since the concentration of hemiacetal groups is very low. However, starches can be readily identified when tested with iodine solution. In water, iodine is yellow-brown; in the presence of starch, iodine forms a deep blue complex with the starch. This complex is not observed with monosaccharides. The acetal linkages in starch are hydrolyzed in hot aqueous acid, giving glucose. Therefore, starches can be distinguished from other reducing sugars by the distinctive reaction with iodine. A positive Benedict's test can only occur after the starch has been hydrolyzed.

Prelaboratory Activity Before coming to lab, look up the structures of glucose, fructose, lactose, maltose, sucrose, and starch; draw them on your laboratory record sheet. Laboratory Activities A. Identify models of sugars. B. Test dilute aqueous solutions of glucose, lactose, fructose, maltose, and sucrose with Benedict's reagent to determine which are reducing sugars C. Determine the effect of hydrolysis on the behavior of sucrose with Benedict's D. Test starch with iodine and with Benedict's both before and after hydrolysis Procedure A. Identifying models of sugars Numbered models of some of the sugars from the prelab activity will be found in the laboratory. Identify the sugar from the model, draw its structure on your data sheet, and write the complete name of the sugar. These sugars may be in the open or ring form. If the sugar is in the ring form, be sure to include the or orientation and the pyranose or furanose designation. To convert from a ring to a Fischer projection, do the following: draw the skeleton of the Fischer projection, placing the aldehyde on the top or the ketone second from the top; place the C6 CH2OH on the bottom; place the C-5 OH group on the right, next to the bottom; finally, place the OH group for each carbon on the right or the left, remembering that an OH that is above the ring will be on the left and an OH that is below the ring will be on the right. B. Identifying reducing and nonreducing sugars To each of 5 clean test tubes, add 1 mL (about 20 drops) of Benedict's reagent. You will test dilute solutions of glucose, fructose, maltose, lactose, and sucrose. Label your tubes; then add 5 to 10 drops of one of the sugar solutions to each tube. Place the tubes in a beaker of boiling water for 5 to 7 minutes. At the end of this time, report your results on the data sheet. Are your results consistent with your expectations? C. Hydrolysis of sucrose Place about 1 mL (about 20 drops) of dilute aqueous sucrose solution in a clean test tube. Add 10 drops of concentrated HCl and heat the tube in boiling water for 10 minutes. Cool the solution and neutralize it with 10% NaOH. It should be just alkaline to litmus paper. Treat the solution as before with Benedict's reagent. Record your results on the data sheet and compare with the prior result when unhydrolyzed sucrose was treated with Benedict's. D. Hydrolysis of starch Place about 1 mL of starch solution in each of 4 numbered test tubes. Add 1 mL Benedict's reagent to tube # 1 and heat in boiling water for 10 minutes. What do you expect to happen? To test tubes # 2 and # 3, add 10 drops of concentrated HCl and heat in boiling water for 10 minutes. What will happen to each of these samples? Tube # 4 will serve as the control. Heat it in boiling water for 10 minutes, without adding any Benedict's or HCl. Cool all of the tubes. Neutralize the solutions in tubes 2, 3, and 4 with NaOH solution until just alkaline. Add 1 mL of Benedict's reagent to tube # 2, and reheat it for several minutes. Add about 20 drops of iodine solution to tubes 3 and 4. Record all your observations on the data sheet.

Experiment 11 Laboratory Record Prelab activity: Structures of carbohydrates Glucose Fructose

Name:______________________________ Date:_______________________________

Lactose

Maltose

Sucrose

Starch

A. Carbohydrate Models Model # 1 Name Structure

B and C. Benedict's tests Carbohydrate Glucose Fructose Lactose Maltose Sucrose Sucrose (after hydrolysis) D. Starch Tests. Indicate which conditions apply to each tube by putting a check in the appropriate boxes. Then record your observations and any conclusions (for example, does the tube contain a reducing sugar? Does it contain intact starch? Did it react with iodine?) HCl Tube # 1 NaOH Benedict's Iodine Observations Conclusions Predicted Result Observations Before Heating Observations After Heating Reducing or Nonreducing?

Tube # 2

Tube # 3

Tube # 4

Questions 1. Write the reaction of Benedict's reagent for any two of your positive tests.

2. Write the hydrolysis reaction for sucrose, using structures.

3. Why does sucrose give a positive Benedict's test after hydrolysis?

4. Examine your results for the starch tests in part D to answer the questions below. a. In which tubes did hydrolysis occur? How do you know?

b. Which tubes contained reducing sugars? How do you know?

c. Can the same sample give a positive Benedict's test and a positive iodine test? Why or why not?

5. a. What is the charge on the copper ions in Benedict's reagent?

b. What is the charge on copper in the copper oxide product?

c. Is Benedict's reagent oxidized or reduced when it reacts with glucose? Explain.