PAMANTASAN NG LUNGSOD NG MAYNILA

COLLEGE OF MEDICINE
Intramuros, Manila, Philippines

EXPERIMENTS ON CARBOHYDRATES
Laboratory Formal Written Report

In Partial Fulfillment of the Requirements in

BIOCHEMISTRY

Presented to:
Department of Biochemistry
PLM College of Medicine

Presented by:
Section 1A
Group # 4

NABUS, Don Karlo R. PAINGCO, Mubarak M. Pascual, Exzel Lens B.

2017- 70001 2017-70069 2017- 70136

RICAFRANCA, John Vincent O. SALVANIA, Rizza Mae V. SAN DIEGO, Kirby D.G.
2017-70045 2017- 70005 2017-70093

October 31, 2017

 I. INTRODUCTION
Carbohydrates are compounds that contain carbon, hydrogen and oxygen. They
can form several compounds like cellulose for structural support, and sugar and starch
for energy source. Carbohydrates are necessary to drive several processes needed by
our body. They can be acquired by several sources and in different forms. The fruits,
vegetables, pastas, dairy products and other food we eat contribute to the increase of
these molecules.
Glucose is the most important carbohydrate in our body. It serves as a precursor
to structures that contain carbohydrates like nucleic acids, glycoproteins, glycolipids and
glycogen. Glucose, together with galactose and fructose, take the simplest form of
sugar called as monosaccharide. They serve as building blocks of more complex sugars
like disaccharides and polysaccharides. Monosaccharides, specifically glucose, serve
as an energy source by different body tissues. Some of these structures include the
brain, red blood cell and some parts of our eyes. Without glucose, mobilization of fat
and proteins might happen thus, proper regulation of glucose is needed.
Disaccharides are the combination of two monosaccharides linked by a
glycosidic bond. Three of the most common examples are maltose, sucrose, and
lactose. These three sugars are mostly common in our diets. Lactose is naturally
available in the milk of mammals. Dissociation of lactose would give glucose and
galactose. Sucrose or table sugar contains glucose and fructose when dissociated. This
is the most abundant type of sugar in human diet. It serves as a natural sweetener of
food the food we eat. Maltose on the other hand, is the combination of two glucose. It is
usually converted to its sugar alcohol form called as maltitol which is used as a bulk
sweetener in syrups.
Carbohydrates must be regulated to maintain balance in our body. Some must
be held low and some must be maintained at a certain level thus identification of sugar
type is very important. Some of the tests include Benedicts test, Seliwanoffs test,
Iodine test, and other more aids in the identification of sugars.
 II. OBJECTIVES
Activity 1 Objective - Molisch Test
To detect the presence of carbohydrates in the given solutions
Activity 2 Objective - Seliwanoffs Test
To determine which of the given solutions are ketoses
Activity 3 Objective - Benedicts Test
To detect the presence of reducing sugars in the given solutions
Activity 3 Objective - Iodine Test
To detect the presence of starch in the given solutions

III. MATERIALS AND METHODS

Six kinds of solutions containing fructose powder, refined sugar, glucose powder,
lactose powder, corn starch and glucosamine powder were initially prepared for the
experiment. The identity of the sugars were known only by the professors. The
carbohydrate tests below were conducted to correctly identify the sugar content of the
solutions. An algorithm of procedures that reflects common properties and tests unique
to sugar was also made by the experimenters.
Molisch Test
Two drops of Molisch reagent (5% solution of alpha-naphthol in alcohol) were
added to 5 mL of each test solution. The solutions were thoroughly mixed and the tubes
were inclined to allow approximately 3 mL of concentrated H2SO4 to run down the walls
of the tube. An acid layer was formed beneath the sugar and the color at the junction of
the liquids were observed. The Molisch test is a general test for carbohydrates.
Seliwanoffs Test
One milliliter of Seliwanoffs reagent (resorcinol) was mixed with 5 drops of each
test sugar solution in test tubes. The tubes were placed in a boiling water batch and the
production of a red colored solution or formation of red precipitate were noted.
Benedicts Test
 Pre-boiled five milliliters of Benedicts reagent were added with 8 drops of sugar
solution to be tested. The solution was placed in a boiling water bath for 3 minutes. The
solution was allowed to cool and formation of precipitate or changes in color of solution
were noted.
Iodine Test
The sugar solutions were added with a drop of Lugols solution in different test
tubes. Changes and intensity of color of the solutions were noted.

