Aimee Symes -1-

Candidate Number: 4610

Biology Coursework

Hypothesis: To investigate the effect of different concentrations of

ethanol on the permeability of beetroot cell membranes.

Prediction: By exposing a membrane to a solvent, ethanol, it will affect

its permeability. Therefore higher the concentration of the solvent, the
more permeable the membrane will be. But by increasing the
concentrations of the ethanol beyond a certain point it may have no
affect on the cell membrane because the ethanol would have broken down
the cell membrane completely. This is because of the effect of ethanol on
the lipids in the membrane. Also, more dye would be present as the lipids
are essential to the structure of the cell membrane as they control the
substances that enter and leave the cell. Ethanol could also destroy some
the proteins or denature the structure because protein has a tertiary
structure. The ethanol would destroy the hydrogen bonds that hold the
protein structure. Therefore, with the lipids and proteins destroyed in
the cell membrane, the pigment is allowed to escape from the cell due to
there being no cell membrane holding substances in the cell.

Scientific Knowledge:
Transfer across cell membrane structure:
There are many ways that ions and molecules are transported across the
cell membrane. By this, the cell must acquire the ions and molecules they
need from their surrounding ‘extracellular fluid’. With the cell membrane
being in contact with ethanol in the extracellular fluid, there is direct
contact with the solvent. Therefore, by diffusion the ethanol is secreted
into the cell and along the way destroys the permeability of the cell
membrane.

The cell transports ions and small molecules across their membranes by
the following means:
Osmosis:
By osmosis the diffusion of water through the plasma membrane is
possible. Since the lipids bilayers are impermeable to essential molecules
and also to a few small molecules like oxygen and carbon dioxide, these
molecules and ions diffuse freely across the cell membrane.
Diffusion or Facilitated diffusion (Passive transport):
By diffusion the ions and molecules move spontaneously across their
concentration gradient (from a region of higher to a region of lower
concentration). Small, polar, hydrophilic molecules, like oxygen, and
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water, can pass through cell membranes by diffusion. Where as larger
hydrophilic molecules, like glucose, can pass through the cell membrane by
facilitated diffusion. In all cases of facilitated diffusion through
channels, the channels are selective. Meaning that the structure of the
protein admits only certain types of molecules through (hydrophilic
pores). Where as non-polar, hydrophobic molecules are not impeded by
the phospholipids in the cell membrane. This means that they are small
enough to fit between the phospholipids layers. The factors that affect
the rate of diffusion are: concentration gradient, temperature, pH (the
charge), surface area, size of molecule and the width of the diffusing
space.
Active transport:
By active transport, ions and molecules are forced by the use of
metabolic energy to move against their concentrated gradient. Forms of
active transport are:
Phagocytosis:
The ingestion of solids from outside of the cell is the process of
phagocytosis. The cell membrane encloses a particle and buds off to
form a food vacuole. Lysosomes with enzymes will fuse with it to enable
digestion of the contents. Soluble products pass out of the vacuole and
are assimilated.
Pinocytosis:
The ingestion of the fluid surrounding the cell is the process of
pinocytosis. The cell membrane encloses some of the fluid and pinches
off to form a vesicle. As the vesicle is closed off the materials are
dispersed in the cell, which is referred to as assimilation.
Exocytosis:
Vesicles budded off from the Golgi apparatus or endoplasmic reticulum
can fuse with the cell membrane, expelling their contents.
Endocytosis:
Where a substance attaches to the cell membrane, inducing it to slide or
flow inwards forming a pouch.

