BIOORGANIC

BIOORGANIC
CHEMISTRY

BioOrganic Chemistry – CH 205 (2010-2011) Experiment #6

Determination of an unknown carbohydrate

based on the results of different chemical tests

Pamela S. de Leon, Manette C. Dejelo*, James Benedict L. Dolojan and Erik Joseph A. Gatdula
Department of Sports Science, College of Rehabilitation Sciences
University of Santo Tomas, España Street, Manila 108

Date Submitted: October 1, 2010

Abstract:

Carbohydrates are the most abundant organic compounds in the plant world. They act as storehouses of
chemical energy (glucose, starch, glycogen); are components of supportive structures in plants (cellulose),
crustacean shells (chitin), and connective tissues in animals (glucosaminoglycans); and are essential components of
nucleic acids (D-ribose and 2-deoxy-D-ribose). Monosaccharides, oligosaccharides, and polysaccharides are the
classifications of carbohydrates. The name carbohydrate arises from the basic molecular formula (CH2O)n where
n=3 or more. (CH2O)n can be rewritten (C*H2O)n to show that these substances are hydrates of carbon.

This experiment involves the following carbohydrates: sucrose, maltose, glucose, fructose, and xylose. The
unknown sample was to be determined with the use of the Barfoed’s test, Benedict’s test, Seliwanoff’s test, and
Bial’s Orcinal test. With the results of these tests, we are able to determine to what the unknown sample is; even
though the results are not exact, they are accurate.

Keywords: Carbohydrates, monosaccharides, oligosaccharides, polysaccharides, sucrose, maltose, glucose,

fructose, xylose, Barfoed’s test, Benedict’s test, Seliwanoff’s test, Bial’s Orcinal test

1. Introduction

Carbohydrates, the most abundant biomolecules in nature, are a direct link between
solar energy and the chemical bond energy of living organisms. More than half of all organic
carbon is found in carbohydrates. They are formed during photosynthesis, a biochemical
process in which light energy is captured and used to drive the biosynthesis of energy-rich
organic molecules from the energy-poor inorganic molecules CO2 and H2O. Most
carbohydrates contain carbon, hydrogen, and oxygen in the ratio (CH 2O)n, hence the name
“hydrate carbon”. They have been adapted for a wide variety of biological functions, which
include energy sources, structural elements, cellular communication and identity, and
precursors in the production of other biomolecules. Certain carbohydrate molecules are
structural elements of nucleotides and nucleic acids.

Carbohydrates are classified as monosaccharides, disaccharides, oligosaccharides,

and polysaccharides according to the number of simple sugar units they contain. In the
experiment, the carbohydrates involved are sucrose, maltose, glucose, fructose, xylose.
Sucrose, common table sugar, is the most abundant of all disaccharides and occurs
throughout the plant kingdom. It is produced commercially from the juice of sugar cane and
sugar beets. Maltose, often called malt sugar, is produced whenever the polysaccharide starch
breaks down, as happens in plants when seeds germinate and in human beings during starch
digestion. It is a common ingredient in baby foods and is found in malted milk. Glucose is
the most abundant monosaccharide in nature and the most important from a human
nutritional standpoint. It is originally called dextrose, and it is the primary fuel for living
cells. Fructose is biochemically the most important ketohexose. It is also known as luvolose
and fruit sugar. The sweetest-tasting of all sugars, fructose is found in many fruits and is
present in honey in equal amounts with glucose. Xylose, also called wood sugar, is a pentose
sugar found in plant tissues; 40% as sweet as sucrose.

In the experiment, the tests involved are the Barfoed’s test, Benedict’s test,
Seliwanoff’s test, and Bial’s Orcinal test. Barfoed’s test shows positive tests for reducing
monosaccharides. Benedict’s test is for reducing sugars such as glucose, sucrose, and
fructose. Seliwanoff’s test shows positive tests for ketoses. Bial’s test shows positive tests for
pentoses.

In this experiment, the group should be able to achieve the following objectives: (1)
to describe the structural features common to all carbohydrates, (2) to give specific chemical
 tests to characterize a given carbohydrate, (3) to identify an unknown carbohydrate based on
the results of the different chemical tests and in comparison with standard carbohydrate, and
(4) to provide the underlying principle involved in carbon chemical test.

2. Methodology
A. Preparation of the solution of the unknown sugar

1g of unknown sugar was given, and then added to10 mL of water. One-fourth of this
was used as the sample for the tests which are the Barfoed’s test, Benedict’s test,
Seliwanoff’s test, and Bial’s Test.

B. Tests

Barfoed’s Test
10 drops of test compounds were placed in separate test tubes. 20 drops of Barfoed’s
reagent were then added. Test tubes were placed in boiling water bath for 10 minutes. The
color of each test tube was recorded and the time of the formation of precipitate was noted.
The color of the reagent (blue) was compared.

