Department of Pure and Applied Chemistry

College of Arts and Sciences

Visayas State University

Date Performed: June 13, 2017 Score: ________

Date Submitted: June 21, 2017 Prepared by: BS Chem-2

Experiment No. 3
Carbohydrates
Objectives:
 Use of chemical tests to identify some physical and chemical characteristics of typical
carbohydrates.
 Differentiate between monosaccharides, disaccharides, and polysaccharides using
above tests.
 Identify an unknown carbohydrate.

Results:
A. Structural Formulas for Carbohydrates
Table 1.1. Structures of Monosaccharides
Structures
Monosaccharides
Fischer Haworth Chair

Glucose

Fructose
Table 1.2. Structures of Disaccharides and Polysaccharides

Disaccharides (Haworth and Chair only)

Lactose Sucrose

Maltose

Polysaccharides

Amylose Glycogen
B. Benedict’s Test for Reducing Sugars
Table 2. Qualitative Test for Reducing Sugars
Compound Color Reducing Sugar (Yes/No)
Water No changes No
Glucose Red Yes
Fructose Red Yes
Sucrose No changes No
Lactose Red Yes
Maltose Red Yes
Starch No changes No

C. Barfoed’s Test for Monosaccharides

Table 3. Qualitative Test for Monosaccharides
Compound Color Monosaccharide (Yes/No)
Water No changes No
Glucose Red Yes
Fructose Red Yes
Sucrose Green-orange No
Lactose Green-orange No
Maltose Green-orange No
Starch No changes No

D. Seliwanoff’s Test for Ketohexoses

Table 4. Qualitative Test for Ketohexose/Aldohexose
Compound Color Ketohexose/Aldohexose
Water No changes Neither of the two
Glucose Light pink Aldohexose
Fructose Red Ketohexose
Sucrose Red Ketohexose
Lactose No changes Neither of the two
Maltose No changes Neither of the two
Starch Light pink Aldohexose

E. Iodine Test for Polysaccharides

Table 5. Qualitative Test for Polysaccharides
Compound Color Polysaccharide (Yes/No)
Water No changes No
Glucose No changes No
Fructose No changes No
Sucrose No changes No
Lactose No changes No
Maltose No changes No
Starch Black Yes

F. Hydrolysis of Di- and Polysaccharides

Table 6. Qualitative Test for Reducing Sugars and Polysaccharides
Compound Benedict’s Test (F-1) Iodine (F-2)
(+) Positive for reducing
Sucrose (Hydrolyzed) (-) Not a polysaccharide.
sugar.
(+) Positive for reducing
Starch (hydrolyzed) (-) Not a polysaccharide.
sugar.
 G. Fermentation Test
Table 7. Fermentation Test of Different Sugars
Compound Observation Fermented( Yes/No)
Glucose 7.500 cm of air was produced. Yes
Fructose 1.500 cm of air was produced. Yes
Sucrose 4.500 cm of air was produced. Yes
Lactose 1.016 cm of air was produced. Yes
Corn Syrup 1.500 cm of air was produced. Yes
Starch 2.500 cm of air was produced. Yes

What gas accumulated in the test tubes? Carbon dioxide gas (CO2(g)).

H. Testing Foods for Carbohydrates

Table 8.1. Qualitative Test for Carbohydrates in Different Kinds of Foods
Test Results
Food Benedict’s Barfoed’s Seliwanoff’s Iodine
Corn Syrup Positive Positive Positive Negative
Brown Sugar Negative Negative Positive Negative
Cereal Negative Negative Negative Positive
Bread Negative Negative Negative Positive

Possible Carbohydrates in the Food Samples

Table 8.2. Carbohydrates Present in the Food Samples
Food Sample
Compound
Corn Syrup Brown Sugar Cereal Bread
Glucose - - - -
Fructose / - - -
Sucrose - /
Lactose - - - -
Maltose - - - -
Starch - - / /
Glycogen - - - -