Function and structure of cell membranes:

All cells have a cell membrane that forms the outer limit of the cell. In
the fluid mosaic model, the membrane structure consists of a double
layer of phospholipid molecules, which is called the lipid bilayer, with a
hydrophilic head which is on the outside of the membrane and a
hydrophobic tail which is on the inside of the membrane. The cell
membrane is considerably quite fluid and this is helped by cholesterol,
which help disturb the close packing of the phospholipids by breaking up
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Candidate Number: 4610
the Van der Waals forces. The proteins that float in this bilayer are
either on the surface or they completely penetrate the lipid layer. The
proteins that penetrate the bilayer may control the entry and removal of
specific molecules from the cell. The proteins that are on the outer edge
or on the surface of the membrane have carbohydrates molecules
attached, usually short sugar chains. These are called glycoproteins. The
carbohydrates part of the glycoproteins is important in cell recognition,
which is the ability of cells in the body to tell whether or not a cell is
from another individual or another organism (invading pathogens), for
example, in the immune system. Some substances, particularly
carbohydrates and ions, are transported across the membrane via the
proteins. Where as some substances, including water, are transported
directly through the lipid layer.

(Picture from: http://www.jdaross.mcmail.com/cell2.htm)

The lipids in the bilayer are soluble to organic solvents with low molecular
weight molecules, such as ethanol; therefore to have beetroot in ethanol
would affect the lipids in the bilayer by destroying or dissolving them.
Hence, more pigment would leak into the solution because there would be
less, if any, structure to the cell membrane to hold in the pigment if the
lipids have been damaged or destroyed.

Biological molecules and cell membrane:

The cell membrane can be seen as a fluid structure in which proteins can
move about depending upon the fluidity of the lipids and the amount of
cholesterol. The amount of fluidity of the membrane influences the
function. By reducing the fluidity in the cell membrane it decreases the
ability of proteins to move and interact, while enhancing the fluidity it
may lead to a break down in the cell membrane. Cell membranes have
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Candidate Number: 4610
been tested upon with ethanol, which have been shown to fluidise the
membrane, which perhaps provides a basis for altered protein function.
Protein in its tertiary structure has been affected and altered or even
denatured which affects the overall fluidity in the cell membrane and the
function or interaction of the proteins. “These influenced cellular
function in the brain and body and led to the various symptoms of alcohol
action.” (Quoted and information from:
http://chemcases.com/alcohol/alc-13.htm)

So not only does ethanol affect the soluble lipids in the cell membrane, it
also affects the proteins in the cell membrane by altering their functions
and interactions between each other. This is because the ethanol affects
the hydrogen bonding in both the lipids and peptide bonds in proteins, as
this is what holds the structure together.

Beetroot:
The betacyanin pigment of beetroot is located in the sap vacuole and, “by
means of the properties of the tonoplast and cell membrane, does not
leak into the cytosol or the extra-cellular sap of the beetroot.” (Quoted
from: www.mrothery.co.uk/cells/resources/betacyanin.doc) When
beetroot is cut, the cells are sliced and naturally pigment spills out. If
the slices were thinner, thus providing a larger surface area it therefore
speeds up the pigment leakage. But if the membrane is destroyed and
the phospholipid bilayer and possibly proteins are altered, more pigment,
betacyanin, and leaks by means of diffusion. The pigment serves as a
marker for scientists who want to isolate intact vacuoles.
(http://www.biologymad.com/resources/BEETROOT%20PIGMENT2.doc)

The basic structure of betacyanins

(Picture from: www.mrothery.co.uk/cells/resources/betacyanin.doc)

Betacyanin: Structure and Solubility:
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Candidate Number: 4610
Betalaines are the red-purple pigments of beetroot, Beta vulgaris.
They are water-soluble and exist as internal salts in the sap vacuoles of
plant cells. Betalaines are made up of red betacyanins and yellow
betaxanthins. Betacyanines include about 90% of beetroot betalaines.
The most important betacyanin is betain. This makes up 75-95% of the
total colouring found in the beetroot. (Statistics found from:
http://www.agsci.ubc.ca/courses/fnh/410/colour/3_70.html)

Ethanol:
The general formula is C2H5OH with the structure as following:

Generally ethanol is an alcohol found in beer, wine, cider, spirits, and

other alcoholic drinks. When pure, it is a colourless liquid with a pleasant
odour, miscible with water or ether. It burns in air with a pale blue
flame. The vapour forms an explosive mixture with air and may be used in
high-compression internal combustion engines. It is produced naturally by
the fermentation of carbohydrates in yeast cells. Industrially, it can be
made by absorption of ethene and sub-sequent reaction with water, or by
the reduction of ethanal in the presence of a catalyst, and is widely used
as a solvent.

