MAPA INSTITUTE OF TECHNOLOGY

School of Chemical Engineering and Chemistry

Experiment 5
CHARACTERIZATION OF CARBOHYDRATES

Introduction
Carbohydrates are classified as polyhydroxy aldehydes or polyhydroxy
ketones. Therefore, they will exhibit chemical properties associated with both
alcohols and carbonyl compounds. In the following series of activities, the student
will be examining the reactivity of some monosaccharides, disaccharides, a
polysaccharide, and their natural samples to .
In the Benedicts test a reducing sugar, which is a sugar with a free or
potentially free aldehyde group, reacts with the blue-colored Cu 2+ ion in the
presence of a base. The copper (II) ion is reduced to a red-orange Cu 2O precipitate
whereas the aldehyde group is oxidized to the carboxylic acid functional group. In
addition to all aldose monosaccharides giving a positive Benedicts test, ketose
monosaccharides, though lacking an aldehyde group, react due to the presence of
an hydroxyl group next to the ketone group. Thus -hydroxy ketones give positive
tests. If there is no potential free aldehyde group, such as when the aldehyde group
is tied up in a glycosidic bond (an acetal bond), the sugar is referred to as a nonreducing sugar.
The Barfoeds test is a variation of the redox reaction mentioned previously.
Copper (II) acetate in acetic acid is not as reactive as the Cu 2+ Benedicts reagent.
Thus, one can distinguish monosaccharides from disaccharides based on how fast
the red-orange precipitate forms. Typically, monosaccharides react within 2-3
minutes, whereas disaccharides take longer.
The Seliwanoff test is used to distinguish ketoses from aldoses using the
aromatic alcohol in the presence of concentrated hydrochloric acid. This is useful for
both monosaccharide ketoses as well as disaccharide ketoses. A positive test is
noted by a red colored solution; a yellowish straw or apricot color is not indicative of
a positive test.
To distinguish pentoses from hexoses, Bials test can be used. Pentoses react
with orcinol in the presence of FeCl 3 and concentrated HCl to give a characteristic
blue-green color. Nonreacting sugars may produce a brown precipitate but the
solution usually remains the yellow color of the FeCl 3.
Molischs test is a sensitive chemical test for all carbohydrates, and some
compounds containing carbohydrates in a combined form, based on the
dehydration of the carbohydrate by sulfuric acid to produce an aldehyde (either
furfural or a derivative), which then condenses with the phenolic structure resulting
in a red or purple-colored compound.
Starch is composed of two fractions; the linear, helical fraction and the
branched amylopectin fraction. When I2 is inserted into the interior of the amylose
fraction, a dark blue color is observed.
Objective

The experiment aims to have the students observe the results of the various
carbohydrate characterization tests on known samples, unknown samples, and their
brought samples. The experimenters should be able to ascertain the differences
between each test. Ideally, the students should be able to glean some information
from their results and have a say about the property of a carbohydrate based on the
test results.
Materials and Methods
Carbohydrate test solutions
5% solutions of glucose, galactose, fructose, maltose, lactose, sucrose, and
starch
An unknown sample for the class
Kamote and potato samples
Test reagents
*The test carbohydrate solution indicated in the steps below refers to the known
carbohydrate solutions, your unknown sample, and your kamote, potato, and rice
samples.
Sample Preparation
1. The known and unknown samples are already prepared, so dont worry about
that.
2. For your kamote, potato, and rice samples, take a piece of each and grind
separately in a mortar and pestle along with roughly 15-20 ml of water.
3. After grinding thoroughly, filter when you think you have produced a liquidy,
sopas-like extract or solution. Save the filtrate and use it as part of the
carbohydrate test solutions. Of course, label it appropriately.
Benedicts Test
1. Prepare a boiling water bath and label eight clean small test tubes.
2. In separate test tubes add 1 mL of the Benedicts reagent. To each test tube add
5 drops of the test carbohydrate solution. Mix the samples.
3. Place all of the test tubes at the same time into the boiling water bath.
4. Note and record how long it takes for the red Cu 2O precipitate to form; also note if
the blue Benedicts reagent color disappears.
5. After 10 minutes remove all the tubes. Keep the boiling water bath going for the
remaining three experiments. Did any sugars not produce the red precipitate?
Which are reducing sugars? Which are not?
Barfoeds Test
1. Use the boiling water bath from before and label a new set of 8 clean small test
tubes.
2. In separate test tubes add 1 mL of the Barfoedss reagent. To each test tube add
10 drops of the test carbohydrate solution. Mix the samples.
3. Place all of the test tubes at the same time into the boiling water bath.
4. Note and record how long it takes for the red Cu 2O precipitate to form.

5. After 10 minutes remove all the tubes. Based on your results which are
monosaccharides? Which are disaccharides?
Seliwanoff Test
1. Use the boiling water bath from before and label a new set of 8 clean small test
tubes.
2. In separate test tubes add 1 mL of the Seliwanoffs reagent. To each test tube add
3 drops of the test carbohydrate solution. Mix the samples.
3. Place all of the test tubes at the same time into the boiling water bath.
4. Note and record how long it takes for the first clear red colored solution to form.
5. Remove all the tubes as soon as the first positive test is seen as prolonged
heating (in excess of 5 minutes) may cause spurious results. Which sugar solution(s)
contain a ketose?
Bials Test
1. Use the boiling water bath from before and label a new set of clean small test
tubes
2. In separate test tubes add 1 mL of the Bials reagent. To each test tube add 10
drops of the test carbohydrate solution. Mix the samples.
3. Place all of the test tubes at the same time into the boiling water bath.
4. Note and record how long it takes for the first clear blue-green solution to form.
5. Remove all the tubes as soon as the first positive test is seen as prolonged
heating may cause spurious results. Which sugar solution(s) contain a pentose?
Molischs Test
1. Place 2 mL of a known carbohydrate solution in a test tube, add 1 drop of
Molischs reagent (10% -naphthol in ethanol).
2. Pour 1-2 mL of conc. H2SO4 down the side of the test tube, so that it forms a
layer at the bottom of the tube.
3. Observe the color at the interface between two layers and compare your result
with a control test.
*A brown color due to charring must be ignored and the test should
be repeated with a more dilute sugar solution.
Iodine test
1. Place 3 drops of each test carbohydrate solution in separate wells of a clean spot
plate.
2. Add 1 drop of the iodine solution to each test carbohydrate solution.
3. Note and record the color of each sample.
4. Did any other solutions besides the starch solution give a positive test?

Some points to consider

Create a table detailing the differences between the tests, especially the
differences in what they detect or what they test for
Create a data table before doing the experiment. Get organized!