Tests for Carbohydrates

Goals

Observe physical and chemical properties of some common carbohydrates.

Use physical and chemical tests to distinguish between monosaccharides, disaccharides, and
polysaccharides.
Identify an unknown carbohydrate.
Relate the process of digestion to the hydrolysis of carbohydrates.

Discussion
A.

Benedicts Test for Reducing Sugars

All of the monosaccharides and most of the disaccharides can be oxidized. When the cyclic structure
opens, the aldehyde group is available for oxidation. Benedicts reagent contains Cu2+ ion that is reduced.
Therefore, all the sugars that react with Benedicts reagent are called reducing sugars. Ketoses also act as
reducing sugars because the ketone group on carbon 2 isomerizes to give an aldehyde group on carbon 1.
H
CH 2 OH
C

OH

Aldose (can be oxidized)

Ketose

When oxidation of a sugar occurs, the Cu2+ is reduced to Cu+, which forms a red precipitate
of cuprous oxide, Cu2O(s). The color of the precipitate varies from green to gold to red depending on
the concentration of the reducing sugar.
O

sugar

CH

Reducing sugar

OH

2Cu 2 +
Blue

sugar

COH

Oxidized sugar

Cu 2 O(s)
Blue

Sucrose is not a reducing sugar because it cannot revert to the open-chain form that would
provide the aldehyde group needed to reduce the cupric ion.
CH 2 OH
O
1

OH
HO

OH
1,2-Glycosidic bond

O
HOCH 2

These carbons cannot open.

Sucrose is not a reducing sugar.

O
HO

CH 2 OH

OH
Sucrose

From Laboratory Manual for General, Organic, and Biological Chemistry, Karen C. Timberlake. Copyright 2007 by Pearson
Education, Inc. Published by Benjamin Cummings. All rights reserved.

Tests for Carbohydrates

B.

Seliwanoffs Test for Ketoses

Seliwanoffs test is used to distinguish between hexoses with a ketone group and hexoses that are
aldehydes. With ketoses, a deep red color is formed rapidly. Aldoses give a light pink color that takes
a longer time to develop. The test is most sensitive for fructose, which is a ketose.
C.

Fermentation Test

Most monosaccharides and disaccharides undergo fermentation in the presence of yeast. The products
of fermentation are ethyl alcohol (CH3CH2OH) and carbon dioxide (CO2). The formation of bubbles
of carbon dioxide is used to confirm the fermentation process.
C6H12O6
Glucose

yeast

2C2H5OH + 2CO2(g)
Ethanol

Although enzymes are present for the hydrolysis of most disaccharides, they are not available for lactose. The enzymes needed for the fermentation of galactose are not present in yeast. Lactose and
galactose give negative results with the fermentation test.
D.

Iodine Test for Polysaccharides

When iodine (I2) is added to amylose, the helical shape of the unbranched polysaccharide traps iodine
molecules, producing a deep blue-black complex. Amylopectin, cellulose, and glycogen react with
iodine to give red to brown colors. Glycogen produces a reddish-purple color. Monosaccharides and
disaccharides are too small to trap iodine molecules and do not form dark colors with iodine.
E.

Hydrolysis of Disaccharides and Polysaccharides

Disaccharides hydrolyze in the presence of an acid to give the individual monosaccharides.

Sucrose + H2O

H+

Glucose + Fructose

In the laboratory, we use water and acid to hydrolyze starches, which produce smaller saccharides
such as maltose. Eventually, the hydrolysis reaction converts maltose to glucose molecules. In the
body, enzymes in our saliva and from the pancreas carry out the hydrolysis. Complete hydrolysis
produces glucose, which provides about 50% of our nutritional calories.
H+ or
H+ or
H+ or
amylase
amylase
maltase
Amylose, amylopectin
maltose

many D-glucose units

dextrins

F.

Testing Foods for Carbohydrates

Several of the tests such as the iodine test can be carried out with food products such as cereals, bread,
crackers, and pasta. Some of the carbohydrates we have discussed can be identified.

