FACULTY OF SCIENCE

BSc (Hons) Biotechnology

May 2014 Trimester

LABORATORY REPORT

Unit Code & Title: UDBB1104 Cell Biology Practical Group: P1

Experiment 4 : Macromolecules

Date of Submission: 8/7/14

Instructor: Dr. Lye Huey Shi

Declaration
I certify that this report is of my own work and does not contain any
unacknowledged work from any other sources.

No. Name Student ID Signature

1 Eswaran 1401660
Objectives:
1. To be able to identify and define monosaccharides, disaccharide and polysaccharides.
2. To be able to identify the monosaccharide components of sucrose and starch
3. Define the test to indicate presence of small sugars, starch, protein and lipids
4. To be able to define and describe hydrolysis

Introduction:
Macromolecules are the very basic building blocks of life. Its what from the simplest
organism to the most complex organisms are made of. Macromolecules consist of four main
classes which are Carbohydrates, Protein, Lipids and Nucleic Acids. In molecular size they are
considered to be huge and this is the reason to their name Macromolecule. This raises an
interesting question. What makes them huge in molecular size? Macromolecules are large in
molecular size because they form polymers. The macromolecules in carbohydrates, proteins, and
nucleic acids are chain-like molecules called polymers. A polymer is a long molecule consisting
of many identical building blocks linked by covalent bonds. The repeating units that serve as the
building blocks of a polymer are smaller molecules known as monomers. These are the aspects
which shape every macromolecule according to the needs of an organism. In this experiment we
will be identifying three macromolecules, which are Carbohydrates, Protein and Lipid.

Carbohydrates may include sugars and polymers of sugars. The simplest carbohydrates
are the monosaccharides or also known as simple sugars. These are the monomers from which
more complex carbohydrates are built on. Disaccharides are double sugars which consist of two
monosaccharides joined by a covalent bond (glycosidic bond). Carbohydrate macromolecules are
polymers called polysaccharides, it is composed of many sugar building blocks such as
monosaccharides and disaccharides. One of the main function of carbohydrate is to store energy.
Two simple tests to identify the presence of carbohydrates are Iodine Test and Benedicts Test.

Proteins are one of the most important macromolecule in our system. This is because the
vital role they play in speeding up chemical reactions (enzyme), defense, storage, transport,
cellular communication, movement, or structural support. Although proteins are diverse, they are
all surprisingly constructed from a simple set of 20 amino acids linked in unbranched polymers,
where the bond between these amino acids are known as peptide bond. This is why a polymer of
amino acids is called a polypeptide. A protein could be defined as a biologically functional
molecule made up of one or more polypeptides, each folded and coiled into a specific three-
dimensional structure. A protein structure has four levels namely Primary Structure, Secondary
Structure, Tertiary Structure and Quaternary Structure. Biuret test could be performed to identify
the presence of peptide bond hence the presence of protein is identified.

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 Lipids are macromolecules that does not include true polymers since they are not big
enough. The reason why lipids are grouped together is because they mix poorly with water
(hydrophobic). They exist in many forms such as fats, phospholipids and steroids. One of their
main function is to store energy as well. Lipids may store twice as much as energy stored in a
carbohydrate, this may be utilized in a long term. A fat is constructed with two components,
glycerol and fatty acid. Three fatty acid molecules are each joined to glycerol by ester linkage. The
resulting fat is called a triacylglycerol. There are two types of fatty acid, the saturated fatty acid
and the unsaturated fatty acid. At room temperature, the molecules of a saturated fat are packed
closely together, this forms a solid. Meanwhile, the molecules of an unsaturated fat cannot pack
together closely enough to solidify because of the kinks in some of their fatty acid hydrocarbon
chains forcing them to exist as liquid (e.g. Vegetable Oil). The Grease spot test/Brown paper test
can be used to identify presence of lipid. (Jane B. Reece, 2013)

Part 1: Carbohydrate
1.1. Monosaccharides and Disaccharides
Materials:
1. Benedicts reagent
2. 1% solution of glucose, fructose, lactose, sucrose and starch
3. Test tube
4. Water Bath
Methodology:

1. A water bath is prepared

2. 1 ml of 1% glucose solution and 5ml of Benedicts solution is placed in the test tube.
3. A control is prepared by adding 5ml of Benedicts solution to distilled water.
4. Both of the test tubes are placed in the water bath for 2-3 minutes.
5. The colour of solution and the precipitate formed are observed.
6. The test is then repeated with 1% sucrose, fructose, lactose and starch.
7. The results were recorded.

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 1.2. Starch
Materials:
1. Starch solution
2. Iodine reagent
3. Test tubes
Methodology:
1. Two test tubes are labelled 1 and 2
2. A positive control is created by putting a few ml of starch solution into test tube 1
3. A negative control is prepared by adding a few ml of distilled water into test tube 2
4. A few drops of iodine reagent are added into each tube.
5. The observations were recorded.

1.3. Hydrolysis of Carbohydrates

Materials:
1. Starch solution
2. Sucrose solution
3. 2N HCl solution
4. Benedicts reagent
5. Iodine reagent
6. Test tubes
7. Water Bath
Methodology:
1. 8 Test tubes were labelled from 1 to 8 and lined up in a test tube rack
2. Two large test tubes are labelled starch and sucrose. An empty beaker was used to hold
these test tubes as they do not fit into the test tube rack
3. 6ml starch solution and 3ml 2N HCl were pipetted into the test tube labelled starch.
4. 5ml sucrose solution and 1ml 2N HCl were pipetted into the test tube labelled sucrose.
5. The contents in this two test tube was then mixed by swirling.
6. 1ml sucrose solution is drawn and put into test tube 1
7. A different pipette is used to draw 1ml of starch solution and put into test tube 3.
8. 1ml of starch solution is added into test tube 4
9. The large starch and sucrose tubes are placed in the water bath. The time was recorded.
10. 2 to 3 minutes later the 1ml of solution from the sucrose test tube is drawn and put into
test tube 2. The sucrose solution was then removed from the water bath.
11. 5 minutes later, 1ml of starch solution is drawn and put into test tube 5
12. 1 ml of starch solution is added to test tube 6
13. Steps 11 and 12 were repeated 10 minutes later by putting the starch solution into test
tube 7 & 8.

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 14. 5ml of Benedicts reagent is added to test tubes 1, 2, 3, 5 and 7. These test tubes were
placed in water bath for 5 minutes
15. 3 drops of iodine reagent was added to test tubes 4, 6 and 8 and placed in the water bath
for 5 minutes as well.
16. The test tubes were then removed and left to cool. The results were recorded.

Part 2: Proteins
Materials:
1. 1% egg albumin
2. Concentrated KOH
3. 0.5% CuSO 4
4. Test tubes
Methodology:
1. Two test tubes were labelled 1 and 2.
2. 3ml of 1% egg albumin was put into test tube 1.
3. Test tube 2 was made control by adding 3ml of distilled water.
4. An equal amount of concentrated KOH was added to both test tubes and mixed
thoroughly.
5. 1ml of 0.5% CuSO 4 is added slowly and mixed together.
6. The colour of solution in each test tube was recorded 2 minutes later.

Part 3: Lipids
Materials:
1. Brown paper
2. Vegetable Oil
3. Water
Methodology:
1. The word Oil is written on one half of a small square brown paper and the word
Water on the other half of the paper.
2. A tiny drop of vegetable oil is added to the half of the paper labelled oil. The oil droplet
was then rubbed gently with fingertip.
3. A negative control was prepared by adding a drop of water to the half labelled water. The
water droplet was rubbed gently.
4. When the droplets have dried off, the paper was hold up to the light and the results were
recorded.

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Results:
Part 1: Carbohydrates
1.1. Monosaccharides and Disaccharides

Sucrose Starch

Distilled Water
(Control)

Lactose

Glucose Fructose
(Figure 1.1: Observation from testing Glucose, Fructose, Lactose, Sucrose,
Starch and distilled water with Benedicts reagent)

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The results obtained are tabulated in the table below:

Solution Observation
1% Glucose The solution formed is brick red in colour
1% Fructose The solution formed is red in colour
1% Lactose The solution formed is brown in colour
1% Sucrose The solution formed is greenish in colour
1% Starch The solution formed is greenish blue in colour
Distilled water Distilled water retains the colour of Benedicts solution, no other changes
observable.

