UNIVERSITY OF NICOSIA – LIFE AND HEALTH SCIENCES
General Biology I – BIOL-101
BIOMOLECULES
Carbohydrates, lipids, proteins and DNA
OBJECTIVES
After the completion of the Laboratory exercise students should be able to:
1. Describe the tests indicating the presence of monosaccharides and disaccharides
2. Describe the test for the presence of starch.
3. Define hydrolysis and give an example of the hydrolysis of carbohydrates.
4. Describe the tests that indicate the presence of lipids.
5. Describe the tests for the presence of proteins.
6. Describe the tests for the presence of DNA
INTRODUCTION
Carbohydrates, lipids, proteins, and nucleic acids are the four major classes of organic
compounds (biomolecules) in cells. Some of these compounds are very large and are called
macromolecules. From these biomolecules, carbohydrates, lipids, and more rarely proteins (when
nutrition is poor in carbohydrates and lipids for prolonged time) are used as fuel sources in the
cell.
Carbohydrates are found in bread, potatoes, cereals, pastas, fruits, vegetables and sweet foods.
They are composed of carbon, hydrogen and oxygen. The hydrogen and oxygen occur in a 2:1
ratio, as in a molecule of water. The general formula for a carbohydrate is (CH2O)n=3-7. There are
three types of carbohydrates:
a. Monosaccharides. Simple sugars with one sugar molecule, e.g. glucose, fructose and
galactose. The general formula for glucose is C6H12O6.
b. Disaccharides. Double sugars with two sugar molecules, e.g. glucose + glucose = maltose,
glucose + fructose = sucrose, glucose + galactose = lactose. (Monosaccharides and
disaccharides dissolve in water and are sweet to the taste).
c. Polysaccharides. Multiple sugars made up of many monosaccharide units linked together to
form a very large molecule, e.g. starch, glycogen and cellulose. Polysaccharides are insoluble
in water and are used as reserves of carbohydrate because they can be stored by the
organism. Plants store their carbohydrates as starch, whilst animals utilize glycogen. Plants
synthesize cellulose, which is a structural polypeptide. It consists entirely of glucose units, but
it has different structure than starch and glycogen.
Lipids or fats are found almost as pure fat in butter, lard and margarine. They are also found in
foods such as milk, nuts and fatty meat. They are composed of carbon, hydrogen and oxygen,
combined together in different proportions than the carbohydrates. Fats are made up of fatty acids
and glycerol. Three molecules of fatty acids and one molecule of glycerol are united to form one
molecule of fat. If there are no double bonds between the carbon atoms composing the fatty acid,
it is called saturated fatty acid. An unsaturated fatty acid has one or more double bonds in its
skeleton. The fatty acids of most animal fats are saturated, whereas liquid fats such as olive oil
contain unsaturated fatty acids. Cell membranes consist mainly of phospholipid bilayer but the
main role of fats is energy storage.
Proteins are found in meat, fish, milk, cheese and eggs. They are composed of carbon, hydrogen,
oxygen, nitrogen and sometimes sulphur and phosphorus. They are molecules which have a high
molecular weight. They are made up of amino acids linked together with peptide linkages (CONH) to form long chains. Each amino acid has a basic amino group-NH2, at one end and an acidic
carboxyl group-COOH, at the other end. The amino acid has also a lateral group-R, which varies
depending on the particular amino acid, e.g. glycine is the simplest amino acid where the R group
is an atom of hydrogen.
Deoxyribonucleic acid or DNA is found in all living organisms. The function of DNA is information
storage. Every living organism is defined by a “blueprint” that dictates what chemical building
blocks are produced to make up the organism and how they are assembled. DNA consists of
nucleotides. In each nucleotide one ring structure known as a nucleotide base is covalently
bonded to a sugar, deoxyribose. There are four different nucleotide bases in DNA named,
Adenine, Thymine, Cytosine and Guanine. The order in which different nucleotides are hooked
together in DNA polymers determines the physical attributes of, and the chemical reactions that
occur in, living organisms.
LABORATORY WORK
(2 hours)
Materials and reagents
Sugar solutions (glucose, fructose, sucrose, starch), I2/KI solution, egg albumin solution, Fehling
solution, Benedict solution, ninhydrin solution, Cu2SO4, dilute HCl, concentrated HNO3, solid
sodium bicarbonate, sodium hydrogen carbonate, Sudan III dye, ethyl alcohol, test tubes,
absorbent paper, hot plates, pH-paper, Pasteur pipettes, glassware.
