CARBOHYDRATES: TESTS IN PLANT ORGANIC COMPONENTS

Arvin Uy – Pacoma
Division of Natural Sciences and Mathematics, University of the Philippines Visayas –
Tacloban College, Magsaysay Boulevard, Tacloban City, 6500, Philippines

ABSTRACT

A carbohydrate is an organic compound which consists only of carbon,

hydrogen and oxygen, with the last two in the 2:1 atom ratio. Carbohydrates make
up the bulk of organic substances on plants and perform numerous roles in living things.
The fruit extract of Dalandan (Citrus sinensis) and the leaf extract of Cabellaro
(Caesalpenia pulcherrima) were tested for the presence of carbohydrates. Both extracts
along with other solutions were tested in color reactions. Also, the hydrolyzed derivatives
were tested for their color reactions. This study aims to differentiate specific reactions on
various carbohydrate groups and their derivatives.
Keywords: carbohydrates, color reactions, hydrolytic products

INTRODUCTION
Carbohydrates are the main energy source for the human body. Chemically,
carbohydrates are organic molecules in which carbon, hydrogen, and oxygen bond together
Animals (including humans) break down carbohydrates during the process of metabolism to
release energy (Chen et al 2008).
All carbohydrates are made up of units of sugar, called saccharide units.
Carbohydrates that contain only one sugar unit (monosaccharides) or two sugar units
(disaccharides) are referred to as simple sugars. Two of the most common
monosaccharides are glucose and fructose. Glucose is the primary form of sugar stored in
the human body for energy. Fructose is the main sugar found in most fruits.
Disaccharides have two sugar units bonded together. Complex carbohydrates are
polymers of the simple sugars. In other words, the complex carbohydrates are long chains of
simple sugar units bonded together (for this reason the complex carbohydrates are often
referred to as polysaccharides).
The carbohydrate content of plants vary and there is little research on the
percentage of carbohydrates present in certain plant species. Nevertheless, carbohydrates
play some very important roles in plants. Starch is the major carbohydrate storage molecule
of plants. They store it when there is an abundance and break it down to glucose for energy
when needed. Cellulose serves a very important role in plant structure. Some carbohydrates
also play important roles in plant immunity as elicitors of plant defenses and signaling
molecules (Trouvelot et al. 2014). Starch is the principal polysaccharide used by plants to
store glucose for later use as energy (Kermode 2011). Starch is often stored in seeds by
plants or other specialized organs; for example, common sources of starch include rice,
beans, wheat, corn, potatoes, and so on.
The presence of carbohydrates can be detected by performing certain tests that
employ the characteristic color reactions of these organic compounds. Carbohydrates can
be characterized as monosaccharides, disaccharides, or polysaccharides; as reducing or

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non-reducing; or as aldoses or ketoses. The aim of this experiment was to perform tests that
differentiate the carbohydrates, aldoses, ketoses and reducing sugars based on those
characteristics. Being familiar with the tests that differentiates these organic macromolecules
will be very helpful in the field of research.

MATERIALS AND METHODS

a. Characteristic color reactions of carbohydrates in some plant materials
First to be done was the Molisch test. The researchers prepared eight clean
test tubes where 1 mL of various solutions were pipetted. Namely, 1 % glucose, 1 %
fructose, 1 % lactose, 1 % sucrose, 1 % starch, leaf extract, fruit extract and distilled
water. A drop of α-napthol was added to each tube and shaken. Then each tube was
added with concentrated hydro-sulfuric acid (H2SO4). A positive result would be a
purple interphase at each solution.

Next was the Seliwanoff’s test. The researchers prepared eight clean test
tubes where 3 mL of various solutions were pipetted. Namely, 1 % glucose, 1 %
fructose, 1 % lactose, 1 % sucrose, 1 % starch, leaf extract, fruit extract and distilled
water. All tubes were then added with 3 mL Seliwanoff’s reagent and heated in a
boiling water bath with the time being recorded. The tubes were taken out of the
water bath as soon as a color change was observed.

Lastly, the researchers performed the Benedict’s test. The researchers

prepared eight clean test tubes where 3 mL of various solutions were pipetted.
Namely, 1 % glucose, 1 % fructose, 1 % lactose, 1 % sucrose, 1 % starch, leaf
extract, fruit extract and distilled water. All tubes were then added with 3 mL
Benedict’s reagent and heated in a boiling water bath with the time being recorded.
The tubes were taken out of the water bath as soon as a brick red precipitate was
observed.

b. Characterization of hydrolytic products of disaccharides and polysaccharides

Action of acid on disaccharides
5 mL of one percent sucrose solution to two test tubes. One test tube would
be added with 5 mL Bendict’s reagent, whereas the other tube was added with 5
drops of hydrochloric acid (HCl). The second test tube was warmed gently over a
flame for 2 minutes after which 2 mL of Benedict’s reagent was added. They were
then placed on a boiling water bath. The tubes were taken out at the appearance of a
brick red precipitate
Action on polysaccharides
The group prepared and labeled 10 clean test tubes. The tubes 1-8 were then
added with 10 mL starch - HCl solution, where 9 had 10 mL one percent starch and
10 had 10 mL one percent glucose. The tubes were then simultaneously added to a
boiling water bath. At the interval of 2 minutes, one test tubes was taken out and
cooled in an ice bath. After all test tubes were taken out, a drop of I2KI was added to
each. The group then compared the color intensities of the achromatic points each
tube attained