Ethanol is used as a raw material in the manufacture of ether, choral, and

iodoform. It can also be added to petrol, where it improves the
performance of the engine. Crops such as sugar cane may be grown to
provide ethanol (by fermentation) for this purpose.
(Information from The Hutchinson: Dictionary of Science)

The pH affects proteins that make up about 70% of most cell

membranes. Proteins are made of amino-acids with a variable number of
nitrogen and oxygen atoms in the protein. These can form hydrogen
bonds with the many hydrogen atoms found in the molecule.

When the pH of a solution is added or changed, then the position of some

of these hydrogen atoms also change. This is because “amino-acids are
amphoteric, and tend to stabilise pH. Thus, they can lose an H+ ion at the
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Candidate Number: 4610
COOH part of the molecule at higher pHs, or gain an H+ ion at the NH2
end of the molecule at lower pHs.” (Quoted from:
http://www.madsci.org/posts/archives/2004-03/1080223472.Cb.r.html)

Therefore the overall shape of the protein molecule changes because of

this change in the pH. However, unlike heat, the denaturing of a protein
by changing its pH is normally reversible.

So, by adding ethanol, betalain in beetroot diffuses out of the cell when
the membrane proteins are damged. This is because of the change in pH
to the cell membrane.

Appropriate equipment:
• Beetroot
• Corer
• Ceramic tile
• Scalpel
• Tweezers
• 10 Test tubes
• 10 Boiling tubes
• Test tube and boiling tube racks
• Measuring cylinder 25cm3
• Colourimeter
• Green filter
• Cuvettes
• Pipette

Choice of equipment:
1. Colourimeter:
The colourimeter is precise, reliable and accurate compared to a natural
eye test comparison or a colour chart comparison. Statistical figures are
given as percentage of transmission of light through the substance. This
also gives a more accurate reading instead an eye test or colour test
comparison. However a chart is considerable easier to read and the
colourimeter is occasionally difficult to make a reading exactly 100% with
a water filled cuvette. These readings fluctuate between 98% and 102%,
which could possibly cause variations with the rest of the experiment
results.

2. Corer:
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Candidate Number: 4610
Provides the same diameter throughout the beetroot and if done
correctly could supply a constant length of beetroot. The length that will
be subsequently used from analysing the preliminary experiment for the
final work will be 9mm. The larger the surface area the more area
exposed to the ethanol.
3. Green filter:
It is the complimentary colour, which would produce accurate readings
compared to a red filter as this would read at 100% transmission of light.
4. Beetroot:
Size: The larger the surface area the greater the leakage of pigment
from the beetroot cell membrane. Therefore with discs this would
increase the surface area of the facings therefore more leakage of
pigment. But with a section at a longer length for example at 2cm, this
would have a smaller surface area on the facings which would initially
cause pigment leakage, but would overall decrease the surface area.
Age: If the beetroot were too old, then several factors would affect the
final results. These could include that the pigment has dried up and
would consequently lead to no pigment leakage. Also if enough of the cell
membrane die, then there would be no structure or support to keep the
pigment in the cell, therefore would lead to more pigment leakage and not
an accurate set of results.

