Experiment No.

3: Qualitative Analysis for Carbohydrates using Glucose, Sucrose, Liver, Paper pulp
and Starch samples

Abstract: Carbohydrates are important because they are the most abundant biological macromolecules on
the Earth and also functions as an essential component of genetic materials and biological membranes.
The goal of the activity is to identify unknown compounds by comparing the reactions of known
carbohydrates using various chemical tests like the Molisch's test, Iodine test and the Benedict's test for the
different classification of carbohydrates like monosaccharide (glucose), disaccharide (sucrose), and
polysaccharides (starch, glycogen in liver, and paper pulp). In the Molisch’s test, exhibited a purple
colored product but it is more appropriate to have a purple ring on each of them which indicates that an -
OH group of sugar are removed in the form of water and furfural is formed from pentose sugar and
hydroxymethyl furfural is formed from hexose sugar when the carbohydrates are reacted and treated with
concentrated sulfuric acid or concentrated hydrochloric acid. While the Iodine test, a positive iodine test
will result to have a dark blue color (starch) while the negative iodine test have a yellow to brown color
that can be seen on glucose, fructose and sucrose. And on the Benedict’s test, resulted to a color change
from blue to brick red, orange or green precipitate which can be seen on glucose while negative Benedict’s
test had no change in color which can be reflected in sucrose after the heating process is conducted.

Balais, Korrine, G.; Carnaje, Ma. Melanie Dalaine, D; Fernando, Elmy Joy, A.; Fresnido, Jeri Anne, O.;
Mamhot, Marco C.; Maramo, Alexandra Therese; Oris, Angela, P.; Group 5, 7:30AM-10:30AM Mondays
& 1:30PM-7:30PM, Mondays, N607 & E610, BS Biology 2-1, Department of Biology, College of Science,
Polytechnic University of the Philippines.

Introduction
Carbohydrates are important because they are the most abundant biological macromolecules on the
Earth. In biochemistry, carbohydrates, having a basic formula of (CH2O)n, are compounds that are
composed of carbon, hydrogen and oxygen. They are organic compounds that contain either an aldehyde
aldose or a ketone. They are also classified as either monosaccharides (e.g. glucose), oligosaccharides (e.g.
sucrose) or polysaccharides (e.g. starch and glycogen). They are linked by glycosidic bonds made by
energy-requiring condensation reactions that release water.
Saccharides, also known as sugars, are mainly known for their significance in energy provision and
storage, structural framework, protection and cell-to-cell interaction. Additionally, it also functions as an
essential component of genetic materials and biological membranes.
In this experiment, the students prepared starch, glucose, sucrose, liver and paper pulp. The goal is
to identify unknown compounds by comparing the reactions of known carbohydrates using various
chemical tests like the Molisch's test, Iodine test and the Benedict's test.

Materials and Methods

1. Equipment and Apparatus

The equipment used upon the accomplishment of this laboratory experiment were hot plate, analytical
balance, water bath, and other glassware.
2. Personal Protective Equipment
Since this experiment involved chemicals, personal protective equipment was worn at all times. These
include laboratory gown, disposable gloves, safety goggles, closed shoes, and neat hair. Those PPEs
asserted were utilized during the experimental procedure.

3. Carbohydrate Sample Preparation

3.1. Glucose and Sucrose Sample Preparation

0.1 M of sugar samples (Glucose and Sucrose) were prepared in a separate container and were
diluted up to 100 mL mark.

3.2. Liver Sample Preparation

Liver sample was triturated in a 10 % Trichloroacetic acid (TCA) to a final proportion of 1 g per
mL TCA. The mixture was strained through ply cheesecloth then the filtrate was collected and was added
two volumes of 95 % ethanol, and a pinch of salt until the creamy white precipitates formed after that, the
mixture was placed in a warm water bath to hasten the precipitation. The upper phase that was layered with
the white precipitate was decanted and filtered through a Whatman filter paper. The precipitate was blotted
and reconstituted in water in order to make 100 g/mL solution.

