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Experiment 4

Title: Macromolecules
Objectives:
After completing this exercise, you should be able to:
1. Define monosaccharide, disaccharide, and polysaccharide and give examples of each.
2. Name the monosaccharide components of sucrose and starch.
3. Describe the test that indicates the presence of most small sugars.
4. Describe the test that indicates the presence of starch.
5. Define hydrolysis and give an example of the hydrolysis of carbohydrates.
Introduction:
Macromolecules are defined as large molecules made up of smaller
organic molecules. There are four classes of macromolecules, which
are carbohydrates, lipids, proteins and nucleic acids. The base
elements of carbohydrates and lipids are carbon (C), hydrogen (H) and
oxygen (O). Proteins are also made up of these base elements but it
also contains nitrogen (N).
Carbohydrates have the general formula [CH2O] n, where n is the
number of repeating units. The main function of carbohydrates is
short-term energy storage, such as glucose. A secondary function is
intermediate-term energy storage, as in starch for plants and glycogen
for animals. In addition, some carbohydrates are involved as structural
components in cells, such as cellulose in the cell walls of plants and
chitin in the exoskeleton of arthropods.
Sugars are structurally the simplest carbohydrates. They are the
structural units which make up the other classes of carbohydrates.
Monosaccharide is single sugars. Important monosaccharide includes
ribose (C5H10O5), glucose (C6H12O6), and fructose which is isomer of
glucose. Disaccharides are formed when two monosaccharide are
chemically bonded together. Monosaccharides are linked into
disaccharides and polysaccharides by a reaction that requires the loss
of one water molecule is called dehydration synthesis. Sucrose is a
common plant disaccharide, which is composed of the monosaccharide
glucose and fructose. Lactose is a sugar, which is found in milk, is a
disaccharide composed of glucose and the monosaccharide galactose.
Polysaccharides are large molecules composed of individual
monosaccharide units. A common plant polysaccharide is starch, which
is made up of many glucose subunits. Glycogen is another
polysaccharide that accumulates in the vertebrate liver and serves as
a storage product. Cellulose is a polysaccharide found in plant cell
walls that forms the fibrous and rigid part of the cell wall. In terms of
human diets, cellulose is indigestible and thus forms an important part
of dietary fiber. Monosaccharide and disaccharide can test by Benedict

reagent. It is dark blue alkaline solution and this can test for presence
of aldahyde functional group, -CHO. When the monosaccharide is
heated with Benedict solution, it will form a brick-red precipitate
indicates presence of the aldehyde group which is positive result. If
disaccharide, it will remain clear which is negative result (Anon., 2016).
Lipids are primarily involved in long-term energy storage. They are
generally insoluble in polar substances such as water. Additionally,
lipids serve as structural components also is major building blocks in
cell membranes and hormones that play roles in communications
within and between cells.
Fats and oils function as an energy source. Animals convert excess
sugars into fats. Most plants store excess sugars as starch, although
some seeds and fruits have energy stored as oils for example, corn oil,
peanut oil, palm oil, canola oil, and sunflower oil. Fat molecules consist
of a glycerol molecule and three fatty acids. Another use of fats is as
insulators and cushions. The human body naturally accumulates some
fats in the posterior area. Subdermal fat plays an important role in
insulation. Waxes are an important structural component for many
organisms, such as the cuticle, a waxy layer covering the leaves and
stems of many land plans, as well as protective coverings on skin and
fur of animals.
Phospholipids and glycolipids are important structural components of
cell membranes. Phospholipids are modified so that a phosphate group
(PO4-) replaces one of the long fatty acid tails. The addition of this
group makes the phospholipid head is polar and the two tails is nonpolar.
Proteins are very important in biological systems as control and
structural elements. Two types of proteins, enzymes and protein
aceous hormones, carry out a number of biological control functions.
Enzymes are chemicals that act as organic catalysts which are a
catalyst are a chemical that speeds up, but is not changed by, a
chemical reaction. Structural proteins function in the cell membrane,
muscle tissues, etc. The building block of any protein is the amino acid,
which has an amino end (NH2) and a carboxylic acid end (COOH). The R
indicates the variable or differing component and also known as the Rgroup of each amino acid. Alanine and valine, for example, are both
non-polar amino acids, but they differ, as do all amino acids, by the
composition of their R-groups. All living things use various
combinations of the same twenty amino acids, which is a powerful
piece of evidence for the phylogenetic connection between all living
things. Protein can test by Biuret reagent which is a 1% of solution of
copper sulphate. This will determine the presence of peptide bond in
protein. If presence of peptide bond it will show violet colour which
mean a positive result for protein present. If solutions turn from blue to
pink, which is peptide are present. If still remain blue, which mean no
protein present which is negative result.