All the carbohydrates tested give a positive result. However the degree of effectiveness of the
test varies from one solution to another.

1.2. Starch
Distilled Water (Control)

Starch

(Figure 1.2: Observation from testing Starch and distilled water with Iodine reagent)

Observation:

The distilled water retains the colour of iodine while Starch solution forms a dark purple
precipitate (positive test indication).

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1.3 Hydrolysis of Carbohydrates

Test Tube 2

Test Tube 3
Test Tube 1

Test Tube 3 Test Tube 4 Test Tube 5 Test Tube 6 Test Tube 7 Test Tube 8

(Figure 1.3: Observation from hydrolysis of starch and sucrose)

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Observation:

The Observation obtained is tabulated in the table below:

Test Tube Number

Sucrose Starch
1 2 3 4 5 6 7 8
Time
(min) 0 2-3 0 0 5 5 15 15
Benedicts Greenish Reddish Blue Blue Blue
Reagent Brown Orange
Iodine Yellow Dark Dark
Reagent with Purple Brown
greenish
brown
precipitate

Part 2: Protein
1% egg albumin

Distilled water
(control)

(Figure 1.4: Observation from testing 1% egg albumin and distilled water with Biuret reagent)

Observation:

Distilled water (negative control) seem to retain the colour of Biurets reagent. However, 1% egg
albumin changes colour to Dark Purple. This is an indication of a positive test

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Part 3: Lipid
Oil

Water (control)

(Figure 1.5: Observation from testing presence of lipid using a brown paper)

Observation:

The part of the brown paper which contains oil creates a translucent spots (grease spots) on
unglazed brown paper (positive test indication). However, the water does not have any effect on
the brown paper.

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Discussion:

Part 1: Carbohydrates
1.1. Monosaccharides and Disaccharides
Benedicts reagent is used to test for simple carbohydrates. Benedict's solution is known to
be a blue colored liquid that contains copper ions. When Benedict's solution and simple
carbohydrates (monosaccharides) are heated, the solution changes to orange red or brick red. This
is a reducing reaction and it is caused by the reducing property of simple carbohydrates. The copper
(II) ions in the Benedict's solution are reduced to Copper (I) ions, which causes the change in
colour of a solution tested. Sometimes a brick red solid, copper oxide, precipitates out of the
solution and collects at the bottom of the test tube. Complex carbohydrates such as starches does
not react positive with the Benedict's test unless they are broken down through hydrolysis which
is done by heating or digestion. Sugar needs to be decomposed into its components such as glucose
and fructose then the glucose test would be positive. That is the reason why sucrose and starch
show a slight indication through colour change because heating the solution with water bath
hydrolyses the polymers of carbohydrates into simple sugars. These sugars are detected by the
Benedicts reagent although they exist in small amount. (Anon., 2009)

(Figure 1.6: Display of colour change in Benedicts reagent depending on amount of simple
sugar found in the solution tested) [Photo obtained from: http://biology-igcse.weebly.com]

The colour change in benedicts solution depends on the amount of non-reducing sugar
present in the solution tested. If there is no non-reducing sugar present in the sample, the solution
would remain blue (no changes). However if the Benedicts reagent turns red then a large amount
of non-reducing sugars are present. There also intermediate groups between this two colours
displayed by the Benedicts solution. These colours and their interpretations are tabulated in the
table below. (IGSCE, 2013)

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 Observation (Final Colour Change) Interpretation
No colour change (Blue) No non-reducing sugar present
Green Trace amount of non-reducing sugar present
Yellow Low amount of non-reducing sugar present
Orange Moderate amount of non-reducing sugar present
Brick-Red Large amount of non-reducing sugar present

We may relate this interpretations to the result obtained and conclude that starch and
sucrose has trace amount of non-reducing sugar present while lactose has moderate amount of non-
reducing sugar present and finally, glucose and fructose has large amount of non-reducing sugar
present.