3.3. Paper Pulp Sample Preparation

5 g of paper was prepared and was teared into pieces. It was macerated in water and diluted up to
100 mL mark.

3.4. Starch Sample Preparation

1 g of starch was prepared and was diluted in 100 mL water. It was then boiled afterwards.

4. Experimental Procedure

4.1. Molisch’s Test

A series of test tubes were prepared. In each test tube, place 5 mL of the carbohydrate sample
(glucose, sucrose, liver, paper pulp, and starch). Two drops of 5% alcoholic alpha napthol was added in the
carbohydrate sample as well as the 1 mL concentration of H2SO4. The change in color was observed after.

4.2. Iodine Test

Five test tubes were prepared. Each of the test tube were contained with 1 mL carbohydrate sample.
In each carbohydrate sample, two drops of 1% iodine solution was added. It was then heated and the change
in color was observed.

4.3. Benedict’s Test

 Test tubes were prepared containing 10 drops of carbohydrate sample. In each carbohydrate sample,
2.5 mL of Benedict’s reagent was added. It was then boiled for 5 minutes. The changes in color of the
samples were then observed after.

Results and Discussion

This section includes the presentation and interpretation of the gathered data during the third
laboratory experiment.
This laboratory experiment merely focused on the carbohydrates wherein several tests were
executed to analyze its qualitative characteristics. Upon accomplishing this, five carbohydrate samples
namely the glucose, sucrose, liver, paper pulp and starch samples were utilized to represent different types
of carbohydrates which some of it comprises monosaccharide (glucose), disaccharide (sucrose), and
polysaccharides (starch, glycogen in liver, and paper pulp). These carbohydrate samples were prepared and
each of it was subjected to Molisch test, Iodine test, and Benedict’s test.

Figure 1. Five formulated carbohydrates sample from left to right: glucose, sucrose, liver, paper pulp, and
starch

A carbohydrate is an organic compound that is, consists only of carbon, hydrogen and oxygen that
makes up the bulk of organic substances on earth and perform numerous roles in living things. The
carbohydrates (saccharides) are catergorized into four chemical groups: monosaccharides, disaccharides,
oligosaccharides and polysaccharides. Saccharides and their derivatives include many other significant
biomolecules that play key roles in the immune system, fertilization, preventing pathogenesis, blood
clotting and development (Qualitative Analysis of Carbohydrates, n. d.).

a. Molisch Test
The first test executed was Molisch test which is a test primarily to detect if a sample contains or
is positive to carbohydrate. It results to a purple ring residue in the sample. It was done by mixing one
carbohydrate sample with concentrated sulfuric acid and .5% alcoholic alpha-naphtol.
Molisch Test is a sensitive chemical test for all carbohydrates, and some compounds containing
carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric acid to produce
an aldehyde (either furfural or a derivative), which then condenses with the phenolic structure resulting in
a purple-colored compound (Qualitative Analysis of Carbohydrates, n. d.).
In addition, according to Karki (2018), in a Molisch test, an -OH group of sugar are removed in
the form of water and furfural is formed from pentose sugar and hydroxymethyl furfural is formed from
hexose sugar when the carbohydrates are reacted and treated with concentrated sulfuric acid or
concentrated hydrochloric acid,. These products reacts with sulphonated α- naphthol to give a purple (violet
red) colored complex.

Figure 2. Five carbohydrates sample from left to right: glucose, sucrose, liver, paper pulp and starch after
Molisch test

Likewise, Karki (2018) stated that for the result interpretation of Molisch test, a positive Molisch
test tend to have a purple color complex while a negative Molisch test had no purple color.
Based on the observations and figures above, all of them had a purple to reddish-violet precipitate
but not all contained an evidently purple ring on it--- they initially all had a purple precipitate and the only
sample that had a long-last purple ring was the paper pulp sample. Thus, paper pulp sample was certainly
tested positive to be a carbohydrate. Unfortunately, those four failed to exhibit that purple ring with longer
duration compare to that of paper pulp that can be seen on the picture above probably due to some errors
but overall, they should be considered to be a carbohydrate derivative since based on a visual observation,
they exhibited a purple colored product but it is more appropriate to have a purple ring on each of them.
 b. Iodine test