IV. RESULTS
Qualitative results of the tests for carbohydrates namely Molisch test,
Seliwanoffs test, Benedicts test and Iodine test are summarized in Table 1.
Table 1. Observable changes for the Tests for Carbohydrates

Solution Molisch test Seliwanoffs test Benedicts Test Iodine Test

A No change No change No change bluish-black soln

B Black soln Red soln No change No change

C Foamy ppt No change Light red ppt No change

D No change Red soln Brick red ppt No change

E No change No change Brick red ppt No change

* soln solution; ppt precipitate

The actual appearance of the tubes are shown in Figure 1. Solutions B and C
formed a black solution and formed a foamy precipitate, respectively, for Molisch Test.
Red solutions were formed by B and D for Seliwanoffs test. In the Benedicts test,
solutions C, D and E formed a characteristic brick red precipitate. Lastly, only solution A
rendered a positive result in iodine test.
Figure 1. Results of Test for Carbohydrates (A Molisch Test; B Seliwanoffs Test; C
Benedicts Test; D Iodine Test)

V. INTERPRETATION AND DISCUSSION

A. MOLISCH TEST (ALPHA- NAPHTHOL REACTION)

The Molisch Test or -naphthol test is a test for the presence of carbohydrates,
forming a purple-colored layer as a positive result. In this test, monosaccharides
generally give a more rapid result while disaccharides and polysaccharides react
slower. However, this test is not specific and can only confirm the presence of
carbohydrates. Further tests should be performed in order to determine the type of
carbohydrate present in the sample.
 Figure 2. Formation of purple-colored dye via Molisch test

The Molisch reagent which was used for this test contains a solution of
-naphthol in 95% ethanol. This reagent forms a five-membered, oxygen-containing ring
(aldehyde) through dehydration reactions with pentoses and hexoses in the presence of
sulfuric acid. Sulfuric acid was used to hydrolyze glycosidic bonds in disaccharides and
polysaccharides in order to yield monosaccharides which will then be dehydrated in the
reaction with the reagent. Figure 3 shows the reaction wherein furfural is formed
through the dehydration of a pentose sugar, while 5-hydroxymethylfurfural is formed
through the dehydration of a hexose sugar in the presence of sulfuric acid. These
furfurals condense with -naphthol to produce a purple-colored dye which forms a layer
that can be theoretically seen in the experiment between the layers of the sample and
the acid (Figure 2).

Figure 3. Dehydration of a pentose to form furfural (left) and hexose to form

5-hydroxymethylfurfural (right)
 The actual results do not show any purple-colored layer in any test sample.
However, all test samples should theoretically yield positive results because all samples
contain carbohydrates. Probable causes for this deviation may either be that there is a
problem with the Molisch reagent used or it was not mixed well enough with the test
samples during the experiment. Since sulfuric acid should form an acid layer below the
layer of the test sample, it can be assumed that it is the denser of the two. However in
the experiment, the sulfuric acid instantly mixed with the test sample even when it was
allowed to flow down the wall of the inclined test tube slowly. This contributes to why
there was no established density difference between the two solutions which caused no
layer of purple-colored dye to form. This may also account for the color change in the
entire solution.

B. SELIWANOFFS TEST (RESORCINOL- HCl TEST)

Seliwanoffs Test is a test for distinguishing sugars containing a ketone group
(ketoses) from sugars which have an aldehyde group (aldoses). It is not specific for
ketohexoses (6-carbon sugars containing a ketone group) as this test also gives a
positive result to disaccharides containing ketohexoses. An example of this is sucrose,
which has fructose, a ketohexose, and glucose, an aldohexose.
This makes use of the fact that ketoses are dehydrated much faster than aldoses
when reacted with the reagent and heated. A ketose, will form a deep red color, while
an aldose, will show a light pink color that takes a longer time to develop when reacted
with Seliwanoff's reagent. The test reagent is composed of hydrochloric acid (HCl),
distilled water, and resorcinol. The HCl is the dehydrating acid while the resorcinol is the
condensation reagent.
 Figure 4. The Seliwanoff test reaction on ketohexoses

