Activity No.

2
Qualitative Analysis of Carbohydrates
(Pilapil, Jean Diane Rose T. BS Chemistry 3)

This experiment aims to introduce you with the identification of unknown carbohydrates. The test
samples were 1% Dextrin, 1% Galactose, 1% Glucose, 1% Lactose, 1% Sucrose, 1% Maltose and 1%
Starch. The test solutions in the Molisch test were treated with Molisch reagent and concentrated
sulfuric acid. In the Orcinol test, test solutions were added with Bial’s reagent and was heated in a flame.
For the Seliwanoff’s test, the test solutions were added with Seliwanoff’s reagent and was heated in a
water bath. And for the Barfoed’s and Benedict’s tests, the test solutions were added with the reagent.
Heated in water bath. In Barfoed’s test, the time was recorded when precipitate forms. Thus, the postive
result for Molisch test is purple liquid layer. In the Orcinol’s test the positive result for pentoses is blue or
green color and hexoses is yellow or brown color. For Seliwanoff’s test, the postive result is red product.
Lastly, for Barfoed’s and Benedicts test were the presence of brick red precipitate.

Introduction

A carbohydrate is an organic compound

with the empirical formula (CH 2O)n where n The 5-carbon monosaccharide ribose is
indicates number of carbons in structure. an important component of coenzymes (e.g.,
Composed only of carbon, hydrogen and ATP, FAD and NAD) and the backbone of the
oxygen, with the last two in the 2:1 atom ratio. genetic molecule known as RNA. The related
deoxyribose is a component of DNA.
Carbohydrates make up the bulk of Saccharides and their derivatives include many
organic substances on earth and perform other important biomolecules that play key
numerous roles in living things. The roles in the immune system, fertilization,
carbohydrates (saccharides) are divided into preventing pathogenesis, blood clotting and
four chemical groups: monosaccharides, development.
disaccharides, oligosaccharides and
polysaccharides. This experiment aims to introduce you
with the identification of carbohydrates. To
Polysaccharides serve for the storage of gain maximum benefit, observations should be
energy (e.g., starch in plants and glycogen in related, as far as possible, to the structure of
animals) and as structural components (e.g., the substances examined (Wiki.
cellulose in plants and chitin in arthropods).
Structural polysaccharides are frequently found
in combination with proteins (glycoproteins or
mucoproteins) or lipids (lipopolysaccharides).
Methodology changed was noted. It was cooled down and the
color observed in each test tubes was recorded.
Chemicals
The chemicals used in the experiment For the Seliwanoff’s test, the test
were 1% Dextrin, 1% Galactose, 1% Glucose, solutions were: 1% Galactose, 1% Glucose and
1% Lactose, 1% Sucrose, 1% Maltose, 1% Starch, distilled water. About 0.5 mL or appoximately
Barfoed’s reagent, Benedict’s reagent, 10 drops of the said test solutions were placed
concentrated Hydrochloric acid and Sulfuric in separate test tubes. 1 mL of Seliwanoff’s
acid, Molisch reagent and Seliwanoff’s reagent. reagent was added and the solution was heated
in a boiling water bath. The time until the color
Materials response was recorded. All the color changes
The materials used in experiment were was recorded in the notebook.
alcohol lamp, 100 mL beaker, 7 pieces droppers,
10 mL graduated cylinder, stirring rod, 11 pieces In the Benedict’s and Barfoed’s tests,
small test tubes, test tube brush, test tube rack, the test solutions were: 1% Glucose, 1%
water bath, iron ring, iron stand and wire gauze. Lactose, 1% Sucrose, 1% Maltose and distilled
water. About 0.5 mL of the test solution and
Procedure 1 mL of each of the reagent was mixed in a
The experiment was divided into four separate test tubes. The mixture was heated in
tests. a boiling water bath and the color of the
The first test was the Molisch test. The precipitate that is formed was recorded. In the
test solutions were: 1% Dextrin, 1% Glucose, 1% Barfoed’s test, the time it takes for the
Sucrose, 1% Starch and distilled water to serve precipitate to form was indicated.
as control or blank test. About 0.5 mL or
appoximately 10 drops of the said test solutions
were placed in separate test tubes. A drop of Results and Dicussion
Molisch regeant was added. The test tube was
slightly tilt, and 0.5 mL of concentrated sulfuric
acid was cautiously added down the side of the
tube. An acid layer forms at the bottom of the
tubes. The color at the interface between the
two layers in each test tube was recorded.

In the Orcinol tests, the test solutions

were: 1% Galactose, 1% Glucose and distilled The Molisch test is a general test for all
water. About 0.5 mL or appoximately 10 drops carbohydarates. The monosaccharides gives a
of the said test solutions were placed in rapid positive test while disaccharides and
separate test tubes. 1 mL of Bial’s reagent was polysaccharides reacts slower.
added. Th solution was carefully heated in a
flame until the mixture boils. The boiling was The Molisch reagent dehydrates
continued for ten (10) seconds or until a color pentoses to furfural and dehydrates hexoses
form hydroxymethylfurfural by the used of
concentrated sulfuric acid. These products The components of Bial’s reagent
condense with α-naphthol to form purple include orcinol, hydrochloric acid, and ferric
condensation product. chloride.
The reaction of this test:
The picture below shows the negative
result (left) and postive result (right).

Source: Google Images

The picture below shows reaction of pentoses

The chemical equation for this test is and hexoses to Bial’s test.

Source: Google Images

Bial's test is used to distinguish

pentoses from hexoses; this distinction is based The test reagent dehydrates
on the color that develops in the presence of
ketohexoses to form 5-hydroxymethylfurfural.
orcinol and iron (III) chloride. Furfural from 5-hydroxymethylfurfural further reacts with
pentoses gives a blue or green color. The
resorcinol present in the test reagent to
related hydroxymethylfurfural from hexoses produce a red product within two minutes
may give a muddy-brown or gray solution, but
(reaction not shown). Aldohexoses react to
this is easily distinguishable from the green form the same product, but do so more slowly.
color of pentoses.
 The photo below shows, the negative more stable than Fehling's reagent. Benedict's
result (on the left), and postive result (on the reagent contains blue copper(II) sulfate
right). (CuSO4)∙ 5H2O which is reduced to red copper(I)
oxide by aldehydes, thus oxidizing the
aldehydes to carboxylic acids. The copper oxide
is insoluble in water and so precipitates. The
colour of the final solution ranges from green to
brick red depending on how many of the copper
(II) ions are present (Al-Ghamdi et al., nd).

The photo below shows the negative

result (on the left) and positive result (on the
Source: Google Images
right) of Barfoed’s test.

Source: Google Images

The photo below is the result of

Benedict’s test for carbohydrates.
Barfoed's test is used to detect the
presence of monosaccharide (reducing) sugars
in solution. Barfoed's reagent, a mixture of
ethanoic (acetic) acid and copper(II) acetate, is
combined with the test solution and boiled. A
red copper(II) oxide precipitate is formed will
indicates the presence of reducing sugar. The
reaction will be negative in the presence of
disaccharide sugars because they are weaker
reducing agents. This test is specific for
monosaccharides. Due to the weakly acidic Source: Google Images
nature of Barfoed's reagent, it is reduced only
by monosaccharides.

This test is used also to differentiate

between reducing and non reducing sugars. It
works on the same principle but Benedict is
Conclusion

It was concluded that carbohydrates

can qualtitative analyze using different test. The
postive result for Molisch test is the presence of
purple liquid layer. In the Orcinol’s test the
positive result for pentoses is blue or green
color and hexoses is yellow or brown color. For
Seliwanoff’s test, the postive result is red
product. Lastly, for Barfoed’s and Benedicts test
were the presence of brick red precipitate.

References

Al-Ghamdi, Sharifa & Al- Shaibi, Huda.

Carbohydrates. Retrieved from
www.kau.edu.sa/Files/0007306/Subjects/3)car
bohydrates.doc

Cox, M. & Nelson, D. Lehninger Principles of

Biochemistry. 4th edition

Experiment 1- Qualitative Test for

Carbohydrates. Retrieved from:
http://www.chem.boun.edu.tr/wp-
content/uploads/2014/04/Chem-415-
Experiment-1.pdf

Ferrier, D. & Harvey, R. Biochemistry (2011).

Lippincott Williams & Wilkins, a Wolters Kluwer
business