Moreover, Iodine test was performed on each carbohydrate sample to test if each formulated sample
contains starch. The sample which tested positive to starch will result to have a blue-black or blue-violet
precipitate. Iodine test uses a carbohydrate sample, 1% iodine solution and heating process to fully
accomplish it.
To support the idea above, iodine test is an indicator for the presence of starch. Iodine solution
(iodine dissolved in an aqueous solution of potassium iodide) reacts with starch producing a blue-black
color (Qualitative Analysis of Carbohydrates, n. d.).
The principle of Iodine test for carbohydrate was that when the starch reacted with iodine, they
formed an absorbed compound that gives blue color and when added by furthered heating process or alkali
like NaOH or KIH, color disappears. This can be supported by a fact that iodine traps in the coiled structure
of polysaccharide. The coiled structure became linear and the iodine molecules were released and the color
disappeared on heating or on addition of alkali (Karki, 2018).
To interpret the results of Iodine test, a positive iodine test will result to have a dark blue color
(starch) while the negative iodine test have a yellow to brown color that can be seen on glucose, fructose
and sucrose (Karki, 2018).

Figure 3. Five carbohydrates sample from left to right: glucose, liver, sucrose, paper pulp, and starch
after Iodine test

As one can see on another figure above, out of five (5) carbohydrate samples, only two (2) of them
which were the paper pulp and superficially, starch made it to be positive in Iodine’s test because of the
blue-black or blue-violet precipitate therefore making them to be concluded that they are composed of
starch. In addition, the other three sample are detected to be negative on iodine test and this can be supported
by what Karki (2018) stated that a negative iodine test have a yellow to brown color that can be seen on
glucose, fructose and sucrose; and that can be seen on liver, glucose and sucrose which resulted to have a
yellow colored sample product in the experiment.

c. Benedict’s test
 Afterwards, the last test demonstrated was Benedict’s test which is a test for reducing sugar.
Typically, this results to a green, orange, or red precipitate. To perform Benedict’s test, a carbohydrate
sample and Benedict’s reagent were combined followed by heating process to obtain a green, orange, or
red precipitate.
Most commonly used tests for detection of reducing sugars are Fehling’s Test, Benedict’s test and
Barfoed’s Test however in this experiment, only the Benedict’s test had been performed.
A reducing sugar is any sugar in a solution that has an aldehyde or a ketone group. The enolization
of sugars under alkaline conditions is an important consideration in reduction tests. The availability of an
aldehyde or keto group helped the sugar to reduce alkaline test reagents for reduction reactions.
Disaccharides or polysaccharides contained glycosidic linkages that bonds a carbohydrate (sugar) molecule
to another one, and thus there is no reducing group on the sugar that can be reflected on the case of sucrose,
glycogen, starch and dextrin. On the other hand, if there is a reducing sugar present, the presence of alkali
causes extensive enolization especially at high pH and temperature. This leads to a higher susceptibility to
oxidation reactions than at neutral or acidic pH. These sugars, therefore, become potential agents capable
of reducing Cu+2 to Cu+, Ag+ to Ag and such (Qualitative Analysis of Carbohydrates, n. d.).
For its principle, the reducing sugar under alkaline condition formed enediols that are powerful
reducing agents and Benedict’s solution happened to contain alkali Na2CO3. Enediols can reduce cupric
ions to cuprous ions which is the basis for Benedict’s reaction and under the heating process, the cuprous
hydroxide heating is converted to red cuprous oxide (Karki, 2018). However, it is not restricted to a red
colored solution, it may be in form with green or orange precipitate that was stated earlier.

