NAME: GROUP 2

UNIT: BIOCHEMISTRY 1
UNIT CODE: ICH 2106
LECTURER: DR. STEVE MUHIA
TITLE: PRACTICAL 1; QUALITATIVE TESTS FOR CARBOHYDRATES

EXPERIMENT3:QUALITATIVE TEST OF CARBOHYDRATES

AIM
To find out different types of carbohydrates using different qualitative tests
Introduction

Carbohydrates are the most abundant bio molecules on Earth. Each year, photosynthesis
converts more than 100 billion metric tons of CO2 and H2O into cellulose and other plant
products. Certain carbohydrates (sugar and starch) are a dietary staple in most parts of the
world, and the oxidation of carbohydrates is the central energy-yielding pathway in most non-
photosynthetic cells. Insoluble carbohydrate polymers serve as structural and protective
elements in the cell walls of bacteria and plants and in the connective tissues of animals.

Carbohydrates are an essential component of our diet and an important source of energy for
us. Most of the things generally included in our diet have a large ratio of carbohydrates present.
Also, the patients suffering from Diabetes excrete huge amounts of sugar in their urine which
needs to be estimated. The purity of carbohydrates can also be checked by methods which can
qualitatively estimate particular sugars. Hence the need for carbohydrate estimation arose.

In this experiment, students will be given solutions containing fructose, glucose, lactose,
galactose, ribose, sucrose, starch and two unknown compounds. Students are required to test
the solutions by using a series of reagent that will yield a color change after reacting with
specific functional groups of the compounds being tested. The unknown solutions will be one of
the above solutions

MATERIALS
 Solutions A, B, C, D, E and F. Molisch’s reagent, Benedict’s reagent, Barfoed’s reagent,
Seliwanoff’s reagent, Lugol’s iodine reagent (IKI)

METHODS

The following qualitative tests are performed on 0.2 M solutions (unless otherwise tested) of
Solutions A, B, C, D, E and F. The devised scheme is used in the prelab section to identify an
unknown solution. The unknown will be one of the above solutions or a mixture of two of the
above solutions.

Molisch test for carbohydrates

The molish test is a general test for the presence of carbohydrates. Molisch reagent is a
solution of α -naphtol in 95% ethanol. This test is useful for identifying any compound which can
be dehydrated to furfural or hydromethylfurfural in the presence of H 2SO4. Furfural is derived
from the dehydration of pentose and pentosans, while hydromethylfurfural is produced from
hexoses and hexosans. Oligosaccharides and polysaccharides are hydrolyzed to yield their
repeating monomers by the acid. The α -naphtol 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 indicated the absence of carbohydrates.

i. 2 ml of the solution is put into a clean test tube.

ii. Two drops of Molisch reagent are added into the test tube and been mixed thoroughly.
iii. The test tube is inclined and 5 ml of concentrated H2SO4 been gently poured down the side
of the test tube.
iv. The purple colour is observed at the interface of the sugar which positive result is indicated

Benedict’s test for reducing sugars

 Alkaline solutions of copper are reduced by sugars having a free aldehyde or ketone
group, with the formation of coloured cuprous oxide. Benedict’s solution is composed of
copper sulphate, sodium carbonate, and sodium citrate (pH 10.5). The citrate will form
soluble complex ions with Cu++, preventing the precipitation of CuCO3 in alkaline solutions.
Benedict’s reagent: Generally, use a commercial reagent, but to make it from scratch, first
100 g sodium carbonate and 173 g sodium citrate dehydrate are dissolved in a final volume
of 850 ml water. Slowly, with stirring, 17.3 g copper sulphate pentahydrate is added in 100
ml of water. The final volume is brought to one litre. The commercial reagent, at least,
seems to be stable for years.
i. 1 ml of the solution is put into a clean test tube
ii. 5 ml of Benedict’s reagent is added into the test tube and been mixed thoroughly.
iii. The test tube is placed in a boiling water bath and been heated for 3 minutes.
iv. The test tube is removed and allowed to be cooled.
v. The colour ranges are observed from green to yellow to orange to brick-red depending on
the amount of reducing sugar in the sample.

Barfoed’s test for monosaccharides

This reaction will detect reducing monosaccharides in the presence of disaccharides. This
reagent uses copper ions to detect reducing sugars in an acidic solution. The same color
changes is looked as in Benedict’s test.

Barfoed’s reagent: This looks like Benedict’s but differs somewhat. The reagent is prepared by
dissolving 70 g copper acetate monohydrate and 9 ml glacial acetic acid (pH 4.6) in water to a
final volume of one liter. The reagent is stable for years.

i. 1 ml of the solution is put into a clean test tube.

ii. 3 ml of Barfoed’s reagent is added into the test tube and been mixed thoroughly.
iii. The test tube is placed in a boiling water and been heated for 3 minutes.
iv. The test tube is removed and allowed it to be cooled.
 v. The formation of green, red, or yellow precipitates are observed which positive result are
indicated.

