QUALITATIVE ANALYSIS OF CARBOHYDRATES

EXPERIMENT 1- QUALITATIVE ANALYSIS OF CARBOHYDRATES

A carbohydrate is an organic compound with the general formula C m(H2O)n, that is,
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 earth and perform
numerous roles in living things.

The carbohydrates (saccharides) are divided into four chemical groups:

monosaccharides, disaccharides, oligosaccharides and polysaccharides.
Polysaccharides serve for the storage of energy (e.g., starch in plants and glycogen
in animals) and as structural components (e.g., cellulose in plants and chitin in
arthropods). Structural polysaccharides are frequently found in combination wit
proteins (glycoproteins or mucoproteins) or lipids (lipopolysaccharides). The 5-carbon
monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD
and NAD) and the backbone of the genetic molecule known as RNA. The related
deoxyribose is a component of DNA. Saccharides and their derivatives include many
other important biomolecules that play key roles in the immune system, fertilization,
preventing pathogenesis, blood clotting and development [1].

This experiment aims to introduce you with the identification of unknown

carbohydrates. To gain maximum benefit, observations should be related, as far
as possible, to the structure of the substances examined.

Some important points:

1. Most of the tests and reactions described are not quantitative and volumes are
approximate, despite these facts some tests do not work if quantities greatly in
excess of those stated are used.
2. DO NOT place your pipettes in reagent bottles as this leads to contamination.
3. In most tests, it is important to apply a control test using water instead of the
solution under examination. If you are in doubt about the result of a test,
perform the reaction with a suitable known compound.
4. In this experiment, sugar samples are given in their solid state. To perform
each procedure, you should prepare your own sugar solution by taking very
small amounts of solid sugars.
5. When you need to boil your sample in a test tube, prepare a hot water in a
large beaker and put your test tube inside the beaker. DO NOT forget to put
boiling chips in the beaker.
TESTS ON CARBOHYDRATES:

1) Molisch’s Test:

Molisch’s 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 red or
purple-colored compound.

Procedure:

2) Carbohydrates as Reducing Sugars:

A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone

group. The enolization of sugars under alkaline conditions is an important
consideration in reduction tests. The ability of a sugar to reduce alkaline test reagents
depends on the availability of an aldehyde or keto group for reduction reactions. A
number of sugars especially disaccharides or polysaccharides have glycosidic
linkages which involve bonding a carbohydrate (sugar) molecule to another one, and
hence there is no reducing group on the sugar; like in the case of sucrose, glycogen,
starch and dextrin. In the case of reducing sugars, 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
so fort. Most commonly used tests for detection of reducing sugars are Fehling’s
Test, Benedict’s Test and Barfoed’s Test.
 a) Fehling’s Test:

Fehling’s Solution (deep blue colored) is used to determine the presence of reducing
sugars and aldehydes. Perform this test with fructose, glucose, maltose and
sucrose.

Procedure:

b) Barfoed’s Test:

Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced so
that is can only be reduced by monosaccharides but not less powerful reducing
sugars. Disaccharides may also react with this reagent, but the reaction is much
slower when compared to monosaccharides. Perform this test with glucose, maltose
and sucrose.
Procedure:

c) Seliwanoff’s Test:

Seliwanoff’s Test distinguishes between aldose and ketose sugars. 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. This test is based on the fact that, when heated, ketoses are more rapidly
dehydrated than aldoses. Perform this test with glucose, fructose, maltose and
sucrose.
Procedure:

Questions:

 Write the reaction(s) involved in Seliwanoff’s Test.

Seliwanoff’s test is normally used to test the presence of ketohexoses in

carbohydrates. The reagents consist of resorcinol and concentrated
hydrochloric acid. The dehydrated ketose then reacts with two equivalents
of resorcinol in a series of condensation reactions to produce a molecule
with a deep cherry red color. Aldose may react slightly to produce a faint pink
color.

 What is the function of resorcinol?

Resorcinol, a complexing agent, is used to provide the color reaction

(cherry red complex) for ketoses.

 What is the aim of using a strong acid?

A strong concentrated acid is a treatment will dehydrate the ketoses more

quickly in order to yield furfural derivatives, and that condensation of
resorcinol yields to a cherry red complex.
  What is the result of testing sucrose with Seliwanoff’s reagent? Explain your
answers by giving reasons and structures.

Sucrose is a disaccharide composed of fructose and glucose; this results

in a positive test. This test is typically performed with 6M HCl. HCl of
Seliwanoff’s reagent cleaves sucrose into fructose (ketose) and glucose
(aldose). This fructose then reacts with resorcinol to yield dark red color
immediately, while the glucose reacts slowly. This is presented and can be
understood better with the help of the structure provided below.

d) Bial’s Test:

Bial’s Test is to determine the presence of pentoses (5C sugars). The components of
this reagent are resorcinol, HCl, and ferric chloride. In this test, the pentose is
dehydrated to form furfural and the solution turns bluish and a precipitate may form.
Perform this test with ribose and glucose.