Figure 4. Five carbohydrates sample from left to right: sucrose, liver, paper pulp, starch, and glucose
after Benedict’s test

A sample that made it positive to Benedict’s test resulted to a color change from blue to brick red,
orange or green precipitate which can be seen on glucose while negative Benedict’s test had no change in
color which can be reflected in sucrose (Karki, 2018).
Based on the results obtained from Benedict’s test and from the figure above, only glucose made it
positive on Benedict’s test since it resulted to a green-colored product indicating that glucose is a reducing
sugar while the four others were not. It was due to the fact that glucose has free aldehyde or ketone group
that when reacted with alkali, it caused an extensive enolization especially at high pH and temperature. This
leads to a higher susceptibility to oxidation reactions than at neutral or acidic pH. These sugars, therefore,
become potential agents capable of reducing Cu+2 to Cu+, Ag+ to Ag and such in Benedict’s solution.

Furthermore, for the answers on the Guide Questions:

1) What is a reducing sugar? What makes it reducing? Explain the principle of Benedict’s and Barfoed’s
Test. Differentiate the two tests.

A reducing sugar is a chemical term that is used to describe a sugar that acts as a reducing agent
and can donate electrons to another molecule. Specifically, a reducing sugar is a type of carbohydrate that
contains a free aldehyde or ketone group (Rizzo, 2019). A sugar will be a redusing sugar if the stereocenter
has an OH group coming off of it. This is because when the sugar is in the open configuration, that alcohol
becomes a ketone or aldehyde which is able to reduce other compounds (Foist, 2017).
Benedict’s test is used to test for simple carbohydrates. The Benedict’s test identifies reducing
sugars (monosaccharide’s and some disaccharides), which have free ketone or aldehyde functional groups.
Benedict’s solution can be used to test for the presence of glucose in urine. Some sugars such as glucose
are called reducing sugars because they are capable of transferring hydrogens (electrons) to other
compounds, a process called reduction. When reducing sugars are mixed with Benedict’s reagent and
heated, a reduction reaction causes the Benedicts reagent to change color. The color varies from green to
dark red (brick) or rusty-brown, depending on the amount of and type of sugar (Aryal, 2019).
Barfoed’s test is used for distinguishing monosaccharides from reducing disaccharides.
Monosaccharides usually react in about 1-2 minute while the reducing disaccharides take much longer
time between 7-12 minutes to react with the reagent. Brick red color is obtained in this test which is due to
formation of cuprous oxide.

2) Explain the principle of Molisch’s Test.

Molisch’s test is a general test for all carbohydrates. In this test, when carbohydrates are reacted
with conc. H2SO4, it becomes dehydrated to form furfural and its derivatives. When monosaccharide are
treated with conc H2SO4 or conc HCl, -OH group of sugar are removed in the form of water and furfural
is formed from pentose sugar and hydroxymethyl furfural is formed from hexose sugar. These products
reacts with sulphonated α- naphthol to give a purple (violet red) colored complex (Karki, 2018).

3) Explain Molisch’s Test. What are the reagents needed and what for?

Molisch’s test is a general test for the presence of carbohydrates. Molisch’s reagent is a solution
of alpha-naphthol in 95% ethanol. The alpha-naphthol reacts with the cyclic aldehydes to form purple
colored condensation products. Although this test will detect compounds other than carbohydrates (i.e.
glycoproteins), a negative result indicates the ABSENCE of carbohydrates (GCL, 2012).

4) Explain why the iodine test can be used to differentiate polysaccharide from di- and monosaccharides.
 Iodine (iodine-potassium iodide, I2KI) staining distinguishes starch (a polysaccharide) from
monosaccharides, disaccharides, and other polysaccharides. The basis for this test is that starch is a coiled
polymer of glucose. Iodine interacts with these coiled molecules and becomes bluish black. Other non-
coiled carbohydrates do not react with iodine. Therefore, a bluish black color is a positive test for starch,
and a yellow-ish brown color (i.e., no color change) is a negative test for starch. Glycogen, the common
polysaccharide in animals, has a slight difference in structure and produces only an intermediate color
reaction (MSU, 2018).