Lab Information

Time:
3 hr
Comments:
Tear out the report sheets and place them next to the matching procedures. .
Related Topics: Carbohydrates, hemiacetals, aldohexoses, ketohexoses, reducing sugars, fermentation

Tests for Carbohydrates

Experimental Procedures
A.

Benedicts Test for Reducing Sugars

Materials: Test tubes, 400-mL beaker, droppers, hot plate or Bunsen burner, 5- or 10-mL
graduated cylinder, Benedicts reagent, 2% carbohydrate solutions: glucose, fructose,
sucrose, lactose, starch, and an unknown
Place 10 drops of solutions of glucose, fructose, sucrose, lactose, starch, water, and unknown in
separate test tubes. Label each test tube. Add 2 mL of Benedicts reagent to each sample. Place the
test tubes in a boiling water bath for 34 minutes. The formation of a greenish to reddish-orange
color indicates the presence of a reducing sugar. If the solution is the same color as the Benedicts
reagent in water (the control), there has been no oxidation reaction. Record your observations.
Classify each as a reducing or nonreducing sugar.

B.

Seliwanoffs Test for Ketoses

Materials: Test tubes, 400-mL beaker, droppers, hot plate or Bunsen burner, 5- or 10-mL
graduated cylinder, Seliwanoffs reagent, 2% carbohydrate solutions: glucose,
fructose, sucrose, lactose, starch, and an unknown
Place 10 drops of solutions of glucose, fructose, sucrose, lactose, starch, water, and unknown in
separate test tubes. Add 2 mL of Seliwanoffs reagent to each. The reagent contains concentrated
HCl. Use carefully.
Place the test tubes in a boiling hot water bath and note the time. After 1 minute, observe the colors
in the test tubes. A rapid formation of a deep red color indicates the presence of a ketose. Record
your results as a fast color change, slow change, or no change.

C.

Fermentation Test
Materials: Fermentation tubes (or small and large test tubes), bakers yeast, 2% carbohydrate
solutions: glucose, fructose, sucrose, lactose, starch, and an unknown
Fill fermentation tubes with a solution of glucose, fructose, sucrose, lactose, starch, water, and
unknown. Add 0.2 g of yeast to each and mix well. See Figure 1.

Figure 1

Fermentation tube filled with a carbohydrate solution

Tests for Carbohydrates

If fermentation tubes are not available, use small test tubes placed upside down in larger test tubes.
Cover the mouth of the large test tube with filter paper or cardboard. Place your hand firmly over
the paper cover and invert. When the small test tube inside has completely filled with the mixture,
return the larger test tube to an upright position. See Figure 2.
Invert small tube

Figure 2

Invert to fill small tube

Turn large tube upright

Test tubes used as fermentation tubes

Set the tubes aside. At the end of the laboratory period, and again at the next laboratory period,
look for gas bubbles in the fermentation tubes or inside the small tubes. Record your observations.
See Figure 3.
CO2 bubble

Figure 3

D.

Fermentation tubes with CO2 bubbles

Iodine Test for Polysaccharides

Materials: Spot plate or test tubes, droppers, iodine reagent, 2% carbohydrate solutions in
dropper bottles: glucose, fructose, sucrose, lactose, starch, and an unknown
Using a spot plate, place 5 drops of each solution of glucose, fructose, sucrose, lactose, starch,
water, and unknown in the wells. (If you do not have a spot plate, use small test tubes.) Add 1 drop
of iodine solution to each sample. A dark blue-black color is a positive test for amylose in starch.
A red or brown color indicates the presence of other polysaccharides. Record your results.
Complete the table to identify your unknown.

E.

Hydrolysis of Disaccharides and Polysaccharides

Materials: Test tubes, 10-mL graduated cylinder, 400-mL beaker (boiling water bath), hot plate
or Bunsen burner, spot plate or watch glass, 10% HCl, 10% NaOH, red litmus paper,
iodine reagent, Benedicts reagent, 2% starch and sucrose solutions in dropper bottles
Place 3 mL of 2% starch in two test tubes and 3 mL of 2% sucrose solution in two more test
tubes. To one sample each of sucrose and starch, add 20 drops of 10% HCl. To the other samples of sucrose and starch, add 20 drops of H2O. Label the test tubes and heat in a boiling water
bath for 10 minutes.