Amino acids are linked together by joining the amino end of one
molecule to the carboxyl end of another. Removal of a water molecule,
which is known as dehydration synthesis, allows the formation of a
specialized type of covalent bond known as a peptide bond.

Part 1: Carbohydrates
Investigation 1: Monosaccharide and Disaccharides
Materials:
i.
ii.
iii.
iv.
v.

Benedicts reagent
1% solutions of glucose, fructose, lactose, sucrose, and starch.
Test tube
Beaker
Hot plate

Methods:
1. A boiling water is made by filling up a beaker about half full of water and heating it on
a hot plate.
2. 1 ml of a 1% glucose solution and 5 ml of Benedicts solution is placed in a test tube.
3. A control is prepared.
4. The two tubes is placed in a boiling water for 2-3 minutes.
5. The colour of the solution is observed and is noted whether precipitate is formed.
6. The test is repeated with 1% solutions of fructose, lactose, sucrose and starch.
7. All results were recorded in a table.
Investigation 2: Starch
Materials:
i.
ii.
iii.

Starch solution
Iodine reagent
Test tubes

Methods:
1. A starch solution was prepared by mixing thoroughly 2g of starch with 10 ml of water
and then this mixture was poured into 200 ml of boiling water.
2. Two test tubes were labeled 1 and 2.

3. A few ml of the starch solution in is added to Tube 1. This is the positive control.
4. Tube 2 was the negative control.
5. A few drops of iodine reagent is added into each tube.
6. The observations were recorded.
Investigation 3: Hydrolysis of Carbohydrates
Materials:
i.
ii.
iii.
iv.
v.
vi.
vii.
viii.

Starch solution
Sucrose solution
2N HCL solution
Benedicts reagent
Iodine reagent
Test tubes
Pipettes
Water bath

Methods:
1. Eight test tubes were labeled 1 through 8. The test tubes were lined up in order in a test
tube rack.
2. Two large test tubes were labeled starch and sucrose respectively.
3. 6 ml starch solution and 3 ml 2N HCl were pipette into the tube labeled starch.
4. 5 ml sucrose solution and 1 ml 2N HCl were pipette into the tube labeled sucrose.
5. Each tube were swirl gently to mix the contents.
Sampling
6. 1 ml of solution from the sucrose tube was drawn and put into tube 1.
7. 1 ml of solution from the starch tube was pipette into tube 3 using a different pipette. 8.
An additional ml of solution from the starch tube was added into tube 4.
9. The extra-large starch and sucrose tubes were placed into the boiling water bath. The
time was noted.
10. After 2 or 3 minutes, 1 ml of solution was drawn from the sucrose tube and put into
tube 2.
11. After 5 minutes, 1 ml of solution was drawn from the starch tube and put into tube 5.
12. A second ml of starch solution was added into tube 6.
13. After 10 more minutes, steps 11 and 12 were repeated, with tube 7 and 8.
14. 5 ml of Benedicts reagent were added to Tubes 1, 2, 3, 5, and 7. The tubes were
placed in the boiling water bath for 5 minutes.
15. 3 or 4 drops of iodine reagent were added to tubes 4, 6, and 8.
16. The tubes were removed from the water bath and was allow to cool for 5 minutes.
The results were recorded.
Part 2: Proteins
Materials:

i.
ii.
iii.
iv.

1% egg albumin
Concentrated KOH
0.5% CuSO4
Test tubes

Methods:
1. Two test tubes were labeled 1 and 2.
2. 3 ml of 1 % egg albumin was added into Tube 1.
3. Tube 2 was the control by using tap water as a substitution.
4. Equal volume of concentrated KOH (~ 20%) was added to both tubes and was mixed
thoroughly.
5. 1 ml of 0.5% CuSO4 was added slowly and mixed.
6. After 2 minutes, the colour was recorded in each tube.
Part 3: Lipids
i.
ii.
iii.
iv.

Materials:
Brown paper
Vegetable oil
Water

Methods:
1. A small square of brown paper was prepared. Oil was written on one half and
water on the other brown paper.
2. A tiny drop of vegetable oil is added on the half of the paper labeled oil and rubbed
gently with fingertip.
3. As a negative control, a tiny drop of water was added on the half of the paper labeled
water and is rubbed gently with a different fingertip to avoid contamination.
4. The spots to be allowed to dry.
5. When the spots are dry, the paper was hold up to the light.
6. The observations were recorded.