Benedict’s Test
10 drops of test compounds were placed in separate test tubes. 20 drops of Benedict’s
reagent were then added. Test tubes were placed in boiling water bath for 5 minutes. The
color of each test tube was recorded and the time of the formation of precipitate was noted.
The color of the reagent (blue) was compared.

Seliwanoff’s Test
10 drops of test compounds were placed in separate test tubes. 20 drops of
Seliwanoff’s reagent were then added. Test tubes were placed in a boiling water bath for 10
minutes. Test tubes were removed from the water bath when there change in color. The color
of each test tube and the time of its formation were recorded. The color of the reagent
(colorless) was compared.
 Bial’s-Orcinol Test
10 drops of test compounds were placed in separate test tubes. 20 drops of Bial’s
reagent were then added. Test tubes were placed in a boiling water bath for 10 minutes. Test
tubes were removed from the water bath when there change in color. The color of each test
tube and the time of its formation were recorded. The color of the reagent (yellow) was
compared.

3. Results and Discussion

BARFOED’S BENEDICT’S SELIWANOFF’S BIAL’S ORCINOL
*Blue *Blue *Colorless *Yellow
o Clear blue sol’n o Clear blue sol’n o Colorless sol’n o Clear yellow sol’n
□ (09:00) red- □ (03:20) orange □ (03:54) clear peach □ (04:40) moss green
Unknown brown ppt; clear ppt; cloudy sol’n sol’n
blue sol’n orange sol’n
o Clear blue sol’n o Clear blue sol’n o Colorless sol’n o Clear yellow sol’n
□ Clear blue sol’n □ Clear blue sol’n □ (02:13) cloudy □ (02:56) dark
Sucrose (no change) (no change) pink sol’n greenish-brown sol’n

o Clear blue sol’n o Clear blue sol’n o Colorless sol’n o Clear yellow sol’n
□ (09:40) red- □ (02:42) orange □ (05:11) clear peach □ (05:15) dark green
Maltose brown ppt; clear ppt; orange sol’n sol’n sol’n
blue sol’n
o Clear blue sol’n o Clear blue sol’n o Colorless sol’n o Clear yellow sol’n
□ (09:17) red- □ (03:30) orange □ (03:54) clear peach □ (05:20) moss green
Glucose brown ppt; clear ppt; cloudy sol’n sol’n
blue sol’n orange sol’n
o Clear blue sol’n o Clear blue sol’n o Colorless sol’n o Orange sol’n
□ (07:35) red- □ (00:41) orange □ (01:46) cloudy □ (02:27) brown-black
Fructose brown ppt; clear ppt; orange sol’n dark pink/red sol’n sol’n
blue sol’n
o Clear blue sol’n o Clear blue sol’n o Colorless sol’n o Clear yellow sol’n
□ (07:30) red- □ (00:22) orange □ (04:56) black sol’n □ (04:22) dark blue
Xylose brown ppt; clear ppt (powder- green sol’n
blue sol’n like); orange
sol’n

o - Before Δ
□ - After Δ

Unknown sample: GLUCOSE

 In Barfoed’s test, all of the samples were originally a clear blue solution. The unknown
sample formed a red-brown precipitate in 9 minutes with a clear blue solution. The sucrose
sample had no changes; it remained to be a clear blue solution. The maltose sample formed a
red-brown precipitate in 9 minutes 40 seconds with a clear blue solution. The glucose sample
formed a red-brown precipitate in 9 minutes 17 seconds with a clear blue solution. The fructose
sample formed a red-brown precipitate in 7 minutes 35 seconds with a clear blue solution. The
xylose sample formed a red-brown precipitate in 7 minutes 30 seconds with a clear blue solution.

In Benedict’s test, all of the samples were originally a clear blue solution. The unknown
sample formed an orange precipitate in 3 minutes 20 seconds with a cloudy orange solution. The
sucrose sample had no changes; it remained a clear blue solution. The maltose sample formed an
orange precipitate in 2 minutes 42 seconds with an orange solution. The glucose sample formed
an orange precipitate in 3 minutes 30 seconds with a cloudy orange solution. The fructose sample
formed an orange precipitate in 41 seconds with an orange solution. The xylose sample formed
an orange precipitate (powder-like) with an orange solution.

In Seliwanoff’s test, all of the samples were originally a colorless solution. The unknown
sample formed a clear peach solution in 3 minutes 54 seconds. The sucrose sample formed a
cloudy pink solution in 2 minutes 13 seconds. The maltose sample formed a clear peach solution
in 5 minutes 11 seconds. The glucose sample formed a clear peach solution in 3 minutes 54
seconds. The fructose sample formed a cloudy dark pink/red solution in 1 minute 46 seconds.
The xylose sample formed a black solution in 4 minutes 56 seconds.

