Chemical Identification of Carbohydrates
Introduction
Carbohydrates are defined as polyhydroxy aldehydes and ketones or materials that upon
hydrolysis produce polyhydroxy aldehydes and ketones. There are different ways of classifying
carbohydrates and tests that differentiate these classes will be performed. Monosaccharides are
simple carbohydrates that can not be broken down into simpler carbohydrates. Monosaccharides
can exist in water in either an open-chain or cyclic form, as is shown for glucose in Figure 1
below. Generally, monosaccharides exist primarily in the cyclic form. The three most common
monosaccharides, shown in cyclic form, are shown in Figure 2.
CHO
H
H
OH
HO
CH2OH H O
H
H
HO
HO
OH
H
H
H
OH
OH
H
OH
CH2OH
Figure 1: Open-chain (shown as a Fisher projection) and Cyclic Forms of Glucose
OH
H
CH2OH H O
CH2OH
CH2OH H O
OH
O
H
HO
H
HO
HO
H
H
H
H
H
HO
H
OH
OH
H
Glucose
CH2OH
OH
OH
OH
Galactose
H
Fructose
Figure 2: Common Monosaccarides
Disaccarides, upon hydrolysis, produce two monosaccharides. Barfoed’s Test
differentiates between monosaccharides and disaccharides. Lactose, a common disaccharide
known as “milk sugar,” is shown in Figure 3 below.
OH
H
CH2OH H O
CH2OH H O
H
O
HO
H
H
OH
H
HO
H
H
H
OH
OH
Figure 3: Lactose, a Disaccharide Composed of Galactose and Glucose
Chemical Identification of Carbohydrates, Page 1
Carbohydrates can exist as either aldoses (containing an aldehydic carbonyl group in its
open-chain form) or ketoses (containing a ketone carbonyl group in its open-chain form)
Seliwanoff’s Test can be used to differentiate aldoses and ketoses. Glucose (an aldose) and
fructose (a ketose) are shown below in Figure 4. Note that these compounds are shown in openchain form for clarity.
CH2OH
H
O
Aldehyde
f unctional group
C
H
C
HO
OH
H
H
H
OH
H
OH
H
OH
H
OH
HO
Ketone
f uctional group
O
CH2OH
CH2OH
Glucose
Fructose
Figure 4: Glucose (an Aldose) and Fructose (a Ketose)—Shown as Fisher Projections.
Carbohydrates that can be oxidized under mild conditions are called reducing sugars.
Reducing sugars include aldoses and ketoses. Ketoses are oxidizable due to an isomerization
called keto-enol tautomerism. Generally, in order to be a reducing sugar, there must be an
available hemiacetal or hemiketal functional group (see Figure 5 below). The most common
example of a nonreducing sugar that you will test is the disaccharaide sucrose, where the
hemiacetal and hemiketal functions of both sugars are tied up. Reducing sugars can be identified
by both Tollen's test and the Benedict's test. In this laboratory, you will use Benedict's test to
identify reducing sugars.
OH
H
C
OH
OR
R
R
C
OR
R
Hemiacetal
Hemiketal
Figure 5: Hemiacetal and Hemital Structures
The general reaction that occurs in the Benedict's Test is as follows:
O
R
O
Cu+2
C
H
+ 2
blue
+ 5
OH
R
C
O
+ Cu2O  + 3 H2O
red
Chemical Identification of Carbohydrates, Page 2
Certain tests are specific for only one specific carbohydrate. As an example the Muceic
Acid Test is specific for galactose and anything that upon hydrolysis produces galactose, such as
lactose. The reaction that occurs in the Muccic Acid Test is as follows:
O
OH
CHO
H
OH
HO
H
HO
H
H
OH
H
HNO3
OH
HO
H
HO
H
H
OH
CH2OH
O
Galactose
OH
Muccic Acid
Muccic acid is insoluble and will be evident as a white precipitate.