Results:
This experiment is to determine the various types of macromolecules such as lipid,
protein and sugar. Five tests have been conducted which is the benedict test, iodine test,
reducing and non-reducing test, biuret test and finally lipid test.
Part 1
-Investigation 1
Benedict test has been conducted on five different sugar. The five sugar are glucose,
fructose, lactose, sucrose and starch.

Glucose

Fructose

Lactose
Sucrose
Starch
Glucose and fructose has a clear brick red precipitate formed while lactose have brick red
precipitate as well but it settle at the bottom after a while. Sucrose has a yellowish

solution with blue tint while starch has a clear blue transparent solution. Glucose,
fructose and lactose has a clear positive indication while starch is clearly having negative
result. Sucrose however is ambiguous but probably a negative result.
-Investigation 2
Iodine has been tested with starch and a negative control which is distilled water.

Starch
distilled water (-ve control)
The figure above is iodine with starch. The solution turns dark blue which indicate
positive result while the water with iodine retain the yellowish colour of iodine which
indicate no or negative results.
-Investigation 3
This test is too see the changes of non-reducing sugar as it was being heated with HCL.

Tube Number
Sucrose
Time
(min)
Benedic
ts
reagent

Starch

1
0

2
2-3

3
0

Light
brick
red
precipit
ate

Brick
red
precipit
ate

Blue
transpar
ent
solution

4
0

5
5
Blue
transpar
ent
solution

6
5

7
15

8
15

Blue
transpar
ent
solution

Iodine
reagent

Dark
Dark
Dark
yello
Blue
Blue
w
soluti
soluti
soluti
on
on
on
For the sucrose tube 1 and 2 with Benedicts test yield positive results having lighter
brick red precipitate on tube 1 and brick red precipitate on tube 2. For starch, tube 3, 5
and 7 still having negative results bearing a blue transparent solution. Tube 4 having
negative results with the iodine test having yellow solution which is the iodines colour.
However tube 6 and 8 yield positive results bearing dark blue solution.
Part 2
The test conducted will be a biuret test. It reacts with protein to form purple colour
solution.

From left to right (-ve control (distilled water), albumin, heated albumin)
The reaction with the negative control is colourless solution with blue sediment. This
indicate negative results. The raw albumin yield a blue purple transparent solution while
the heated raw albumin yield a darker purple opaque solution. Either way, both the raw
albumin and heated raw albumin yield positive results.

Part 3
The third test is a simple lipid test. It is to test the presence of lipid since it yield a greasy
appearance.

From left to right (water, vegetable oil)


The brown paper has been split to two sides. One is stain with water and the other has
been stain with vegetable oil. The water side leave no marks as it dried up after a few
minutes. The vegetable oil however leave a dark greasy spot the other side. This indicate
positive results.

Discussion:
Part 1
Monosaccharaides are the most basic units of carbohydrates. Examples of the
monosaccharaides are glucose, fructose and galactose. They are fundamental units of
carbohydrates and cannot be further hydrolysed to simpler compounds. They are the
simplest form of sugar and are usually colorless, water-soluble, and
crystalline solidsMonosaccharaides have a sweet taste, this is because all of the
monosaccharaides are reducing sugar, which contain free aldehyde group or free ketone
group. The monosaccharaides can be divided into two groups: the aldoses, which have
an aldehyde group, and the ketoses, which have a ketone group. Ketoses must
first tautomerize to aldoses before they can act as reducing sugars (Anon., september
2013).
Starch can be separated into two fractions--amylose and amylopectin. Natural starches
are mixtures of amylose (10-20%) and amylopectin (80-90%).Amylose forms a colloidal
dispersion in hot water whereas amylopectin is completely insoluble. The structure of
amylose consists of long polymer chains of glucose units connected by an alpha
acetyl linkage. Iodine is a polysaccharide made up of many glucose molecules. Do to
starch's long structure, a shorter polymer (and component of starch) is stuck in the middle
of starch. The Iodine then binds to this amylose in the starch molecule and turns it black.
Iodine needs to be hundreds if not thousands of glucose molecules to create these starchamylose-amylopectin molecule complexes to which iodine will bind (Larsen, 2012).
Part 2
The biuret test is a chemical test used for detecting the presence of peptide bonds, it can
be used to assess the concentration of proteins because peptide bonds occur with the same
frequency per amino acid in the peptide. The reagent used in the Biuret Test is a solution
of copper sulphate (CuSO4) and potassium hydroxide (KOH).the time are taken when
doing the test , this is because the KOH is there to raise the pH of the solution to alkaline
levels; the crucial component is the copper (II) ion from the CuSO4 (Anon., n.d.).
After the time taken, when peptide bonds are present in this alkaline solution, the
copper (II) ions will form a coordination complex with four nitrogen atoms involved in
peptide bonds, as described in the figure below.