In Bial’s test, all of the samples were originally a yellow solution except fructose with an
orange solution. The unknown sample formed a moss green solution in 4 minutes 40 seconds.
The sucrose sample formed a dark greenish brown solution in 2 minutes 56 seconds. The
maltose sample formed a dark green solution in 5 minutes 15 seconds. The glucose sample
formed a moss green solution in 5 minutes 20 seconds. The fructose sample formed a brown-
black solution in 2 minutes 27 seconds. The xylose sample formed a dark blue-green solution in
4 minutes 22 seconds.
 With the help of the results, the unknown sample was determined. The tests show that the
unknown sample is glucose. Going back to the results, in the Barfoed’s test, the unknown sample
formed a red-brown precipitate in 9 minutes with a clear blue solution. The glucose sample
formed a red-brown precipitate in 9 minutes 17 seconds with a clear blue solution. In the
Benedict’s test, the unknown sample formed an orange precipitate in 3 minutes 20 seconds with a
cloudy orange solution. The glucose sample formed an orange precipitate in 3 minutes 30
seconds with a cloudy orange solution. In the Seliwanoff’s test, the unknown sample and the
glucose sample formed a clear peach solution in 3 minutes 54 seconds. In the Bial’s test, the
unknown sample formed a moss green solution in 4 minutes 40 seconds. The glucose sample
formed a moss green solution in 5 minutes 20 seconds. These results are accurate enough to say
that the unknown sample is glucose.

4. Conclusion

Carbohydrates are the most abundant class of bioorganic molecules on planet Earth.
Although their abundance in the human body is relatively low, carbohydrates constitute about
75% by mass of dry plant materials. Green (chlorophyll-containing) plants produce
carbohydrates via photosynthesis. In this process, carbon dioxide from the air and water from the
soil are the reactants, and sunlight absorbed by chlorophyll is the energy source.

CO2 + H2O + solar energy → carbohydrates + O2

Plants have two main uses for the carbohydrates they produce. In the form of cellulose,
carbohydrates serve as structural elements, and in the form of starch, they provide energy
reserves for plants.

A carbohydrate is a polyhydroxy aldehyde, a polyhydroxy ketone, or a compound that

yields polyhydroxy aldehydes or polyhydroxy ketones upon hydrolysis. The carbohydrate
glucose is a polyhydroxy aldehyde, and the carbohydrate fructose is a polyhydroxy ketone. A
monosaccharide is a carbohydrate that contains a single polyhydroxy aldehyde or polyhydroxy
ketone unit. Monosaccharides cannot be broken down into simpler units by hydrolysis reactions.
Pure monosaccharides are water-soluble, white, crystalline solids. Oligosaccharide is a
carbohydrate that contains two to ten monosaccharide units covalently bonded to each other.
Disaccharides are the most common type of oligosaccharide; it is a carbohydrate that contains
two monosaccharide units covalently bonded to each other. Like monosaccharides, disaccharides
are crystalline, water-soluble substances.

Sucrose Maltose

Glucose Fructose

Xylose

The unknown sample in the experiment is glucose. D-glucose, originally called dextrose,
is found in large quantities throughout the living world. In animals, glucose is the preferred
energy source of brain cells and cells that have few or no mitochondria, such as erythrocytes.
Cells that have limited oxygen supply, such as those in the eyeball, also use large amounts of
glucose to generate energy. Dietary sources include plant starch and the disaccharides lactose,
maltose, and sucrose. Glucose is one of the products of photosynthesis in plants and some
prokaryotes. In animals and fungi, glucose is the result of the breakdown of glycogen, a process
known as glycogenolysis. In plants, the breakdown substrate is starch. In animals, glucose is
synthesized in the liver and kidneys from non-carbohydrate intermediates, such as pyruvate and
glycerol, by a process known as gluconeogenesis. Glucose, when undergoes the Barfoed’s test,
forms a red-brown precipitate with a clear blue solution. When it undergoes the Benedict’s test,
it forms an orange precipitate with a cloudy orange solution. When it undergoes the Seliwanoff’s
test, it forms a clear peach solution. When it undergoes the Bial’s test, it produces a moss green
solution.

Glucose (Fischer Projection)

5. Acknowledgements

I would like to thank the following persons:

Mrs. Gelvero and Mr. Layco, our lab instructors in Chemistry Lab 205, who guided us in
conducting the experiment.

The librarians of the Medicine Library and the Miguel de Benavides Library for helping
me find the sources I need to support my results in the experiment.
 My fellow group mates, Pamela S. De Leon, James Benedict L. Dolojan, and Erik Joseph
A. Gatdula, for being dedicated to the experiment and showing unity, cooperation, and team
work.

6. References

Stoker, S. (2010). Exploring general, organic, and biological chemistry.

Philippines: ESP Printers Inc.

Foote, C., Iverson, B., & Ansyln, E. (2008). Organic chemistry. (5th ed.).
USA: Brooks/Cole Pub Co.

Garrett, R., Grisham, C. (2009). Biochemistry. (4h ed.).

USA: Chelsea House Publications
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*Corresponding author (Dejelo, Manette C.)