Hypothesis:
Prediction:
Part One
Tests for sugars
Fehling’s Test
Monosaccharides
1. To 1 ml of glucose solution (use 0.1-1% for sugar solutions) add sufficient Fehling’s solution
to give the contents of the test tube blue color. (Fehling’s solution is made by mixing equal
volumes of Fehling’s 1 and 2 together). Boil for 1 minute. An orange-red precipitate of copper
oxide indicates the presence of a reducing sugar. The copper sulphate in the Fehling’s
solution has, therefore, been reduced to copper oxide. Hence glucose is often referred to as
a reducing sugar.
Disaccharides
2. Now test for the disaccharide sucrose (a non-reducing sugar). Proceed as in step 1. Did you
observe any changes?
Sucrose is a non-reducing sugar, so it must be broken down into its constituent monosaccharide
units before Fehling’s test can be carried out. If sucrose is boiled with diluted hydrochloric acid, it
will be hydrolyzed to glucose and fructose.
3. Add 2 ml of sucrose solution to a test tube. Add 0.5 ml diluted hydrochloric acid. Put the tube
into boiling water for 1 minute. Then, carry out Fehling’s test.
Iodine test for starch
To 1 ml of a fresh solution of 1% starch add a few drops of iodine solution. A blue-black color
indicates the presence of starch.
Part Two
Tests for lipids
Emulsion test
Take 2 ml of ethyl alcohol (ethanol) in a test tube and add 1 ml of olive oil. Shake well so that the
oil mixes in well with the alcohol. Now add 2 ml of cold water. The presence of fats is indicated
by the formation of small droplets or globules of fat in suspension. These droplets give a milky
appearance. The formation of droplets and globules is due to hydrophobic interactions with water.
Lipids are immiscible with water. Adding water to a solution of a lipid in alcohol, results in an
emulsion of tiny lipid droplets which reflect light and give a white appearance.
Sudan III test
Sudan III is a red dye. Add 2 ml of oil to 2 ml of water in a test tube. Add 4-5 drops of Sudan III
and shake. A red stained oil layer separates on the surface of the water, which remains uncolored.
Fat globules are stained red and are less dense than water.
Part Three
Tests for proteins
Biuret test
To 2 ml of a 1% solution of egg albumin (BSA) add 3 ml of 40% sodium hydroxide and shake the
test tube well. Then add 5 drops of 1% copper sulphate. A violet color indicates the presence of
protein. The Biuret test indicates the presence of peptide linkages between amino acids. In the
presence of dilute copper sulphate in alkaline solution, nitrogen atoms in the peptide chain form
a purple complex with copper (II) ions (Cu2+). Biuret is a compound derived from urea, which also
contains the –CO-NH- group and gives a positive result.
Xanthoproteic test
To 3 ml of 1% egg albumin (BSA) add 1 ml of concentrated nitric acid. A white precipitate should
appear. Boil until the solution is yellow. Cool and add 2 ml of concentrated 40% NaOH to give an
orange color. This indicates the presence of protein. The xanthoproteic test indicates the
presence of specific amino acids, e.g. tyrosine, tryptophan and phenylalanine due to specific
groups on them.
Part four
Test for DNA
Diphenylamine test
DNA can be specifically detected using the Dische diphenylamine reagent. The deoxyribose
moeity of DNA is converted to a molecule which binds with diphenylamine to form a blue colour,
which is proportional to the concentration of DNA. The diphenylamine test is thus a quantitative
test for DNA.
Obtain two test tubes and add 1 ml of DNA solution to #1, and add 1 ml of water to #2. Then add
2 ml diphenylamine reagent to each, place in a boiling water bath for ten minutes and look for a
light blue colour forming.

Identify the unknown samples of various biomolecules solutions you are given in the table.
Questions for discussion
 What subunits make up Carbohydrates, Proteins, DNA
 You have been given an unknown solution. Describe how you would test it for the presence
of: Starch, Lipid, Monosaccharides, Protein, DNA.
 You have tested an unknown sample containing only one type of macromolecule with biuret
and Fehling’s tests. The solution when mixed with biuret reagent becomes violet. The same
solution when boiled with Fehling’s reagent changes to Brownish-orange. What does this tell
you about the sample?
 Whole butter gives negative test for protein. When the same butter is clarified, however, the
liquid lower layer is definitely positive for protein. Explain why these different results might
have been obtained.
References
1. Campbell, N.A. 1999. Biology. (5th ed.). Benjamin-Cummings.
2. Robinson, M.A. and Wiggins, J.F. 1989. Animal Types 2: Vertebrates. Hutchinson & Co.
(Publishers)
3. Fragoulis, E.G. 1992. Laboratories of Biochemistry I & II. University of Athens, Department of
Biology