Seliwanoff’s test for ketoses

Seliwanoff’s Reagent: 1 g resorcinol is dissolved in 330 ml concentrated HCl, dilute to one liter
(approximately 4M HCl final). This reagent seems to be stable for more than a year, though we
usually make less than the recipe specifies.

i. 1 ml of the solution is put into a clean test tube.

ii. 5 ml of Seliwanoff’s reagent is added into the test tube and been mixed thoroughly.
iii. The test tube is placed in boiling water bath and been heated for 4 minutes
iv. The test tube is removed and allowed to be cooled.
v. The formation of orange or red colour (not precipitate) is observed which the positive
results are indicated.

Iodine test for starch and glycogen

The use of Lugol’s iodine reagent (IKI) is useful to distinguish starch and glycogen from other
polysaccharides. Lugol’s iodine yields blue-black color in the presence of starch. Glycogen reacts
with Lugol’s reagent to give a brown-blue color. Other polysaccharides and monosaccharides
yield no change; the test solutions remains the characteristics brown-yellow of the reagent. It is
thought that starch and glycogen from helical coils. Iodine atoms can then fit into the helices to
form a starch–iodine complex. Starch in the form of amylose and amylopectin has less branches
than glycogen. This means the helices of starch are longer than glycogen, therefore binding
more iodine atoms. The result is that the color produced by a starch-iodine complex is more
intense than that obtained with a glycogen-iodine complex.

i. 5 ml of the solution is put into a clean test tube.

 ii. 2-3 drops of Lugol’s iodine reagent are added into the test tube and been mixed
thoroughly.
iii. A brown-blue colour is observed which the positive result is indicated for glycogen while
a blue-black colour is for starch. The brown colour of the test reagent is the negative
test.

RESULTS

Type of CHO Barfoed’s Test

Molisch Test

Seliwanoff’s
Benedict’s

Iodine
Test

Test

Test

Solution + + + + -
A(Fructose)
Solution + + + - +
B(Glucose)
Solution + + - - -
C(Lactose)
Solution + - + + -
E(Galactose)
Solution + - - + -
D(Sucrose)
Solution + - - - -
F(Starch)
Discussion

Based on this experiment, we conducted different type of carbohydrate test which are
Molisch, Benedicts, Barfoed and Iodine tests on different carbohydrates. For Molisch test, the
result shown are positive on all types of carbohydrates. 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.

For Benedict’s test, based on the table, the positive results only shown on fructose, glucose and
lactose. Theoretically, in the presence of even small quantities of reducing sugars the entire
body of the solution will be filled with a precipitate which is red. But in the case of non-reducing
sugar like sucrose, the solution will remain perfectly clean.

For Barfoed’s test, experimentally mostly the results shown are positive, but the negative
results shown only on lactose, sucrose and starch also on solution unknown 1. 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.

For Seliwanoff’s test only fructose, galactose and sucrose shows the positive results based on
the table. Theoretically the 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.
Example of ketose is monosaccharide such as glucose, fructose and galactose which will gives
rapid positive result but unfortunately
 For Iodine test, based on the table only glucose gave a positive result. 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. This is due to there might be
a slightly starch contained in glucose which gave a positive result on this test.

Conclusion

Carbohydrates are important biomolecules, in the sense that they are pretty abundant on the
surface of the planet. Having a wide variety of industrial applications as well as already naturally
occurring applications, the actual importance of carbohydrates is already quite obvious.
Ranging from the glycogen that exists as the energy storage within animals, to the cellulose that
forms the integral structure of plants, carbohydrates can definitely be seen as something that is
akin to the basic make up of life as we know it. This experimental procedure has aided in being
able to understand the structures of these carbohydrates and how these physical properties
dictate how the sugar will behave in different situations and the like. Like how sugars are
considered to be reducing sugars if they have an aldehyde group that can be oxidized or if they
are able to create one through isomerism (tautomeric conversion of fructose to an aldose).
Moreover, the knowledge of the fundamental classification test for these compounds has been
invaluable, given that the knowledge and familiarity with these tests also reinforce the
knowledge of the properties of the carbohydrate samples tested.

References

1. Dreywood, R. (1946). Qualitative Test for Carbohydrate Material. Industrial & Engineering Chemistry
Analytical Edition, 18(8), 499-499. http://dx.doi.org/10.1021/i560156a015
2. http://www.biologydiscussion.com/carbohydrates/test/qualitative-and-quantitative-tests-for-
carbohydrates/13042