Variables:
1. Volume of the solution:
The same ratio of ethanol and distilled water must be accurate and
constant. This is because the results would not average scientifically well
neither would it be considered a fair test as a larger amount of ethanol
may have a larger affect on the cell membrane possible at a faster rate
than it would be with a smaller amount.
2. Constant and compromised timing:
If the beetroot was left at a longer length of the time in one experiment
and then at a shorter time the next, would not be considered accurate or
constant. With the beetroot in at a longer length of time eventually the
ethanol would have totally destroyed the cell membrane whatever the
size of the beetroot or ratio with distilled water. This is because of the
chemical properties of the solvent would affect the lipids in the cell
membrane. If the beetroot was left in a shorter amount of time, then
the ethanol would not have had it’s full affect on the cell membrane to
gain any efficient results from.

3. Repeated experiment:
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Candidate Number: 4610
This experiment would be repeated three times overall to gain a correct
scientific average and so the timing can be exact.
4. Filter colour:
Originally a colourimeter comes with three different filters, red, green
and blue. For this experiment the green or blue filter would be
acceptable. This would be because blue and green are the complimentary
colours and if the red filter would be used then this would have an overall
affect on the final results. This would not give correct readings as the
colour of the natural pigment in beetroot is red and for the colourimeter
to measure the light transmission, red liquid through a red filter would
just read at 100% because it would not distinguish between the colour
changes. Where as blue and green are the opposite colours could
distinguish this. For accuracy reasons a green filter will be used for the
final experiment as this was the same filter used in the preliminary
experiment.
5. Accuracy:
No spillages or loss of solution from splashes as this would affect the
volume and ratio of the ethanol and distilled water
6. Diameter of beetroot:
From preliminary results the diameter of the beetroot was changed
between 8mm and 9mm, this didn’t affect the overall results, which
suggests that for the final experiment would be to keep the diameter of
the beetroot a constant factor at 9mm. This diameter was chosen for
practical reasons so that it would comfortably fit into a boiling tube. If
the diameter was changed and not kept at a constant variable then it
would be considered that with a larger surface area the pigment leakage
would be sped up, but this is not what is being measured and not needed
to be furthered experimented with.
7. Size of the beetroot:
From the practical aspect of the preliminary experiment it would be
considered very difficult to cut the beetroot at a long length, e.g. 60mm,
as this would be because of the natural sizes of the beetroot which are
small naturally and that the entire experiment may require more than one
beetroot. Therefore for the final experiment lengths of 2mm are
acceptable. From my preliminary results different lengths do affect the
light transmission percentages, but considering that to cut beetroots at
specific lengths accurately is time consuming. This might affect the
speed at which the experiment is completed at and whether all aspects of
safety and accuracy are induced.

Measurements:
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Candidate Number: 4610
Transmission of light through the cuvettes with the final results of the
solution and colouring are being measured. Three consecutive
experiments are carried out to find the final average. Each experiment
would consist of 20 to 30 minutes each. For the final experiment, the
concentrations of ethanol would be between 0 and 20: the range being 0,
5, 10, 15, and 20.

Safe working environment:

1. Ethanol:
Corrosive and highly flammable. Irritating to skin and eyes. Can be
damaging if large amount is splashed into eyes. Intoxicating if ingested.
If ingested in undiluted form, it has a severe drying effect on mucous
membranes of mouth and throat. Intoxicating if continuously inhaled for
a long period of time. (Information from:
http://www.distill.com/materialsafety/msds-eu.html)
2. Spillages:
Safety hazard, inhalation if not cleared, corrosive and room needs to be
well ventilated even without spillages.
3. Scalpels:
Cutting the beetroot – white tile needed and cautious of a sharp
implement
4. Gloves:
Beetroot pigment stains clothes, therefore gloves are useful with the use
of beetroot as well the usage of ethanol, as it is skin irritant.