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 RESULTS AND DISCUSSION
Eight samples, namely one percent glucose, one percent fructose, one percent
lactose, one percent sucrose, one percent starch, Caesalpenia pulcherrima leaf extract,
Citrus sinesis fruit extract and distilled water (as standard), were analyzed in various test for
the presence of carbohydrates.
The first test to be done was the Molisch Test, which is a general test for carbohydrates. All
samples underwent the test (see Figure 1). This test is specific for all carbohydrates;
monosaccharides gives a rapid test, while disaccahrides and polysaccharides react slower.
This test uses concentrated sulfuric acid and α-napthol. This test will give a purple colored
product on the interface of the sulotion; shown chemically in Figure 1. This test showed
positive results to all tested solutions, except distilled water (see Table 1), which denotes the
presence of a carbohydrate. Since the fruit and leaf extracts gave a positive result to this
test, this indicates that both extracts have carbohydrates (“Qualitative Analysis of
Carbohydrates (Theory),” n.d.).

a b
Figure 1. Experimental set-up for the Molisch test. Positive results is indicated by the
formation of a purple product or interphase. Arranged from M1 to M8 are one percent
glucose, one percent fructose, one percent lactose, one percent sucrose, one percent
starch, Caesalpenia pulcherrima leaf extract, Citrus sinesis fruit extract and distilled water

Figure 2. Chemical equation formation of violet-colored dye in Molisch Test.

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 Second test performed was the Seliwanoff’s test. This test is a test for the presence
of ketoses and aldoses. Ketoses are distinguished from aldoses via their ketone/aldehyde
functionality. If the sugar contains a ketone group, it is a ketose and if it contains an
aldehyde group, it is an aldose. All samples mentioned underwent this test (see Figure 3).
This test is based on the fact that, when heated, ketoses are more rapidly
dehydrated than aldoses. This test will result to a cherry red solution as a positive; it must
be noted that ketoses produce a much darker cherry red color, as compared to a faint pink
color produced by an aldose. This cherry red color is due to the formation of a molecule with
resorcinol attached to it (see Figure 4). Solutions of one percent lactose, one percent
glucose, one percent starch and distilled water did not yield a positive result (see Table 1).
The extracts from the C. pulcherrima leaves and C. sinensis fruit also gave a positive result
to the test. This indicates that the extracts contained either or both sucrose and/or fructose
(“Carbohydrates - Seliwanoff’s Test,” n.d.).

Figure 3. Experimental set-up for the Seliwanoff’s test. Positive results is indicated by the
formation of a purple product or interphase. Arranged from M1 to M8 are one percent
glucose, one percent fructose, one percent lactose, one percent sucrose, one percent
starch, Caesalpenia pulcherrima leaf extract, Citrus sinesis fruit extract and distilled water.

Figure 4. Chemical equation showing formation of red solution in Seliwanoff's test.

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 Benedict's test is used to test for simple carbohydrates. The Benedict's test identifies
reducing sugars (monosaccharide's and some disaccharides). A reducing sugar is any
sugar that, in a solution, has an aldehyde or a ketone group. . All samples were treated with
the Benedit’s reagent and produced a variety of results, as shown in Figure 5. Generally,
Benedict's test detects the presence of aldehydes and alpha-hydroxy-ketones, also by
hemiacetal, including those that occur in certain ketoses. Thus, although the ketose fructose
is not strictly a reducing sugar, it is an alpha-hydroxy-ketone, and gives a positive test
because it is converted to the aldoses glucose and mannose by the base in the reagent. The
brick red precipitate is due to the combination of copper (II) sulfate pentahydrate+ reducing
sugar which is a brick red substance (see Figure 6). A green color is also a positive result
but this indicates that the concentration of the carbohydrates are low (“Carbohydrates -
Benedict’s Test,” n.d.).

Figure 5. Experimental set-up for the Benedict’s test. Positive results is indicated by the
formation of a purple product or interphase. Arranged from S1 to S8 are one percent
glucose, one percent fructose, one percent lactose, one percent sucrose, one percent
starch, Caesalpenia pulcherrima leaf extract, Citrus sinesis fruit extract and distilled water

Figure 6. Chemical equation in formation of red precipitate in Benedict's test.

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 Based on the results obtained, both the leaf and fruit extracts may contain sucrose,
glucose, and/or fructose since both extracts gave positive results to Molisch test indicating
that they contain carbohydrates, to the Seliwanoff’s test indicating that a ketose is present
which could be fructose, and to the Benedict’s test indicating that reducing sugars are
present. The fructose may be present as a monomer of sucrose or as an isomer of glucose.
A compilation of the three tests for all the tests is summarized in Table 1.

Table 1 Characteristic color reactions of carbohydrates. Results are indicated as + for

positive, - for negative and 0 for unperformed.

