When the potatoes are ready to use, they should be kept with labels showing which intensity the potato was at. Using Petri dishes with the labelled intensities, the potatoes will be cut into their disks. Using a chip cutter, the potatoes will be cut into the same cross sectional area. To finish off the disks, the depth of the disks will be cut, to the edge nearest the surface to ensure, the section of the potato used is the part with the extra levels of light induced solanine. Four disks per potato will be cut in this way for each potato, which will result in four equal sized potato disks for each potato under each of the four light intensities.
20cm³ of the hydrogen peroxide volume will be measured using a pipette and put into a small beaker. A water bath will be made and a measuring cylinder will be filled to with water. The water filled measuring cylinder will be placed upside down into the water bath without any water escaping. This will then clamp into position using a clamp and stand. The glass tubing from a conical flask will be positioned so that it comes in underneath the measuring cylinder. Finally, the conical flask will be positioned by using wooden blocks if necessary.
The four potato discs of the first light intensity will be placed within the conical flask. The 20cm³ of the hydrogen peroxide solution will be added to the discs and the bung replaced on the conical flask as the stop clock is started. The reaction will be left over a certain period of time, and measurements of the dependent variable, the volume of oxygen will be taken from the measuring cylinder. After this, the conical flask will be washed out with distilled water, and the water within the measuring cylinder replaced before repeating the experiment for the potato discs of the other light intensities.
Choice of materials and quantities
As stated within my research, the production of the glycoalkaloid solanine increases within potato tissue when exposed within light. This also accumulates the production of chlorophyll, which is why the potato goes green in colour. However, certain types of light cause this as an effect in different ways. As the aim of this investigation is to see how the production of chlorophyll, and increased concentration of solanine effects the rate of catalase activity as a result of light exposure, it is important that the type of light used for the storage conditions is one which causes these effects to a high level, so that a significant change in rate can be observed. This therefore means that an incandescent or an ultraviolet light source cannot be used for the experiment. It is therefore more appropriate to use a fluorescent light source, such as a fluorescent lamp.
Potatoes come in many different varieties, changing in both size and even colour. A variety readily available in supermarkets and suitable for this experiment is the King Edward variety. This is a medium sized potato with a light skin colour which could easily show green surface coloration. This is important, as a green surface coloration is an indication of increased levels of solanine, and will need to be seen before the start of the experiment. The increased solanine levels will be found mostly at the surface where this green colour will appear as a result of chlorophyll production. Therefore to ensure that the results obtained are from potato tissue exposed to light, it is important that the disks used are cut from the surface of the potato. This means that the depth of the potato disks need to be small, as only one eighth of an inch of the surface stores solanine and chlorophyll. The diameter of all disks will be kept the same using a chip cutter, and therefore the depth has to be done by hand. A depth of 5mm is one which should be able to be achieved by hand, and is one which should contain light induced solanine.
Hydrogen peroxide concentration is an important part of the experiment to be conducted. The concentration has be strong enough to show a clear progressing catalase-hydrogen peroxide reaction without being too fast or slow, so that a good set of results cannot be obtained. 20 volume (80% hydrogen peroxide) is a suitable concentration, as this is one which is strong enough to show good progress of the catalase reaction despite the high pH conditions of the peroxisomes caused by the increased solanine. As discussed within my experimental hypothesis, the high pH conditions caused by the increased solanine shifts away from the optimum pH of catalase and the reaction would be expected to be slower. It is for this reason why a high hydrogen peroxide concentration would be ideal to use for this experiment.
The time period allowed for the catalase-hydrogen peroxide reaction is important to consider. This has to be just about long enough to show a rate of reaction, any longer than this would be a waste of materials and time. For this particular experiment, a time period of four minutes is long enough to show this, with a volume recording taken at every minute. This will show the rate of the catalase-hydrogen peroxide reaction for the potato tissue of each light intensity.
The number of different light intensities required to store the King Edward potatoes is another factor needed to be considered. Four light intensities are needed to compare the rate of reaction for, to come a firm conclusion from the results. The first potato will be right next to the lamp, and therefore be fully exposed to the whole intensity. The other potatoes will follow this by being 30cm behind each other, so that the second potato will have half the intensity, the third will have a quarter the intensity, and the last an eighth. Comparison within ratios between the light intensity is enough to draw a strong conclusion from the results to be obtained.