1.2. Starch

Iodine test is used to indicate the presence of starch. A positive test is indicated when the
iodine turn dark purple. The iodine test is so effective that the colour can be detected visually with
concentrations of iodine as low as 0.00002M at 20C. When starch is mixed with iodine in water,
an intensely colored starch-iodine complex is formed. The iodine is trapped in the coils of beta
amylose molecules of a soluble starch. The starch forces the iodine atoms into a linear arrangement
in the central groove of the amylose coil where some transfer of charge between the starch and the
iodine is. This changes the way electrons are confined and in a way contributes to the changes in
spacing of the energy levels. The iodine/starch complex has energy level spacing that are just
enough for absorbing visible light- giving the complex its intense purple color. (Senese, 2010)

1.3 Hydrolysis of Carbohydrates

From the results obtained we have to closely examine observations from test tube 1 to test
tube 8. Test tube 1 and 2 shows positive result for Benedict test. However, the presence of reducing
sugar is more visible in test tube 2. This is because the presence of HCl in the solution hydrolyses
the sucrose solution into reducing sugars. Although in test tube 1 these reducing sugars are present
in a small amount, they are still indicated by the Benedicts reagent.

Test tube 3, 5 and 7 gives negative reaction for Benedicts reagent since starch is not a
reducing sugar and the addition of HCl does not hydrolyze the big starch molecules into reducing
sugars. However, test tube 4, 6 and 8 seem to display a positive indication for starch. Iodine reagent
in test tube 4 turns yellow with brown precipitate, this might indicate that some starch molecules
may have been hydrolyzed into simple sugars while a big amount of starch molecules still remain
in the solution. Test tube 6 indicates the presence of starch since the Iodine reagent turns dark

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purple. This is a clear indication that starch solution is present. Benedicts solution in test tube 7
turns dark brown this might indicate that there are more amount of starch present and some starch
molecules are hydrolyzed, giving the brown colour.

Part 2: Protein

The biuret test is a chemical test that detects the presence of proteins in a solution. The test
relies on a color change to confirm the presence of proteins. If proteins are found, the sample will
turn violet or dark purple. A common biuret reagent uses an alkaline mixture or reagent, composed
of potassium hydroxide and copper sulfate. The normal color of biuret reagent is blue and the
reagent turns violet in the presence of peptide bonds. Peptide are the chemical bonds that hold two
amino acids together. The proteins detected must have at least three amino acids, which means
that the protein must have at least two peptide bonds in order for the biuret test to display a positive
indication. The reagents copper ions, with a charge of +2, are reduced to a charge of +1 in the
presence of peptide bonds, causing their color to change.

However there are other ways to increase the sensitivity of biuret test. The Smith assay
increases sensitivity a hundredfold, this technique uses bicinchoninic acid as the source of copper
and turns purple when protein is present. A spectroscopic reading at 562 nm would indicate the
amount of protein in the sample. (Bank, 2010)

Part 3: Lipids

The brown paper test is used to identify presence of lipids. When vegetable oil is rubbed
on the brown paper, it creates a translucent spots (grease spots) on the unglazed brown paper. This
is because fats penetrate and get trapped within the paper fibers creating a permanent translucent
spot. Meanwhile for the control, the water on the brown paper will dry off and does not leave
behind any effect on the brown paper. A more accurate test to identify presence of lipids would be
the Sudan test. In this method, a fat soluble dye (Sudan red) stains the lipid molecules red in colour.
This enables us to identify the amount and location of lipids in a test.

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References

Anon., 2009. Testing for Lipids, Proteins and Carbohydrates. [Online]

Available at: http://www.seplessons.org/node/362
[Accessed 3 July 2014].

Bank, E., 2010. What Does a Biuret Test Mean in Biology?. [Online]
Available at: http://education.seattlepi.com/biuret-test-mean-biology-4659.html
[Accessed 4 July 2014].

IGSCE, 2013. Food test 2 - Benedict's test for Reducing Sugars. [Online]
Available at: http://biology-igcse.weebly.com/food-test-2---benedicts-test-for-reducing-sugars.html
[Accessed 3 July 2014].

Jane B. Reece, L. A. U. M. L. C., 2013. Campbell Biology pg 68-81. 10th ed. United States of America:
Benjamin Cummings; 10 edition.

Senese, F., 2010. How does starch indicate iodine?. [Online]

Available at: http://antoine.frostburg.edu/chem/senese/101/redox/faq/starch-as-redox-indicator.shtml
[Accessed 3 July 2014].

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