Discussion
As it can be seen in table 1.1, fructose has no chair conformation. This is because fructose is only
a five membered ring when drawn in Haworth projection. One requirement in order for a sugar to have
a chair conformation is its structure must be a six membered ring when drawn in Haworth projection
and since fructose did not conform that requirement, therefore, it has no chair conformation at all.
Unlike glucose which is a six membered ring in Haworth projection. And also there is no chair
conformation for sucrose since one compound present in its molecule is fructose.
In Benedict’s test, only the glucose, fructose, lactose, and maltose had reacted with the coppric
sulfate, CuSO4, in the Benedict’s reagent which produced solid red precipitate, Cu 2O. Thus, the four
compounds are reducing sugars while the rest are not. Glucose, fructose, lactose, and maltose are
termed as reducing sugars because, by looking at their Haworth structures in tables 1.1 and 1.2, they
all have hydroxyl groups on their anomeric carbons which are all not linked to another compounds by
a glycosidic bond, which makes the ring open upon the reaction with Benedict’s reagent and caused
the reagent to be reduced and colored. Sucrose is not a reducing sugar because of the glycosidic
bond present between the glucose and fructose molecules which does not allow the ring(s) to open
with presence of the CuSO4 in Benedict’s reagent.
 In Barfoed’st test, only glucose and fructose produced red precipitate while sucrose, lactose,
and maltose produced a green-orange solution, and no changes happened in the starch. Barfoed’s
test is used to distinguish monosaccharides from other types of carbohydrates. Since glucose and
fructose are the only known monosaccharides in the group, therefore, they are the ones that react
with the Barfoed’s reagent at a fast rate of reaction and reduced the CuSO4 into solid Cu2O.
Seliwanoff’s test is used to determine the presence of a ketohexose compound with a red
solution produced as the positive result for ketohexose and light pink for the aldohexose. With the
resorcinol solution (resorcinol crystals in equal amount of H2O and HCl), only fructose and sucrose
produced red solutions while glucose and starch produced light pink solutions, and the remaining
solutions had no notable changes in them. By looking again their structures in tables 1.1 and 1.2, it can
already be predicted upon performing the Seliwanoff’s test that fructose is a ketohexose and
therefore, produce a red precipitate, while in the case of sucrose which is drawn in Haworth cyclic
structure, if drawn in Fischer projection, the carbonyl group (keto group) in its molecule will be visible,
thus, it can also be said that sucrose is really a ketohexose compound.

Since starch is a long chain of monosaccharides which is about thousands of monosaccharide

molecules and is termed as a polysaccharide compound, so, it was the only carbohydrate that gave
positive result for the presence of polysaccharide in Iodine test by producing a black solution. The rest
of the solutions produced red-orange solutions which had no changes in the color of the iodine at all
and it indicated a negative result for the presence of polysaccharide. This was because the solutions
that were tested with iodine solution, aside from starch, were monosaccharides (glucose and fructose)
and disaccharides (sucrose, lactose and maltose), and water, which is actually not a carbohydrate.
Unhydrolyzed sucrose produced a negative result for reducing sugars, while hydrolyzed sucrose
produced a positive for reducing sugars in Benedict’s test. This difference of results was due to the
presence of the strong acid, HCl, in the heated sucrose-HCl solution, which broke the glycosidic bond
between sugar molecules namely glucose and fructose. The breaking of the bond causes the release
of the two sugar molecules which are monosaccharides and reducing sugars by nature. Also the
hydrolyzed starch gave a positive result for reducing sugar in Benedict’s test. This was also because of
HCl which broke the –O- bond between the sugar molecules forming the starch chain and starch was
broken down into simple sugars, which is actually glucose. Both of the hydrolyzed solutions, sucrose
and starch, gave negative results in iodine test since they were already broken down into simple units
of sugars which are monosaccharides.
After the addition of yeast to the solutions of glucose, fructose, lactose, sucrose, corn syrup, and
starch, it can said that all of the solutions of the carbohydrates were fermented since there was an
observable evolution of bubbles in the petit tubes in the larger test tubes of the solutions. These bubbles
indicated the presence of carbon dioxide gas, CO29g0), and each solution produced air with
corresponding length after the fermentation has completed. For glucose, 7.500 cm of air was
produced, 1.500 cm for fructose, 4.500 cm for sucrose, 1.016 cm for lactose, 1.500 cm for corn syrup,and
2.500 cm for starch.
Four food samples, corn syrup, brown sugar, cereal, and bread, were tested in Benedict’s,
Barfoed’s, Seliwanoff’s, and Iodine tests for the presence different types of carbohydrates. Corn syrup
has produced positive results in Benedict’s, Barfoed’s, and Seliwanoff’s tests while it produced a
negative result in iodine test. The brown sugar has only showed a positive result in Seliwanoff’s test while
it showed negative results in the rest of the tests. While cereal and bread both showed positive results
in iodine test and showed negative results in the other three tests. From table 8.2, it is shown that fructose
is the carbohydrate that is present in corn syrup. For brown sugar it is sucrose that is present in it. For
cereal and bread, it is the starch that is present in both food. All of the carbohydrates present in the
different food samples were proven and confirmed by the Benedict’s, Barfoed’s, Seliwanoff’s, and
iodine tests.

Conclusion
The Benedict’s, Barfoed’s, Seliwanoff’s, and Iodine tests are effect qualitative test in identifying
the physical and chemical characteristics of carbohydrates. In Benedict’s and Barfoed’s tests a red
precipitate will be produced as an indication for positive results of reducing sugar(s) and
monosaccharide(s). In Seliwanoff’s test a red solution will be produced as an indication for the positive
result of a ketohexose compound and light pink solution for an aldohexose compound. And in Iodine
test, a black solution will be observable when a polysaccharide carbohydrate is present in the solution.
Monosaccharides are only determined through Barfoerd’s test where they react very fast with
the CuSO4 producing the brick-red precipitate of Cu2O and the other types of carbohydrates do not
react. When in doubt if the other solution contains disaccharide or polysaccharide compounds, Iodine
test can directly be performed using iodine solution, usually in the form of potassium iodide (KI). In this
test, the polysaccharide will be determined as it will produce a black solution right away.
In identifying an unknown carbohydrates, one can actually start with Iodine test to determine
whether the compound is a polysaccharide or not, followed by Benedict’s test to identify if the
compound is a reducing sugar, next with Barfoed’s test in order to identify if the compound is a
monosaccharide. And lastly, the Seliwanoff’s test which will help identify if the compound is a
ketohexose, aldohexose, or neither of the two. If ever the compound is only positive in Seliwanoff’s, it
can be predicted that the compound contains sucrose and a hydrolysis process can be further
performed to confirm the deduction.

Reference:
Ferrier, Denise R. Lippincott’s Illustrated Reviews Biochemistry. Sixth Edition.

Answers to Questions.
1. Compare the results of the Benedict’s test with sucrose before and after hydrolysis. Why? Are
they different?
Before hydrolysis, sucrose produced a blue solution with Benedict’s reagent which
indicate a negative result for reducing sugar. But after hydrolysis, a red precipitate was
produced indicating a positive result for reducing sugar. This happened because the HCl that
was added into the solution broke the glycosidic bond that linked the glucose and fructose
molecules. Thus, glucose and fructose, which are monosaccharides and reducing sugars, were
released and reacted with the CuSO4 producing the red precipitate, Cu2O(s), which was visible
at the bottom of the test tube.

2. Compare the results of the Benedict’s test with starch before and after hydrolysis. Why are they
different?
The same things happened in starch, negative result was produced before hydrolysis and
positive after hydrolysis. This was because HCl broke the bonds at the –o- linkages, which formed
the starch chain, which led to the release of sugar molecules, specifically glucose and reacted
with the CuSO4 which produced solid Cu2O.
3. Compare the results of the iodine test with starch before and after hydrolysis. Why is there a
difference?
A black solution was observed when starch was tested with iodine solution, which is a
positive result for the presence of polysaccharide. After starch was hydrolyzed, a red-orange
solution was produced because, as mentioned above, HCl broke the –O- linkages between the
sugar molecules that make up the starch. So, the starch was broken down into simple sugars,
specifically glucose which is a monosaccharide.
Appendix

Heating of the Solutions Benedicts’s Test for Barfoed’s Test for

for Benedict’s, Barfoed’s Reducing Sugar Monosaccharide
and Seliwanoff’s Tests

Seliwanoff’s Test for Iodine Test for Fermentation of Sucrose

ketohexoses Polysaccharides (Left) and Corn Syrup
(Right)

Testing Food (Corn Syrup)

for Carbohydrates