It is certain that low light intensity achieved by a longer distance from the florescent lamp, will cause less light induced solanine to be produced. This lower content will mean that the pH of the peroxisomes whence the catalase is present will be increased by only a little. With contact with 20 volume hydrogen peroxide concentration, the reaction will be quite fast. It is therefore ideal that a 20ml measuring cylinder be used to measure the dependent variable, the volume of oxygen produced, as this is large enough to measure the volume over the four minute period allowed.
Variables
The independent variable here is the light intensity upon which the same variety of King Edward potatoes will be exposed to. This will be done within a darkened room where only a fluorescent lamp will be a light source, and the potatoes will be exposed to different light sources due to their distance from it. To ensure that the potatoes are exposed to the same light intensity at all times, and that the chemical changes within the potato tissue is due to the same exposure, it is important that the fluorescent lamp maintains the same power throughout the time needed for the potatoes to turn green. This could be over three days, and therefore there is a likely chance that the fuse could go, or the lamp could get overheated. For this reason, the lamp to be used should have a new bulb within it, and be a lamp which does not transfer heat to the surroundings to a large extent.
The dependent variable within this experiment is the volume of oxygen collected within the measuring cylinder, as it is this reflection of the catalase-hydrogen peroxide reaction, it will be used as the results, and therefore be what the conclusion is drawn from. It is therefore important that this is controlled in the most accurate possible way by preventing the oxygen from escaping by any other route. Oxygen may escape between the bung and the neck of the conical flask. To ensure this does not cause inaccuracy within the dependent variable and therefore an inaccuracy within the results, this has to be prevented. This will be done using a gelatinous substance such as petroleum jelly around the bung after it has been replaced. This will physically stop any oxygen gas from escaping and the volume will have to enter the measuring cylinder where it will be recorded.
Within this experiment, there are no confounding variables which could alter with the light intensity and alter the true relationship between this and the volume of oxygen collected. This is a straightforward experiment with no variable blocking the actual outcome. This means that the results obtained from this experiment should give a reflection of how the light intensity effects the catalase activity within potato tissue.
Accuracy and ensuring reliability of the results to be obtained
As with most results obtained from a practical experiment, the main source of error lies within the experimental procedure, or the apparatus used. It is therefore important to ensure accuracy within these, sodium carbonate solution that the results obtained are reliable to draw a firm conclusion.
Within this experiment, an error within the results can occur if there are any leaks within the apparatus, which could allow oxygen to escape. As identified earlier, this can occur between a bung and the neck of a conical flask. To prevent any air from escaping, a gelatinous substance like petroleum jelly can be used to surround the bung after it has been replaced. This should physically block any oxygen gas particles from escaping.
Another potential source of error is a forced amount of oxygen being forced into the measuring cylinder as the bung is pushed down too hard. It is important to avoid doing this within the experiment as sometimes in order to replace the bung quickly, unintentionally the bung does get pushed down hard. Errors within the readings obtained for the results could therefore be due to a result of air being present within the measuring cylinder. Air being allowed to enter the measuring cylinder whilst placing it within the water bath should not be allowed, and this also applies when being positioned by the clamp.
Accuracy of the readings for the volumes of oxygen produced should be taken at eye level to the apparatus. This will ensure that exact volume from the same position is obtained and therefore reducing the inaccuracy of human error. Eye level with apparatus should also be ensured when filling the pipette with the hydrogen peroxide solution. This is to ensure the meniscus is resting above the line each time. This will reduce the any errors within results due to inaccurate volumes of hydrogen peroxide present within the conical flask.
To ensure that no residue of the previous experiment remains within the conical flask before using it again, it is important to wash the flask out with distilled water. This will also remove any impurities from the flask as a result to washing with tap water. This will ensure the same accuracy for the next experiment.
To be able to be sure that the results obtained have no error within them, it is important to repeat the experiment at least twice. This will not only give more than one set of results to compare so that erroneous results can be identified, but also give enough results to take an average value. This average value can be used to base the conclusion, and will portray a good representation of the results obtained by all three experiments.