Solution
Molisch
one percent glucose
one percent fructose
one percent lactose
one percent sucrose
one percent starch
Leaf Extract
Fruit extract
Distilled Water
Tests
Seliwanoff’s Benedict’s
+ - + (brick red ppt)
+ + + (orange ppt)
+ - + (brick red ppt)
+ + -
+ - -
+ + + (green ppt)
+ + + (yellow ppt)
- - -

This part of the experiment has the same principle as that of the previous. Here, however, it
is shown that the addition of HCl to the solution causes the sample to give a positive result.
Sucrose is a non-reducing sugar so it does not give a positive result to the Benedict’s test.
Adding HCl to sucrose, however, causes the sucrose to hydrolyze into its component
monosaccharides glucose and fructose. The glucose and fructose then reduce the copper
(II) in the reagent to copper (I) oxide which is the brick red precipitate.

Figure 7. Experimental set-up for the characterization of hydrolytic products on

disaccharides. Positive results is indicated by the formation of a brick red precipitate.
Table 2 Characterization of hydrolytic products on disaccharides
Reaction
Sucrose + Benedict’s Clear blue solution
Sucrose + HCl + Benedict’s Clear solution with brick red precipitate

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 The solution used for this test was designed such that the HCl will hydrolyze the
starch in the solution upon heating in the boiling water bath. The solution turned blue upon
the addition of I2KI because the starch polysaccharide forms a complex with I3 which is dark
blue to violet in color (Figure 9). Once the starch is hydrolyzed into glucose monomers it will
no longer be able to form a starch-iodine complex and no blue solution is formed.

Figure 8. Experimental set-up for the characterization of hydrolytic products on

polysaccharides.

Table 3 Characterization of hydrolytic products on polysaccharides

Test tube no. Color Reaction

1 Indigo (light blue violet)
2 Indigo (light blue violet)
3 Dark Violet
4 Violet
5 Violet
6 Light Violet
7 Faded Violet
8 Clear Solution
9 Clear Solution
10 Clear Solution

CONCLUSION
Color reactions can be performed to differentiate reducing sugars from non-reducing
ones and monosaccharides from disaccharides and polysaccharides. Acid hydrolyzes
oligosaccharides and polysaccharides into their monosaccharide components which can
then be identified using the various tests performed. The results showed that the C.
pulcherriima leaf extract and C. sinensis fruit extract has carbohydrates present in both the
leaf extracts and fruit extracts.

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 LITERATURE CITED

Carbohydrates - Benedict’s Test. (n.d.). Retrieved February 20, 2017, from

http://www.harpercollege.edu/tm-
ps/chm/100/dgodambe/thedisk/carbo/bened/benedict.htm
Carbohydrates - Seliwanoff’s Test. (n.d.). Retrieved February 20, 2017, from
http://www.harpercollege.edu/tm-
ps/chm/100/dgodambe/thedisk/carbo/seli/seli.htm
Carbohydrates - The Molisch Test. (n.d.). Retrieved February 20, 2017, from
http://www.harpercollege.edu/tm-
ps/chm/100/dgodambe/thedisk/carbo/molisch/molisch.htm
Chen, P. Y., Lin, A. Y. M., McKittrick, J. and Meyers, M. A. (2008) Structure and mechanical
properties of crab exoskeleton. Acta Biomaterialia, 4(3), 587-596.
https://doi.org/10.1016/j.act.bio.2007.12.010
Kermode, A. R. (2011). Plant Stotrage Products (Carbohydrates, Oils and Proteins). eLS, 1-
8. https://doi.org/101002/9780470015902.a0001325.pub2
Qualitative Analysis of Carbohydrates (Theory) : Biochemistry Virtual Lab I : Biotechnology
and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab. (n.d.).
Retrieved February 20, 2017, from
http://vlab.amrita.edu/?sub=3&brch=63&sim=631&cnt=1
Taiz, L., & Zeiger, E. (2002). Plant Physiology (3rd ed.). Sinauer Associates.
Trouvelot, S., Héloir, M.-C., Poinssot, B., Gauthier, A., Paris, F., Guillier, C., … Adrian, M.
(2014). Carbohydrates in plant immunity and plant protection: roles and potential
application as foliar sprays. Frontiers in Plant Science, 5.
https://doi.org/10.3389/fpls.2014.00592

LIST OF TABLES AND FIGURES

Figures
No. Title Page
1 Experimental set-up for the Molisch test 3
2 Chemical equation formation of violet-colored dye in Molisch Test 4
3 Experimental set-up for the Seliwanoff’s test 4
4 Chemical equation showing formation of red solution in Seliwanoff's test 5
5 Experimental set-up for the Benedict’s test 5
6 Chemical equation in formation of red precipitate in Benedict's test 6
7 Experimental set-up for the characterization of hydrolytic products on 6
disaccharides
8 Experimental set-up for the characterization of hydrolytic products on 7
polysaccharides.

Tables
No. Title Page
1 Characteristic color reactions of carbohydrates 6
2 Characterization of hydrolytic products on disaccharides 6
3 Characterization of hydrolytic products on polysaccharides 8