Procedure:
 3) Action of Alkali on Sugars:

Procedure:

4) The Inversion of Sucrose:

Sucrose is a disaccharide, which means that it is a molecule that is derived from two
simple sugars (monosaccharides). In the case of sucrose, these simple sugars are
glucose and fructose. Inverted sugar is a mixture of glucose and fructose. It is
obtained by splitting sucrose into these two components. The splitting of sucrose is a
hydrolysis reaction which can be induced simply by heating an aqueous solution of
sucrose. Acid also accelerates the conversion of sucrose to invert.
Procedure:

5) Iodine Test:

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. Apply this test to all the polysaccharides provided.

Procedure:
Post Lab Discussions

Questions:

1. What is the chemical reaction involved in Molisch Test?

Dehydration is involved in Molisch test, the dehydration of the carbohydrate by

sulfuric acid produce an aldehyde.

2. Write the reaction(s) involved in Fehling’s Test.

Fehling's solution can be used to distinguish aldehyde vs ketone functional

groups. Aldehydes are oxidized, giving a positive result, but ketones do not react.

3. Some disaccharides such as maltose are reducing agents, whereas others, such
as sucrose are not. Explain briefly by including the structures of the sugars.

Maltose is a reducing disaccharide while sucrose is a non-reducing one

because of the absence of free aldehyde or ketone group in sucrose. While in
maltose, there are two glucoses present. So, this aldehydic group allows the sugar
to act as reducing sugar.

4. Write the reaction(s) involved in the Barfoed’s Test.

The aldehyde group of the monosaccharide is oxidized to the carboxylate.

5. When you test starch with Barfoed’s reagent, what would be the answer, positive
or negative? Explain your answer by giving reasons and structures.

Positive because the red precipitate is formed meaning the monosaccharide is

present.

6. Write the reaction(s) involved in Bial’s Test.

Dehydration is present in Bial’s test, the reason for that is the pentose is
dehydrated to form furfural and the solution have generated a colored substance
which turns bluish and a precipitate may form.

7. Is it possible to distinguish DNA and RNA structures by using Bial’s Test?

Bial’s test is performed to detect the presence of pentoses and

pentosans. This test can also be used for the quantification of RNA in a sample.
8. The boiling step is common for each test for the reducing sugars. Why boiling is
necessary for the reduction to take place?

The presence of alkali causes significant enolization of reducing sugars,

especially at high pH and temperature; sugar enolization in alkaline
circumstances is a crucial factor in reduction tests. Boiling is a physical heating
procedure that hydrolyzes some of the carbohydrate and fiber content in raw
materials into fermentable sugars or reducing sugars.

9. Name the complex formed by the addition of concentrated sulfuric acid to a sugar
solution and explain the reaction?

Carbon snake is a demonstration of the dehydration reaction of sugar by

concentrated sulfuric acid. An exothermic reaction takes places during which a
column of carbon rises from the beaker with a cloud of steam and sulfur dioxide. In
the presence of concentrated sulfuric acid sucrose is dehydrated to produce
carbon and water. The heat of the reaction vaporizes the water causing the column
of carbon to form.

10. Why sucrose gives negative Benedict test?

Sucrose is thus a non-reducing sugar which does not react with Benedict's
reagent. Sucrose indirectly produces a positive result with Benedict's reagent if
heated with dilute hydrochloric acid prior to the test, although after this treatment it will
no longer be sucrose.

11. Explain, although starch has free hemiacetal bond it gives negative Benedict test?

Starches do not react or react very poorly with Benedict's reagent due to the
relatively small number of reducing sugar moieties which occur only at the ends
of carbohydrate chains. Other carbohydrates which produce a negative result
include inositol. One hemiacetal “needle” in a haystack of “acetals” is not enough to
give a positive test for reducing sugars. Therefore these polysaccharides are not
considered reducing sugars.

12. Why glucose (monosaccharide) and maltose (disaccharide) give positive Benedict
test?

As we’ve seen, glucose is in equilibrium with an open-chain (or “linear”) form

containing an aldehyde. Maltose is a reducing sugar because its two glucose
 molecules are connected in such a way that one anomeric carbon can open
to form an aldehyde group. Sugars with a hemiacetal functional group give positive
tests since they are in equilibrium with an open-chain aldehyde. As a result, like
glucose, maltose produces a positive Benedict's test result.

13. What is the difference between Benedict and Barfoed's reaction?

The pH of Barfoed's reagent is lower (about 4.5) and the heating time is
shortened to two minutes, compared to Benedict's reagent. Benedict's test
determines whether the sample is a reducing sugar, and Barfoed's test determines
if it is a monosaccharide or disaccharide.

14. What are the carbohydrates that give positive result with Seliwanoff? Why?

Seliwanoff’s test is used to differentiate between sugars that have a ketone

group (ketose) and sugars that have an aldehyde group (aldoses). This test is a
timed color reaction specific to ketohexoses. Fructose and sucrose are two
common sugars which give a positive test. Fructose produces positive results since
it contains a ketose sugar. Sucrose gives a positive test as it is a disaccharide
consisting of fructose and glucose.