Risk assessment
The main risk or danger here is the chemical hydrogen peroxide. Hydrogen peroxide is an irritating substance which can be irritating to you eyes, nose and skin. If smelt, the vapours can be very irritating, especially to those who have asthmatic or breathing problems. Anyone who encounters this problem is advised to leave the room and find surroundings where they may breathe with ease. If it comes in contact with your eyes it is especially dangerous as it can cause the cornea to burn very easily. If such as situation was to happen it would be advised to wash the substance out with water, and to consult a physician immediately. It is therefore advised to wear goggles at all times. If the substance comes in contact with the skin it may sting or be irritant, and you are advised to wash your hands with warm water.
Another safety precaution that should be followed is to be aware of the apparatus. There is a lot of glass equipment to be used and this should be handled with care when in use. The equipment should be placed away from the desk, and stools should be tucked under the desk, sodium carbonate solution that if an incident were to occur, they would not act as a safety hazard.
Equipment
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25cm³ conical flask with extended glass tubing and rubber bung – Where the potato disks and hydrogen peroxide will react
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12mm diameter potato chip cutter – To cut the potato tubes
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20 volume hydrogen peroxide – Concentration of hydrogen peroxide solution for potato disks to react with
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White tiles – To cut the potato disks
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Scalpel – To cut the potato disks
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Small rule – To measure 5mm width when cutting the potato disks
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60W Fluorescent lamp – To use as the source of light intensity for the potatoes
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4 King Edward potatoes – The variety of potatoes to be used
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Metre rule – To measure the 30cm distance between the potatoes during storage
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Water cylinder – To fill with water and act as a base to the measuring cylinder
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20ml measuring cylinder – The measuring of the volume of oxygen will be done using this
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Clamp and stand – To hold the measuring cylinder upside down
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10ml pipette – To use to transfer the hydrogen peroxide
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2 100ml beaker – To fill with the measured 20ml hydrogen peroxide and hydrogen peroxide solution with.
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Wooden blocks – To use as a base for the conical flask
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Vaseline – To cover the rubber bung after it has been replaced. To prevent oxygen from escaping
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Stop clock – To use to measure the four minute interval
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4 Petri dishes – To hold the potato disks after they are cut
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Marker pen – To label apparatus
Method
Storage of the potatoes
- In a darkened room, place a 60W fluorescent lamp at one end of a bench. Turn it on, so that light fills the room. Place one of the potatoes right next to the lamp. Place the others 30cm behind each other by using a metre rule.
- Leave the potatoes for approximately four days or until the first potato has turned completely green. Remove them from there positions and turn the fluorescent light off.
Experimental method
- Using the 12mm potato cutter, chop potato tubes from one of the potatoes. Cut 12 5mm thick potato disks ensuring they are cut next to the edge. Put them in a labelled Petri dish. Do the same for the other three potatoes.
- Fill the water bath to half full. Fill the measuring cylinder with water. Place upside down into the water bath so that no air is let in. Clamp this into position.
- Position the conical flask with the wooden blocks, and ensure that the end of the glass tubing enters the measuring cylinder.
- Put four disks of the first light intensity within the conical flask. Pour some of the hydrogen peroxide solution within a beaker. Using a pipette transfer 20ml of the solution to a labelled beaker.
- Pour the hydrogen peroxide solution into the conical flask. Put the rubber bung on and start the stop clock immediately.
- Add the vaseline to the rubber bung
- Record the volume of oxygen collected within the measuring cylinder at every minute for four minutes.
- After four minutes, remove the conical flask. Empty the contents, and wash out the flask and bung using tap water. Then wash out using distilled water.
- Repeat steps 2-8 for to obtain all the results for the repeats and the other potato disks of different intensities. Ensure you repeat each light intensity potato disk experiment twice to obtain three sets of results for each one.
Hypothesis
When exposed to light, the solanine concentration on the surface of the skin will increase. The light will also accumulate the formation of chlorophyll. The increased solanine, like any other glycoalkaloid will cause the cells to become alkaline. This will cause the peroxisomes where the catalase is stored to also become a high pH.
Catalase, whose optimum pH is around 7.6, will slow down the rate at which it decomposes the hydrogen peroxide due to this change in high pH.
Those potatoes closer to the fluorescent lamp will experience a higher light intensity. This will cause more chlorophyll and solanine formation faster than those potatoes further away.
