Hypothesis: To investigate the effect of different concentrations of ethanol on the permeability of beetroot cell membranes.
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by
izzyd1995 (student)
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 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 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 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: ...
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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:
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 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
2. Corer
3. Ceramic tile
4. Scalpel
5. Tweezers
6. 10 Test tubes
7. 10 Boiling tubes
8. Test tube and boiling tube racks
9. Measuring cylinder 25cm3
0. Colourimeter
1. Green filter
2. Cuvettes
3. Pipette
Choice of equipment:
. 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.
. Corer:
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.
. 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.
. 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:
. 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.
. 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.
. Repeated experiment:
This experiment would be repeated three times overall to gain a correct scientific average and so the timing can be exact.
. 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.
. Accuracy:
No spillages or loss of solution from splashes as this would affect the volume and ratio of the ethanol and distilled water
. 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.
. 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:
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:
. 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)
. Spillages:
Safety hazard, inhalation if not cleared, corrosive and room needs to be well ventilated even without spillages.
. Scalpels:
Cutting the beetroot – white tile needed and cautious of a sharp implement
. 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 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
2. Corer
3. Ceramic tile
4. Scalpel
5. Tweezers
6. 10 Test tubes
7. 10 Boiling tubes
8. Test tube and boiling tube racks
9. Measuring cylinder 25cm3
0. Colourimeter
1. Cuvettes
2. Pipette
Preliminary Method:
. Using a corer, cut strips of beetroot with the corers at 8mm and 9mm
2. Then using a ruler cut these sections into the following:
. Five 8mm diameter at 2cm in length
2. Five 8mm diameter at 0.5cm in length
3. Five 9mm diameter at 2cm in length
4. Five 9mm diameter at 0.5cm in length
. Thoroughly rinse cut beetroot so that excess pigment will not affect the overall results
2. 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
3. Label clearly which test tubes and boiling tubes contain which ratio of concentration of the ethanol
4. Accurately measure with a pipette for the following percentages and correct measuring ratio of distilled water and ethanol
. 100% ethanol 0% water, ratio 12cm3 of ethanol : 0cm3 of water
2. 75% ethanol 25% water, ratio 9cm3 of ethanol : 3cm3 of water
3. 50% ethanol 50% water, ratio 6cm3 of ethanol : 6cm3 of water
4. 25% ethanol 27% water, ratio 3cm3 of ethanol : 9cm3 of water
5. 0% ethanol 100% water, ratio 0cm3 of ethanol : 12cm3 of water
. Leave for 40 to 60 minutes for effects to happen
2. After this period of time, take out the beetroots from the boiling tubes or test tubes
3. Using a colourimeter, set up by placing complementary green or blue light filter into the designated position
4. 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
5. By using the dial, set the percentage of transmittance of light on the reading to 100%, or get as close to 100% as possible
6. Then at one at a time fill a cuvette with the final results individually with each test tube or boiling tube substance
7. 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.
Preliminary Results measuring the percentage of light transmittance:
Concentration of Ethanol
Beetroot:
9mm diameter 2cm length
Beetroot:
9mm diameter 0.5cm length
Beetroot:
8mm diameter 2cm length
Beetroot:
8mm diameter 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 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:
. 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
Experimental Method:
. Using a corer, cut strips of beetroot with the corers with a 9mm diameter
2. Then using a ruler cut ALL the beetroot at the length of 2cm
3. Thoroughly rinse cut beetroot so that excess pigment will not affect the overall results
4. Label clearly which boiling tubes contain which ratio of concentration of the ethanol
5. Accurately measure for the following percentages and correct measuring ratio of distilled water and ethanol
. 20% ethanol 80% water, ratio 2.5cm3 of ethanol : 9.5cm3 of water
2. 15% ethanol 85% water, ratio 2cm3 of ethanol : 10cm3 of water
3. 10% ethanol 90% water, ratio 1.5cm3 of ethanol : 10.5cm3 of water
4. 5% ethanol 95% water, ratio 1cm3 of ethanol : 11cm3 of water
5. 0% ethanol 100% water, ratio 0cm3 of ethanol : 12cm3 of water
. Leave for 30 to 40 minutes for effects to happen
2. After this period of time, take out the beetroots from the boiling tubes
3. Using a colourimeter, set up by placing complementary green light filter into the designated position
4. 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
5. By using the dial, set the percentage of transmittance of light on the reading to 100%, or get as close to 100% as possible
6. Then at one at a time fill a cuvette with the final results individually with each boiling tube substance
7. Record results by noting the percentage reading of the light transmittance
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
Method
Equipment:
. Test tube rack
2. Boiling tubes
3. Graduated syringe
4. Measuring cylinders
5. Beakers
6. Ceramic tile
7. Cork borer
8. Razor blade
9. Thermometer
0. Forceps
1. Stop clock
2. Colourimeter
Method:
. 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 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).
0. Repeat the experiment three times and calculate an average for each solution.
1. Carefully record all of your results in a table.
by
izzyd1995 (student)