5) Sucrose and Fructose yields positive results in the Seliwanoff’s Test. What is the principle behind this
test?
Seliwanoff’s test is used to distinguish aldoses from ketoses. This test is used to distinguish aldoses
from ketoses. On treatment with conc. Acid, ketoses are dehydrated more rapidly to give furfural derivatives
and on condensation with resorcinol give cherry red complex. The test will be answered by fructose, sucrose
and other keto containing carbohydrates. On treatment with conc. Acid, ketoses are dehydrated more
rapidly to give furfural derivatives and on condensation with resorcinol give cherry red complex. The test
will be answered by fructose, sucrose and other keto containing carbohydrates. (Karki, 2018)
Ketoses are differentiated from aldoses by means of their ketone/aldehyde usefulness. If the sugar
contains a ketone bunch, it is a ketose. If the sugar contains an aldehyde bunch, it is an aldose. This test is
much like Bial’s test. This test depends on the rule that, when heated, ketones are more quickly dried out
than aldoses.

Conclusion
It is concluded that each test yield different result for each carbohydrates since each test generates
different compounds- Molisch test (general CHO), Benedict’s test (reducing sugar), and Iodine test
(amylose).
Molisch test is a chemical test to detect carbohydrates which undergoes dehydration when heated
with concentrated H2SO4 to form furfural derivatives. Furfural derivatives obtained are condensed with
alpha naphthol to give colored compounds and confirms the presence of carbohydrates (Sharma, 2017).
Glucose is a monosaccharide which gives rapid positive test as well as sucrose which is a disaccharide and
starch, a polysaccharide.
The Benedict’s test is a test used to determine the existence of reducing sugar. All monosaccharides
are reducing sugars. Some disaccharides are reducing sugar due to their exposed carbonyl group. Other
disaccharides used in the experiment, starch and sucrose, are non-reducing sugars which did not react with
Benedict’s solution. Glucose is a reducing sugar which give positive test. Sucrose and starch are non-
reducing sugar- hydrochloric acid is added for positive result.
The Iodine test is used for the presence of starch. It is a type of polysaccharide carbohydrate which
is made up of amylose and amylopectin. Starch is the only carbohydrate in the experiment that gave positive
result in Iodine test as the color of the solution change from yellow to dark blue- the formation of dark blue
color indicates the presence of amylose. This is because the iodine molecules slip inside the amylase coil
and forms helices where iodine molecules assemble.
It is also concluded that each test yield different result for each carbohydrates since each test
generates different compounds- Molisch test (general CHO), Benedict’s test (reducing sugar), and Iodine
test (amylose).
REFERENCES

Foist, L. (2017). Reducing vs. Non-Reducing Sugars: Definition and Comparison.

Gaurab, K. (May 1, 2018). Benedict’s test: Objective, Principle, Reagents, Procedure, and Result.
Retrieved from: https://www.onlinebiologynotes.com/benedicts-test-objective-principle-reagents-
procedure-and-result/

Gaurab, K. (May 1, 2018). Iodine Test: Objective, Principle, Reagents, Procedure, and Result.
Retrieved from: https://www.onlinebiologynotes.com/tests-for-specific-carbohydrates-seliwanoffs-
test-bials-test-and-iodine-test

Gaurab, K. (May 1, 2018). Molisch test: Objective, Principle, Reagents, Procedure, and Result.
Retrieved from: https://www.onlinebiologynotes.com/molischs-test-objectives-principle-reagents-
procedure-and-result/

GCL. (2012). Molisch's Test for Carbohydrates.

(n. a.). (2018). Experiment 1- Qualitative Analysis of Carbohydrates. Retrieved from:

https://www.google.com/url?sa=t&source=web&rct=j&url=http://www.chem.boun.edu.tr/wp-
content/uploads/2014/04/Chem-415-
Experiment1.pdf&ved=2ahUKEwjIl7eulOPkAhVPfd4KHd5kBlQQFjAQegQIARAB&usg=AOvVa
w0jLZOCF-Y4-b7pg0Gdx18N

Rizzo, N. (2019). Live Strong. The Definition of Reducing Sugars.

Sharma, A. (2017, December 16) What is the principle of the Molisch test for carbohydrates? Retrieved
from https://www.quora.com/What-is-the-principle-of-the-Molisch-test-for-carbohydrates