In this figure, the nitrogen on the left are adjacent in the sequence of one peptide, and
the nitrogen on the right are adjacent in the sequence of another peptide (or another
section of the same peptide). The longer a peptide, the more of these complexes can form.
Copper Sulphate solution is a blue color, but when the copper (II) ions are coordinated
with the nitrogen atoms of these peptide bonds, the color of the solution changes from
blue to violet. This color change is dependent on the number of peptide bonds in the
solution, so the more protein, the more intense the change. When the peptides are very
short, the solution turns a pink color, rather than violet.
Part 3
Monosaccharides can be linked together to form disaccharides and polysaccharides by
glycosidic bonds through condensation processes (Mc Murry, 2008). The glycosidic
bonds holding the disaccharides and polysaccharides can be break down to form their
constituent monosaccharides through hydrolysis. In this experiment, the hydrochloric
acid is used as the hydrolyse agent for sucrose and starch. The sucrose and starch
disintegrated to their monosaccharide after being heated with hydrochloric acid.
Sucrose + Hydrochloric acid + Heat Glucose + Fructose
Starch + Hydrochloric acid + Heat Disaccharide maltose
The Biuret test is a test used for detecting the presence of peptide bond. In the presence
of peptide bonds, a copper (II) ion forms violet coordination complexes in an alkaline
solution (Gold., 1990). Both raw albumin and heated albumin gives positive result after
added and mixed thoroughly with equal volume of concentrated KOH and 1 ml of 0.5%
CuSO4. The raw albumin yields blue purple transparent solution white the heated albumin
yields a darker purple opaque solution. On high temperature, the tertiary structures of
albumin changed due to the breaking down of hydrogen bond. Hence, the heated albumin
gives more peptide bond and so darker in colour when tested with Biuret test.
Hydrogen atoms can be added to the fatty acids chain and removes the double bonds
through hydrogenation. One of the test is Bromine test which is a qualitative test for the
presence of unsaturation (R.L. Shrinder & , 1997). The lipid will be tested with a small
amount of elemental bromine in an organic solvent such as dichloromethane or carbon
tetrachloride. Presence of double bond in the lipid will lead to decolourization of the deep
brown colour of bromine, the more the double bond present, the more the disappearance
of the deep brown colour of bromine.

One of the test used to indicate the rancidity of cooking oils is peroxide value which is
a measure of the concentration peroxides and hydro peroxides formed in the lipid
oxidation. Peroxide value is measured by the titration of iodide ion. When the peroxide
values are high, this indicated the lipid is a rancid fat.

Conclusion:
At the end of the experiment, the conclusion of the experiment is glucose, fructose and
lactose are reducing sugar and benedict test is able to indicate reducing sugar by having a
brick red precipitate color solution. Starch and sucrose however are non-reducing sugar
hence the benedict solution remain blue in color. Also starch can be identify by reacting
with iodine reagent to get a dark blue solution which indicate presence of starch.
However by heating starch with hydrochloric acid maltose will be hydrolyzed and will
not react with iodine. It will react after being hydrolyze and form deep blue colour
solution. Also, by reacting sucrose which is a non-reducing sugar with HCL it will form
reducing monomer hence it will react with benedict reagent to form brick red precipitate.
For the protein test, biuret is able to react with protein to form purple solution and
further heating will form a darker purple colour indicate more peptide bond due to
breaking of protein polymer into monomer. Lastly, lipid is able to be identify by grease
leave on the brown paper with the lipid test.

References
Anon., 2016. Benedict's solution. [Online]
Available at: http://www.infoplease.com/encyclopedia/science/benedictsolution.html
[Accessed 8 7 2016].
Anon., n.d. Proteins Test. [Online]
Available at: https://fulltimes.wordpress.com/protein/
[Accessed 11 July 2016].
Anon., september 2013. wikipedia. [Online]
Available at: https://en.wikipedia.org/wiki/Monosaccharide)
Gold., 1990. Organic Compounds In Biological Systems,2nd edition. s.l.:John
Wiley and Sons,Inc.
Larsen, P. D., 2012. ucdavis chemwiki. [Online]
Available at:
http://chemwiki.ucdavis.edu/Core/Biological_Chemistry/Carbohydrates/Case_S
tudies/Starch_and_Iodine
Mc Murry, J., 2008. Organic Chemistry, 7th edition. Belmont: Thomson
Brook/Cole.
R.L. Shrinder, C. H. T. M. & R. F. J. W. &. S. D. C., 1997. The Systematic
Identification of Organic Compounds. s.l.:s.n.