Chemical Identification of Carbohydrates, Page 3
Procedure:
All tests will be performed on solutions of the following carbohydrates: glucose, fructose,
galactose, sucrose, lactose, and an unknown sugar. 5% solutions of the known carbohydrates will
be provided. If the unknown carbohydrate solutions are not already prepared for you, a 5%
solution of your unknown may be prepared by dissolving 50 mg of unknown in 1 ml of
deionized water.
1. Muccic Acid Test: Place 10 drops of each 5% carbohydrate solution in separate 10 x 75
mm test tubes. Add 10 drops of concentrated nitric acid and heat at 100°C in a boiling
water bath for one hour. Replenish the nitric acid as needed to keep a constant volume.
Note that concentrated nitric acid is very corrosive and perform this test under the hood.
At the end of the heating period cool slowly for one hour and note the formation of a fine
white precipitate. If time permits, allow the test tubes to sit for one week before
observing the precipitate. This indicates the presence of galactose. Record your results on
your data sheet.
2. Seliwanoff's Test: Place 5 drops of each 5% carbohydrate solution in separate 10 x 75
mm test tubes. Add 10 drops of deionized water and 2.5 ml of the Seliwanoff reagent to
each test tube. Heat at 100°C in a boiling water bath for 5 minutes. Ketoses produce a red
solution in about 2 minutes. Aldoses produce a red solution much more slowly. Record
your results on your data sheet.
3. Benedict's Test: Place 5 drops of each 5% carbohydrate solution in separate 10 x 75 mm
test tubes. Add 25 drops of Benedict's reagent to each test tube. Heat at 100°C in a
boiling water bath for 3 minutes. Reducing sugars form a red precipitate. Record your
results on your data sheet.
4. Barfoed's Test: Place 5 drops of each 5% carbohydrate solution in separate 10 x 75 mm
test tubes. Add 15 drops of Barfoed reagent to each test tube. Heat at 100°C in a boiling
water bath for 5 minutes. Monosaccharides form a red precipitate in 2 to 3 minutes.
Disaccharides require more than 10 minutes. Record your results on your data sheet. Note
that under some circumstances, the time required may be a bit longer. In fact, sometimes
the disaccharide response may not occur in the time allotted for the lab.
Chemical Identification of Carbohydrates, Page 4
Name ____________________________________
Chemical Identification of Carbohydrates Report
Muccic Acid Test
Compound
Observation
Conclusion
Observation
Conclusion
Glucose
Fructose
Galactose
Sucrose
Lactose
Unknown # ____
Seliwanoff’s Test
Compound
Glucose
Fructose
Galactose
Sucrose
Lactose
Unknown # ____
Chemical Identification of Carbohydrates, Page 5
Benedict’s Test
Compound
Observation
Conclusion
Observation
Conclusion
Glucose
Fructose
Galactose
Sucrose
Lactose
Unknown # ____
Barfoed’s Test
Compound
Glucose
Fructose
Galactose
Sucrose
Lactose
Unknown # ____
Chemical Identification of Carbohydrates, Page 6
What are your conclusions about and your identification of your unknown?
Chemical Identification of Carbohydrates, Page 7
Questions and Problems:
1. Give structures for each of the known carbohydrates:
2. A carbohydrate upon treatment with Seliwanoff's reagent gave a red solution in about 1.5
minutes. Treatment with Barfoed's reagent gave a red precipitate in 2 minutes. Which of
the known carbohydrates could it be? Defend your choice.
3. A carbohydrate upon treatment with Benedict's reagent gave no red precipitate. The
solution remained blue. Which of the known carbohydrates could it be? Defend your
choice.
4. A carbohydrate solution gave the following test results:
Muccic acid test
Benedict's test
Barfoed's test
Seliwanoffs test
+
+
 (after 5 minutes)
 (after 5 minutes)
Which of the known carbohydrate solutions could it be?
Chemical Identification of Carbohydrates, Page 8