Secondary source information to inform strategy:

The previous testing on the effect of temperature showed that at higher
temperatures more pigment was left in the solution. This previous testing
provided a basis for a method and suitable equipment that may have been
needed for the preliminary and final experiment. For the preliminary
work, a similar experiment will be investigating the effects of ethanol on
the cell membrane of the beetroot instead of temperature. After
completing the temperature investigation, a suitable prediction could be
presented by employing the results. To justify length and shape of
beetroot, the beetroot shape will be cylindrical because a large surface
area to volume ratio would provide more cells exposed to the ethanol.
The smaller the length the larger the surface area, however if the
beetroot was too small then not enough pigment would be left to analysis.
A colourimeter was used for the previous testing on the affects of the
temperature and was reliable for statistical information instead of an eye
test comparison. The qualitative information given from the colourimeter
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Candidate Number: 4610
is precise; therefore a colourimeter will be used for the final experiment
with the affects of ethanol on the beetroot membranes.

Why ethanol?
By studying the effects of ethanol or organic solvents on beetroot cell
membranes can give scientists an idea how ethanol or organic solvents
may have on a persons cell membrane. From this experiment the cell
membrane from the beetroot would show how much ethanol would affect
it’s cell membrane and the level of damage at what concentration or ratio
with distilled water. From the results it would give scientist predictions
of the effect of ethanol on a persons stomach or digestive lining.
Therefore knowing these effects would help provide a wider knowledge of
alcohol on the body’s cell membranes and perhaps how to overcome the
dangers of drinking.

Preliminary Apparatus:
• Beetroot
• Corer
• Ceramic tile
• Scalpel
• Tweezers
• 10 Test tubes
• 10 Boiling tubes
• Test tube and boiling tube racks
• Measuring cylinder 25cm3
• Colourimeter
• Cuvettes
• Pipette

Preliminary Method:
• Using a corer, cut strips of beetroot with the corers at 8mm and
9mm
• Then using a ruler cut these sections into the following:
o Five 8mm diameter at 2cm in length
o Five 8mm diameter at 0.5cm in length
o Five 9mm diameter at 2cm in length
o Five 9mm diameter at 0.5cm in length
• Thoroughly rinse cut beetroot so that excess pigment will not
affect the overall results
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Candidate Number: 4610
• Prepare boiling tubes for the sections of the diameter of 9mm as
these do not fit into a normal test tube, and prepare test tubes for
the sections of 8mm
• Label clearly which test tubes and boiling tubes contain which ratio
of concentration of the ethanol
• Accurately measure with a pipette for the following percentages
and correct measuring ratio of distilled water and ethanol
o 100% ethanol 0% water, ratio 12cm3 of ethanol : 0cm3 of water
o 75% ethanol 25% water, ratio 9cm3 of ethanol : 3cm3 of water
o 50% ethanol 50% water, ratio 6cm3 of ethanol : 6cm3 of water
o 25% ethanol 27% water, ratio 3cm3 of ethanol : 9cm3 of water
o 0% ethanol 100% water, ratio 0cm3 of ethanol : 12cm3 of water
• Leave for 40 to 60 minutes for effects to happen
• After this period of time, take out the beetroots from the boiling
tubes or test tubes
• Using a colourimeter, set up by placing complementary green or
blue light filter into the designated position
• Fill a cuvette of distilled water and place into the colourimeter
with the clear sides facing the reader and the ridged edges facing
the sides
• By using the dial, set the percentage of transmittance of light on
the reading to 100%, or get as close to 100% as possible
• Then at one at a time fill a cuvette with the final results
individually with each test tube or boiling tube substance
• Record results by noting the percentage reading of the light
transmittance

Preliminary Prediction:
By exposing a membrane to a solvent, ethanol, it will affect its
permeability. The higher the concentration of the solvent, the more
permeable the membrane will be. For preliminary results, the ranges of
concentrations are going to range between 0% and 100%. From these
results, observations may evidently show that at 50%, 75% and 100%
concentrations the cell membrane of the beetroot has been totally
broken down. This is because by increasing the concentrations of the
ethanol beyond a certain point it may have no effect on the cell
membrane.
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Candidate Number: 4610

Preliminary Results measuring the percentage of light transmittance:

Concentration of Beetroot: Beetroot: Beetroot: Beetroot:

Ethanol 9mm diameter 9mm diameter 8mm diameter 8mm diameter
2cm length 0.5cm length 2cm length 0.5cm length
100% 10.2% 21.0% 30.0% 20.4%
75% 13.3% 16.8% 28.1% 59.8%
50% 16.0% 20.9% 49.0% 11.3%
25% 36.3% 80.7% 98.3% 26.0%
0% 40.0% 91.6% 90.3% 92%
Water = 101.8%

Preliminary Analysis:
From analysis of the results, it is to conclude that there is too large a
range between concentrations and that the effects on the beetroot cell
membrane happens between the concentrations 25 and 0. Therefore
possible for the final experiment the range will be between 0 and 20: 0,
5, 10, 15, and 20. Consequently there will an average range between the
stages where the cell membrane is most affected from the ethanol. To
overcome these anomalies more accuracy and timing would be a factor,
also possible only having one length and one diameter measurement. This
would then provide a concentrated measurement and comparison. Also
when returning to the beaker holding the cut beetroot, a lot of the
pigment was still present which might have had an affect on the overall
results. To overcome this, the cut beetroot would need all of the excess
pigment absent from the beaker whilst in storage purposes. However
from this experiment, it is clear that the length and possible even the
diameter does effect the final pigment leakage.

From the preliminary work the final investigation with only need boiling
tubes because in the preliminary the beetroot was 8mm in diameter,
which fitted into test tubes but a 9mm diameter needed boiling tubes,
now that for the final the beetroot is only a 9mm diameter and at 2cm
length boiling tubes would be required.

Preliminary Graph Analysis:

From analysis and comparison of the graphs, it is apparent that the
general trend of each graph is decreasing, ignoring any anomalies. The
beetroot with the diameter of 9mm and the length of 2cm were devoid of
anomalies, this could be because this was the first of the four
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Candidate Number: 4610
experiments, and therefore accuracy and suitable timing were required,
hence a better set of results compared to the other three. The results
with the beetroot at 9mm in diameter went up at the end of the graph
line; this could be accounted for as not the correct timing or enough
accuracy. Similar inconsistent results occurred in both sets of results
for the beetroot at 8mm in diameter. In particular with the length at
0.5cm, this sudden increase and then decrease is could also be because of
timing or accuracy.

Experimental Graph Analysis:

From these results and general graph trends decreasing, for the final
experiment, a similar pattern should occur with a decreasing trend. This
is apparent with the original prediction made that the higher the
percentage of ethanol exposed to the cell membrane, the permeability is
affected enough to allow further pigment leakage.

Method Preparation:
Taking into consideration the final results of the preliminary experiment
and the inadequate methods used during that experiment, the method will
be altered slightly from the original preliminary made.
The changes will include the following:
1. Only need 15 boiling tubes rather than 10 boiling tubes and 10 test
tubes, this is because the beetroot at a 9mm only fits in a boiling
tube where as the 8mm fits in a test tube. Since the
concentrations have been lowered to the range between 0 and 25,
with a gap of 5 between each, then only 5 boiling tubes shall be
needed for each experiment hence 15 altogether.
2. A corer for a diameter of 9mm instead of 8mm for the above
reasoning.
3. Lengths only at 2cm instead at 0.5cm and 2cm, this would provide a
constancy and accuracy.
4. Found that leaving the substances in for only 30-40 minutes was
sufficient enough rather than 40-60 minutes. If the beetroot was
left in for too long then no matter what concentration the
beetroot was in with ethanol, all of the pigment would have
naturally leaked out, or the ethanol would eventually have
destroyed the entire membrane
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Candidate Number: 4610