Figure 5. The Seliwanoff test reaction on aldohexoses

Ketohexoses are dehydrated when reacted with concentrated acids to yield
furfurals or their subsidiaries. The HCl in the Seliwanoff reagent dehydrates
ketohexoses to form 5-hydroxymethylfurfural. Then, 5-hydroxymethylfurfural further
reacts with resorcinol in the test reagent to produce a cherry-red product within seconds
to a few minutes. On the other hand, ketopentoses yield blue-green series. Aldoses
take time to form a red color since they are further rearranged when dehydrated by a
concentrated acid.
 In the experiment, the test tubes 2 and 4 gave a positive reaction to the test
which indicates that the carbohydrates present in the test are ketone-containing. The
sugar samples given to the class which are or contains ketoses are fructose powder
and refined sugar. Further tests are needed to distinguish one from the other.

C. BENEDICTS TEST
The Benedicts test is done to find out if a solution or substance contains the
presence of a reducing sugar. The reagent will detect aldehydes, except aromatic ones,
as well as alpha-hydroxyketones. Reducing sugars include all monosaccharides, such
as fructose, galactose, and especially glucose, as well as disaccharides and
polysaccharides that also contain free aldehyde or ketone groups. In reducing
disaccharides and polysaccharides, their free anomeric carbon, i.e. not involved in a
glycosidic bond, is referred to as the reducing end. Common disaccharides detected by
the test include lactose and maltose. Sucrose is not detected by the test since it does
not contain any free aldehyde or ketone group, and that the anomeric carbons of the
glucose and fructose forming the sucrose are bound by a glycosidic bond, making it
hard for sucrose to open up its structure in order to react with other molecules.
The reagent is made up of sodium citrate, sodium carbonate, and copper (II)
sulfate dissolved in water. When a reducing sugar is exposed to the reagent, the copper
ions in the solution will be reduced by the sugar, forming copper (I) oxide, which
presents as a brick-red precipitate, and carboxylic acid.

Figure 6. The Benedicts test reaction on reducing sugars

The test is very similar to that of the Fehlings test, in the sense that they both
test for reducing sugars and make use of copper ions. Their difference however, lies in
the choice of complexing agent. Fehlings reagent makes use of aqueous sodium
potassium tartrate as its complexing agent, while Benedicts reagent uses aqueous
sodium citrate. By using sodium citrate as a complexing agent, the Benedicts reagent
became an improved version of the Fehlings reagent since the citrate complex is more
stable than that of the tartrate complex of the Fehlings reagent, in that the former does
not form a precipitate when standing for long. In fact, the Fehlings reagent comes in
two separate solutions, with one solution being the aqueous copper (II) sulfate, while
the other one being the aqueous sodium potassium tartrate. These two solutions are
only mixed when the Fehlings reagent would already be used for testing, since mixing it
early would just cause the complex to precipitate and lead to the wastage of the
reagent. The Benedicts reagent is then much more convenient and advantageous to
use compared to the Fehlings reagent.
The reaction of the solution to the Benedicts reagent would produce a wide
range of colors in the solution, from blue to green to yellow, orange, and finally
brick-red. A blue colored solution would indicate that the solution has no presence of
reducing sugars. A green solution would indicate traces of reducing sugars, while a
green precipitate would indicate the presence of reducing sugars, albeit in small
amounts. The color of the precipitate formed would move from green to yellow to
orange as the amount of reducing sugars in the solution increases, and finally to
brick-red, in solutions with high amounts of reducing sugars.
In the experiment, solutions C, D, and E produced positive results, with solution
C forming a light red precipitate while solutions D and E forming brick-red precipitates,
indicating the presence of reducing sugars in those solutions.

D. IODINE TEST
Iodine test is used in testing for the presence of starch. A yellow-ish brown color
(i.e. no color change) is a negative test for starch while a positive result yields a
purple-black or bluish-black coloration on the solution when added with Lugols solution,
which is a mixture of water insoluble iodine (I2) and water soluble potassium iodide (KI).
Molecular I2 is not easily soluble in water, which is why KI is added. Together, they form
triiodide ions which forms complex with the starch to create a bluish-black coloration as
shown in the process: . The exact
structure of starch-iodine complex which causes the bluish-black coloration is not clear
since it is hard to study due to its amorphous structure. It has been proposed and

believed that the iodine species inside the helix are repeated or units. However,

recent studies have found evidence that infinite polyiodide chains are contained in
the starch-iodine complex (Madhu et al., 2016), which means that the iodine which

forms complex with starch could be in the form of or .

Starch is a homopolymer of glucose forming an -glycosidic chain produced by
plants as its energy storage. It is composed of 13-20% amylose, a helical non-branching
structure, and 80%-87% amylopectin , which consists of branched chains composed of
24 to 30 glucose residues with 1 4 linkages in the chains and by 1 6 linkages at
the branch points (Rodwell et al., 2015). Amylose is the one that is involved in the
change of color in reaction with polyiodide ions. The amylose acts as a charge donor
and the polyiodide as an acceptor, forming a charge-transfer complex which causes the
change in color (Saenger, W., 1984). This complex absorbs light of a different
wavelength than polyiodide, and the color turns bluish-black. The helical structure of the
amylose is the key factor for the color change. The helical structure is formed because
all the alpha acetal links connect C1 of one glucose to C4 of the next glucose forming
bond angles in the acetal linkage shown in figure 7 (Ophardt, 2017).

Figure 7. A portion of amylose helical chain showing the bending of amylose

chain due to the bond angles formed by alpha acetal links between C1 and C4.
 The polyiodide ions which is in linear conformation slip into the coil of starch
causing an intense bluish-black color as (figure 8). Thus, in the experiment, only
solution A was positive for starch content.

Figure 8. Graphic illustration of a triiodide ion slipping into the helical structure of
amylose observed from top view. Illustration adapted from C. Ophardt. c 2003.

Though iodine test is mainly used for the detection of starch, other
polysaccharide solutions could be qualitatively tested with Lugols solution (Chhabra,
2016). Glycogen, which is the storage polysaccharide in animals, gives a reddish brown
color, amylopectin gives a reddish violet color, inulin gives yellow color, and the
intermediates of starch hydrolysis, namely amylodextrin, erythrodextrin, and
achrodextrin give violet, red, and no color respectively.

VI. CONCLUSION
Carbohydrates are macromolecules which serves as the major source of energy
of our body. Glucose, a monosaccharide carbohydrate, drives the start of several
processes to produce energy. Other carbohydrates enter our body and follows a
specific pathway as it is being metabolised. Like any other molecules, carbohydrates
must be maintained at a certain level. An imbalance concentration of different
carbohydrates may lead into several complications, thus regulation is needed. In order
for us to regulate carbohydrates, one must identify first the type of sugar it possesses.
In the experiment, one of the tests done to classify carbohydrates is the Molisch
Test or also known as the alpha-naphthol reaction. Primarily, it does not classify
carbohydrates instead it verifies the presence of carbohydrates in the sample. It can be
observed that monosaccharides react faster than disaccharides and polysaccharides. In
the experiment, neither of the test tubes gave a positive result (Purple colored layer) it
may be due to a problem with the Molisch reagent.
Seliwanoffs test was also done to distinguish ketoses and aldoses. The
presence of ketoses will give off a deep red color while a light result would mean that
aldose is present in the sugar. In the experiment, test tubes B and D gave a deep red
color which indicated the presence of ketose in the carbohydrate sample.
Another test used in the experiment is the Benedicts test. It is done to identify
which among the samples are reducing sugars. Reducing sugars are carbohydrates
with free aldehyde group or free ketone groups. In the experiment, test tubes C, D, E
gave a positive result marked by a brick red precipitate.
Lastly, Iodine test was also done in the experiment in order to identify which
among the samples given are starch containing. A purple-black or bluish black
coloration would mean a positive result and yellowish brown color would indicate
negative. In the experiment, only test tube A gave a positive result. This means that the
other samples are starch free.
Based from the results of the experiment, the researchers were able to come up
with a flow chart in order to determine the identity of the sample solutions (Figure 9).
The researchers concluded that samples A to E are cornstarch, sucrose, lactose,
fructose, and glucose respectively.
 Figure 9. Flowchart for Determining the Identity of the Unknown Solutions

In general, all the objectives of the experiment are met. Although some results
from the experiment are deviant from the theoretical results as seen in Molisch test, the
mechanism of test was discussed.
 The researcher would like to recommend to be mindful of the purity of the
reagents to be used in the experiment. Contamination of reagents may deviate the
result of the experiment from the theoretical results.

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