Tests for Carbohydrates

Remove the test tubes from the water bath and let them cool. To the samples containing HCl, add
10% NaOH (about 20 drops) until one drop of the mixture turns litmus paper blue, indicating the
HCl has been neutralized. Test the samples for hydrolysis as follows:
Iodine Test Place 5 drops of each solution on a spot plate or watch glass. Add 1 drop of iodine
reagent to each. Record observations. Determine if hydrolysis has occurred in each.
Benedicts Test Add 2 mL of Benedicts reagent to each of the samples and heat in a boiling water
bath for 34 minutes. Determine if hydrolysis has occurred in each.
F.

Testing Foods for Carbohydrates

Materials: Sugar samples (refined, brown, natural, powdered), honey, syrups (corn, maple,
fruit), foods with starches: cereals, pasta, bread, crackers, potato, Benedicts
solution, Seliwanoffs reagent, iodine reagent
Obtain two carbohydrate samples to test. Perform the Benedicts, Seliwanoffs, and iodine tests
on each. Describe the kinds of carbohydrates you identify in each sample.

Tests for Carbohydrates

Report Sheet
Date ___________________________

Name _________________________________

Section _________________________

Team

_________________________________

Instructor _______________________
Pre-Lab Study Questions
1.

What happens to glucose or galactose when the Cu2+ in Benedicts is reduced?

2.

Would you expect fructose or glucose to form a red color rapidly with Seliwanoffs reagent?

3.

Why dont all the disaccharides undergo fermentation with yeast?

4.

How can the iodine test be used to distinguish between amylose and glycogen?

Results of Carbohydrate Tests

A. Benedicts Test B. Seliwanoffs Test
Glucose
Fructose
Sucrose
Lactose
Starch
Water
Unknown
#_______

C. Fermentation Test

D. Iodine Test

Tests for Carbohydrates

Report Sheet
Questions and Problems
Q.1 From the results in part A, list the sugars that are reducing sugars and those that are not.

Reducing sugars

Nonreducing sugars

Q.2 What sugars are ketoses?

Q.3 What sugars give a positive fermentation test?

Q.4 Which carbohydrates give a blue-black color in the iodine test?

Identifying an Unknown Carbohydrate

Unknown No.__________
Results with Unknown
Benedicts
(A)
Seliwanoffs
(B)
Fermentation
(C)
Iodine
(D)
What carbohydrate(s) is/are in your unknown?

Possible Sugars Present

Tests for Carbohydrates

Report Sheet
Questions and Problems
Q.5 What carbohydrate(s) would have the following test results?
a. Produces a reddish-orange solid with Benedicts and a red color with Seliwanoffs
reagent in 1 minute

b. Gives a color change with Benedicts test, a light orange color with Seliwanoffs reagent
after 5 minutes, and produces no bubbles during fermentation

c. Gives no color change with Benedicts or Seliwanoffs test, but turns a blue-black color with
iodine reagent

E.

Hydrolysis of Disaccharides and Polysaccharides

Results
Iodine test

Sucrose + H2O

Sucrose + HCl

Starch + H2O

Starch + HCl

Benedicts test
Hydrolysis
products
present
Questions and Problems
Q.6 How do the results of the Benedicts test indicate that hydrolysis of sucrose and starch occurred?

Q.7 How do the results of the iodine test indicate that hydrolysis of starch occurred?

Tests for Carbohydrates

Report Sheet
Q.8

Indicate whether the following carbohydrates will give a positive (+) or a negative () result in
each type of test listed below:
Benedicts Test

Seliwanoffs Test

Fermentation Test

Glucose
Fructose
Galactose
Sucrose
Lactose
Maltose
Amylose
Amylopectin
F.

Testing Foods for Carbohydrates

Food Item 1
Benedicts
test
Seliwanoffs
test
Iodine
test
Possible
carbohydrates
present

Food Item 2

Iodine Test