Experimental Method:
• Using a corer, cut strips of beetroot with the corers with a 9mm
diameter
• Then using a ruler cut ALL the beetroot at the length of 2cm
• Thoroughly rinse cut beetroot so that excess pigment will not
affect the overall results
• Label clearly which boiling tubes contain which ratio of
concentration of the ethanol
• Accurately measure for the following percentages and correct
measuring ratio of distilled water and ethanol
o 20% ethanol 80% water, ratio 2.5cm3 of ethanol : 9.5cm3 of water
o 15% ethanol 85% water, ratio 2cm3 of ethanol : 10cm3 of water
o 10% ethanol 90% water, ratio 1.5cm3 of ethanol : 10.5cm3 of water
o 5% ethanol 95% water, ratio 1cm3 of ethanol : 11cm3 of water
o 0% ethanol 100% water, ratio 0cm3 of ethanol : 12cm3 of water
• Leave for 30 to 40 minutes for effects to happen
• After this period of time, take out the beetroots from the boiling
tubes
• Using a colourimeter, set up by placing complementary green light
filter into the designated position
• Fill a cuvette of distilled water and place into the colourimeter
with the clear sides facing the reader and the ridged edges facing
the sides
• By using the dial, set the percentage of transmittance of light on
the reading to 100%, or get as close to 100% as possible
• Then at one at a time fill a cuvette with the final results
individually with each boiling tube substance
• Record results by noting the percentage reading of the light
transmittance
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Candidate Number: 4610

Bibliography:
http://www.jdaross.mcmail.com/cell2.htm
http://www.distill.com/materialsafety/msds-eu.html
www.mrothery.co.uk/cells/resources/betacyanin.doc
http://www.biologymad.com/resources/BEETROOT%20PIGMENT2.doc
http://www.agsci.ubc.ca/courses/fnh/410/colour/3_70.html
http://www.madsci.org/posts/archives/2004-03/1080223472.Cb.r.html
The Hutchinson: Dictionary of Science (1994)
Handouts from school

Pictures from:
www.mrothery.co.uk/cells/resources/betacyanin.doc
http://www.agsci.ubc.ca/courses/fnh/410/colour/3_70.html
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Candidate Number: 4610

Method

Equipment:
• Test tube rack
• Boiling tubes
• Graduated syringe
• Measuring cylinders
• Beakers
• Ceramic tile
• Cork borer
• Razor blade
• Thermometer
• Forceps
• Stop clock
• Colourimeter

Method:
1. Cut enough cylinders from the taproots of fresh beetroot with a
number 8 mm (diameter width) cork borer to make 35, 30mm
cylinders.
2. Place the cylinders into a large beaker and rinse under running tap
water until no more pigment is released into the water.
3. While the cylinders are rinsing collect 11 clean, dry boiling tubes
and label them with the ethanol %’s you will be using; these should
be 0% (distilled water), 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%,
50%.
4. Place 30ml of distilled into the corresponding tube and measure
the temperature. Place 30ml of each solution of ethanol into the
other tubes. These can be prepared by diluting the supplied 100%
solution of ethanol.
5. Place one cylinder of rinsed beetroot into each of the tubes and
start the stop clock. (It may be necessary to stagger the start
time for each dilution to give you time to remove the cylinders at
the end of the test) Stagger by 30 seconds.
6. Leave the cylinders in the boiling tubes for exactly 30 minutes
7. After 30 minutes have elapsed, shake the tubes and carefully
remove the cylinders from the tubes taking care not the damage
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Candidate Number: 4610
the cylinders. This will give you 11 tubes containing solutions
stained with red pigment.
8. Calibrate a colourimeter using a clean cuvette of distilled water
and a blue filter.
9. Transfer each of the solutions to a clean, dry cuvette and measure
the transmittance using a colourimeter. (It may be necessary to
recalibrate the colourimeter regularly with distilled water to
ensure the accuracy of your results).
10. Repeat the experiment three times and calculate an average for
each solution.
11. Carefully record all of your results in a table.
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Candidate Number: 4610

Analysis: