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Tweezers- [What? This is used to pick the beetroot up from the beetroot solution, Why? I must be careful not to squeeze the beetroot too much or this could lead to damaging the beetroot hence damaging the cell membrane of the beetroot. The beetroot discs must not be held with a human hand as excess chemicals and bacteria can come in contact with the beetroot and disrupt the experiment]
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Stopwatch- [What? This is used to make sure each experiment is carried out for the same amount of time at each temperature Why? To ensure it is a fair experiment. I will be using this to time accurately; I will be timing for 10 minutes as the beetroot is in the water bath.]
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Ruler- [What? This equipment is going to be used for measuring the size of beetroot; I will be measuring 4cm of the beetroot length and width wise accurately Why? So they are the same length and surface area for each experiment.]
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Boiling tubes [What? This is used to place the suited number of beetroot discs and distilled water during the experiment. A boiling tube is used instead of a test tube as a test tube can break in extreme temperatures. Why? I will be using three boiling tubes where the beetroot will be going in, I will be using three as in each boiling-tube, the beetroot can have more space and so they are not all squashed in]
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Tile- [What? This is used to place the beetroots on in order to cut them in the accurate length .I will be using a while tile to place my beetroot onto, Why? The reason why I am using a white tile instead of black or any other is because using a white is easier to see.]
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Colour Emitter- [What? This is used to measure the light absorbency through the solution containing the anthocyanin in the cuvette so that I can work out how much diffusion took place Why? This is a accurate way of measuring the amount of light absorbed
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Measuring Scales- [This is a very accurate piece of equipment as it measures the mass of the beetroot accurately].
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Tissue Paper – [This is used for taking any excess solution of the beetroot and to make sure the beetroot is dry.]
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Cuvette- [This is used to hold the sample of coloured water from each temperature so it can be placed in a colorimeter.]
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Boiling Tube Rack –[This is used to place the boiling tubes for the experiment in while something else needs to be done.]
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Pipette- [This will be used to collect the samples containing water and anthocyanin which will then be transferred into cuvettes.]
Method
First heat the water baths up to 20,40, 60 degrees Celsius and ensure that the baths are ready to use for the experiment. Cut the beetroot of 4cm long and 4cm width with a scalpel on a white tile using a ruler. This will make it a fair experiment because uneven pieces of beetroot can mean that big pieces will leak more than smaller pieces.
Weigh the beetroot using a balance at 1g.
Wash the beetroot with distilled water because it is purer than tap water. This is done to get rid of excess pigment and leakage from the beetroot, the beetroot is cut into smaller pieces rather than a bigger size so that the surface area is maximised and therefore faster diffusion will take place. A ruler is used to ensure that the beetroot and disks are the same size so that the results are reliable and accurate. Water baths will be used as it’s an accurate apparatus and ensures the water is at the temperature required.
A water bath is better than a Bunsen burner because the apparatus keeps the temperature constant even though the temperature slightly rises or lowers but it is generally reliable. The Bunsen burner cannot keep the constant temperature without having to switch it off and then turning it back on when the water has cooled too much. The Bunsen burner if heating a test-tube will continuously heat the test-tube up and then temperature will consistently rise as it gets hotter.
Use a thermometer to check if the water baths are at the required temperatures, once they are set up at the required temperature use a measuring cylinder to obtain 15cm cubed of water at the meniscus level, and then pour the distilled water to the boiling-tubes. I will use this volume of water because too much water will cause the solution to be diluted and light absorbance levels will be low. Place each of the boiling-tubes into each of the water baths.
Place the discs of beetroot into the boiling tubes at all at the same time and immediately start the stopwatch until ten minutes. This will be sufficient enough time to make a difference to the permeability of the membrane because the colour is released during that period of time and leaving it in the water for further periods of time will not make any difference. Once the 10 minutes are over, take the boiling –tubes out of the water baths. Remove the beetroot from the boiling-tube with a tweezer so that no more pigment diffuses into the water. Removing the beetroot with a tweezer will ensure that the solution does not get contaminated out of the test tube with our hands, also leaving the beetroot for long periods of time will mean that more pigment is released and less light will pass through the solution which produces inconsistent and unreliable results. Stir the pigment and water 5 times for each boiling tube so that the colour is equally distributed throughout the boiling-tube, use a pipette to collect the sample from the boiling-tubes and place the samples into the cuvettes, place all the cuvettes into a colorimeter and measure the absorption of light through the water, repeat the experiment two more times to obtain fair and accurate results.
Safety
The only potential hazard is the scalpel as they can damage the skin if used incorrectly, however the position of the first aid kit was known in case of an emergency, an apron was used to protect the clothes from the red pigment anthocyanin which stains clothes
Control Experiment
A control experiment is required to ensure that nothing else apart from the independent variable will affect the results. The control in this particular experiment will be the calibration of the colorimeter. This is due to the fact that the colorimeter reads the light absorbance. Therefore according to this the distilled water will be used to make sure that temperature is the only variable that will affect the final results. I must ensure that the distilled water shows a reading of 0.00 on the colorimeter.
Things I Must keep constant
There are many things I must keep constant in my experiment, this should be done carefully and accurately in order to ensure that I get positive and reliable results. If I get unreliable results then I can know that my factors that need to be kept constant were not kept constant accurately.
These are the factors that need to be kept constant
Surface Area Of Beetroot
Mass Of Beetroot
Volume Of Water Beetroot is in
Time For Each Test Made
Number of Swirls
Removal Of Beetroot
Surface Area Of Beetroot
I must keep the surface area of the beetroot constant by cutting the beetroot with a scalpel of length and thickness of 4 cm. I should also keep the weight of the beetroot constant which should be 1g. The rate at which a pigment diffuses through a membrane depends on the surface area in contact with the substance. The rate at which the pigment diffuses is found out by using the equation: Rate of Diffusion=Surface Area x Concentration difference divided by thickness of membrane
This equation is called Ficks law.
Volume of water beetroot is in
The volume of water that the beetroot is in should also be kept constant. This is kept constant by measuring 20 cm cubed of water using a measuring cylinder. This is because the concentration of the water must be kept constant at all time as it affects the diffusion rate and may give inaccurate and unreliable results.
Time for each test made
The time for each test made should be kept constant as the amount of time the beetroot is left in the solution does affect the rate of diffusion. The longer the beetroot is left, the larger amount of dye will leak out. So I must ensure that I use a stopwatch to time the experiment for 10 minutes and take the boiling tube out of the water bath as well as taking the beetroot out of the boiling tube.
Equipment
I must ensure that all the equipment is clean and not contaminated, as this will affect my results because the substances might get into the sample that may affect the light absorbency and this may affect the reading of the spectrophotometer. To control this variable I will check the equipment before using it in my experiment and I will always wash any equipment before reuse.
Temperature of the water baths
The temperature of the water bath must be kept constant because if the temperature is different to the temperature you are recording then you might produce different results e.g. if you test the diffusion of a beetroot at 300C but the temperature of the water bath is set at 250C, the sample may be less concentrated to how it would have been if it were at 300C. I have taken this variable into account and will keep it controlled by checking the temperature of the water baths to make sure they are at the temperatures I require.
Other variables to consider:
- Keep temperature the same throughout the diffusion process. This will be achieved by placing the boiling tubes in the water baths until the time is up.
- The number of beetroots must be the same for each experiment as then the experiment will not be accurate as if there are more pieces of beetroot more diffusion can take place therefore more anthocyanin being released. This will not affect my experiment as I am only using one 4cm long beetroot.
- The pipette needs to be washed before a sample of solution is taken out as from the previous experiment, some excess pigment may remain in the pipette and if this is used for another sample then it may alter the results. I will wash the pipette out thoroughly using distilled waster, which will remove the excess pigment and will insure the results are not going to be affected.
The colorimeter is a very highly sensitive device so any fingerprints or liquid on the outside of the cuvettes would decrease the accuracy of my results. I will wipe the cuvette with a paper towel to wipe of any liquid and will only hold it with the tips of my fingertips at the very top so as to minimise the error in accuracy when the cuvette is placed in the colorimeter.
Independent and dependent variables
Independent Variable is the variable that I will be changing whilst dependent variable is something that does not change.
The independent variable in my experiment will be the temperature; I will have different temperatures such as 0, 20, 40, and 60 degrees Celsius
The dependent variable will be the amount of pigment in beetroot, which will be measured at the % of absorption of light.
Prediction
I predict that as the temperature increases the rate of diffusion also increases. I also predict that the absorbance rate increases, increasing the temperature will increase the kinetic energy of the phospholipids in the cell membrane so more dye is released. I also predict that larger amounts of dye will diffuse at denaturing temperatures of 60 degrees Celsius. As the membrane is damaged, the protein structure that makes up the membrane is also damaged. The disulphide, hydrogen bonds, peptide bonds all get disrupted and so more pigment diffuses from the vacuole of the membrane.
High temperatures can disrupt the active site of the carrier which affects the shape of the fluid mosaic model membrane, I predict as the temperature increases from 0 to 40degrees Celsius the kinetic energy increases which in turn speeds the rate of diffusion of the pigment.
When the temperature reaches over 37 degrees Celsius the temperature will cause complex polypeptide chains to break down and this deforms the proteins, which leaves even larger gaps in the cell membrane. This will greatly increase the amount of anthocyanin in the sample so after 37 degrees Celsius we can expect the result to rise sharply; an adequate amount of anthocyanin will pass into the sample as the structure of the cell membrane can diffuse out of the cell.
At temperatures of 60 degrees Celsius, I predict also that the structural damage occurs at the membrane and denatures proteins will therefore raise the amount of red pigment released out of the cells. I also hypothesis that at temperatures of 60 degrees Celsius, the fluidity and stability of the membrane which is normally a rigid structure weakens and becomes a fluid. This causes perforations along the length of the membrane and hence the red pigment anthocyanin leaks out by diffusion. The high concentration of the red pigment inside the cell must be in equilibrilium to the concentration outside the cell. I predict further that beyond 60, the membrane will continue to break down rapidly. As I have predicted before that as the temperature increases to 40 the diffusion rate increases, but permeability of the plasma membrane does not alter.
When the experiment is conducted at temperatures of 60 degrees Celsius the water expands the cholesterol, glycoproteins and phospholipids, which apply pressure on the membrane from the inside. The protein structure and the membrane is denatured and hence increases the surface permeability. All this suggests that high temperatures will allow the molecules to shake and vibrate more frequently. The molecules will collide more frequently and result in successful collisions. I also think that as the molecules enter the cell by osmosis, then the cells will swell up and eventually bust which releases the pigment. Osmosis is the movement of water molecules down a concentration gradient through a selectively permeable membrane, between 20 to 40 degrees Celsius, the tonoplasts which are the vascular membranes leak but at 60 degrees Celsius the tonoplasts will disrupt completely, so overall I predict that as temperatures increases from 20-40 degrees Celsius, the rate of diffusion and absorbance rate increases but at 60 degrees Celsius diffusion and absorbance rates rise dramatically.
Results
Calculations From Results
Conclusion
The graphs and the data show that as the temperature increases, the amount of light absorbance increases. From 0 to 20 degrees Celsius, there is a gradual increases of absorbency as the line of the graph between these two temperatures has a gentle gradient, so this means that the diffusion rate of the pigment increases as the temperature increases, diffusion is the net movement of molecules from a region of high concentration (beetroot sample) to a region of low concentration (distilled water). Also as the temperature rises, the permeability of the cell membrane increases. Increasing the temperature raises the kinetic energy of the phospholipids in the cell membrane, so more pigment is released. From 20 to 40 degrees Celsius, the line has a steep gradient, which demonstrates a further increase in the amount of light absorbed. The average absorbance rise from 0.163Au to 0.56Au between temperatures 20-40 degrees Celsius.
As the temperature rose, the anthocyanin molecule gained a lot of kinetic energy and diffuses quickly out of the cell.
The bonds holding membrane proteins together vibrate such as the hydrogen, hydrophobic and peptide bonds. Within the phospholipid bi-layer there are proteins and these proteins consist of polypeptide chains. As temperature increases from 40-60 degrees Celsius the average absorbance increases and the molecules vibrate so energetically that these bonds break easily and produces holes within the cell wall and phospholipid bilayer.
Furthermore high temperatures denature intrinsic proteins in the membrane so more pigment escapes.
High temperatures also denature the tertiary 3D structure of the protein. Proteins are hydrophilic channels for ions and polar molecules and also allow certain substances to enter and leave the membrane, so if the proteins specific structure is altered as in the case of high temperatures, the cytoplasm and other compounds within the membrane will leak out, this has been demonstrated by a steady increase in anthocyanin which is a polar molecule to leak out of the beetroot as the temperature increases.
So between temperatures of 0-40 degrees Celsius, the pigment escapes due to faster diffusion rate associated with molecules having more kinetic energy. But from 40-60 degrees Celsius there is increased diffusion of pigment because of the denaturation of the membrane proteins.
Between 40 to 60 degrees Celsius, the phospholipids do not fit together because the membrane becomes more fluid. So more pigment escapes through gaps between the phospholipid bilayer, which acts as a “barrier” to stop the anthocyanin pigment molecules from escaping.
Beetroot also contains large amounts of betacyanin, a red pigment, situated in the large internal membrane vacuoles. At 60 degrees Celsius, when the membrane structure is disrupted, the pigment can cross the vacuole membrane and cell membrane and leaks out into the surrounding environment. High temperatures also distorts the active site of the carrier affecting the shape of the fluid mosaic model membrane, so even if you lower down the temperature, the cell will not regain its original 3D shape and bonds cannot reform.
At 60 degrees Celsius, the lipids that make up the membrane lose their rigid structure and weaken, which leads to perforations along the membrane. This in turn causes the leakage of anthocyanin by diffusion as the concentration of anthocyanin inside the cells seeks equilibrium with the low concentration outside.
But after 60 degrees Celsius, lipids in the phospholipid bi-layer simply melt. The rise in temperature causes a rise in kinetic energy; this change in kinetic energy causes the fluidity to increase.
Weaknesses begin to form in the membrane and as the kinetic energy was further increased, perforations formed along the membrane structure. This resulted in a tilt in the diffusion gradient, which caused an increase of anthocyanin leakage.
The glycolipids, glycoproteins and membrane proteins stop functioning at higher temperature and these all give structural advantages to the membrane so their loss would weaken the membrane structure significantly.
The glycolipids and glycoproteins would collapse and so hydrogen bonds formed with the external solution would be non-existent and also the proteins would denature, so more anthocyanin can diffuse out of the cell.
From the gradients I can tell that from 0-40 degrees Celsius, the gradient increases because of the almost straight vertical line. At 60 degrees Celsius the gradient of the graph decreases. So at about 60 degrees Celsius, this relates to when the lipids in the cell surface membrane begin to break down producing holes in the membrane through which the red pigment anthocyanin leaks out
From 0-40 degrees Celsius, the pigment molecules have more energy to move about and so diffuse out of the cell.
From 0-20 degrees Celsius, membrane proteins are intact and the hydrogen bonds, that hold the proteins are vibrating.
So overall my results and prediction support my conclusion, the rise in temperature is positively correlated to the amount of light absorbed.
As temperature rises from 0-40 degrees Celsius, the kinetic energy of the pigment molecules increases and the pigment molecules more rapidly and diffuse out of the cell. From 40 to 60 degrees Celsius the bonds that hold the polypeptide chains within the protein vibrate and break. Hence the proteins get denatured which leaks holes in the phospholipid bilayer allowing the anthocyanin to diffuse out of the cell.
Evaluation
The results I obtained from this experiment was quite accurate as I had repeated the experiment three times, from the graphs I had no anomalous results, but this may be due to not many readings being recorded. There were also certain limitations, which made the experiment harder than it could have been and without these limitations the results, would be much better and produce the best absorbances.
There may be same sources of experimental error; one source of error was extracting the beetroot from the distilled water with the tweezers. The rough use of a tweezer could have pierced the cell membranes and the tonoplasts membranes surrounding the vacuole containing the red pigment, this causes more of the red pigment to be squeezed out into the distilled water, so more pigment is released into the distilled water than should have been.
I could have measured different amounts of water each time using a measuring cylinder. More water can dilute the pigment of the beetroot, having less water could produce a dark colour which would produced different absorbance’s and hence are less likely to produce better results. I also could have left the boiling tubes in the water baths longer/shorter than the intended time. This means that it is not a fair test as the boiling tube is not left for enough time. So the pigment that should have been released was in plentiful amounts or not be in sufficient amounts, which could mean inaccurate reading on the colorimeter.
I also found by cutting the beetroot to all the same sizes were pretty difficult when using a scalpel, so I might have at different sizes of beetroot each time, so if a beetroot is cut too big, then excess amounts of pigment is released and produce inaccurate readings in the colorimeter. Also it was harder to maintain the temperature of the water baths so this must produce unreliable results on the colorimeter. I might not also have drained the excess fluid off the beetroot after I cut it, so this means that more pigment is released that should have been which could lead to unfair results.
To improve the reliability of the results I would repeat the experiment again, to slightly change it, I would also use a different colorimeter, which measures light over a narrower range of wavelength. I could use a cork borer to cut the beetroot all the same size, so that the beetroot has all the same length, width and surface area. I should use a stop watch carefully to check that the 10 minutes was up and remove the beetroot from the boiling-tube, but I could have let the stop watch run a bit over the time or under the time and so the beetroot may not be taken out at the exact time.
This could possibly indicate that as the beetroot was left in the heated water for a longer period, more pigment could have leaked out.
Furthermore after I had cut the beetroot into pieces and washed them to clean the red pigment leakage, I found it difficult not to disrupt the membrane as I tried to dry the pieces off. I think that a wider range of temperatures should be used because I think it is appropriate for the experiment. Smaller intervals could have been used to find out at what specific temperature the membrane intensity was breached. So I would repeat the experiment at temperatures of 0,10, 20,30,40,50,60,70 and 80 degrees Celsius. I could have repeated the experiment more than three times for each temperature but overall I tried to do my best as time was on my shoulder, I also tried my best of ability to cut the beetroot all at the same size and used a water bath to keep the temperature consistent. The samples were left in the water bath for a same period of time.
The boiling tubes may have been dirty so this could have affected the colour of the sample also.
The thermometer was used to check the temperature at the start of when the beetroot was put in the water and at the end of the intended time when taking the beetroot out.
I also washed the beetroot each time I put it in the boiling tubes inside the water bath.
A thermostatic water bath is much more accurate as it retains water at the required temperature, it can also be said that the results will be more reliable as a better piece of equipment used.
Some of the disadvantages about this experiment that I carried out was I did not always cut the beetroot straight and sometimes I cut them vertically which causes the surface area to be different on the cylinders.
Some beetroot cylinders were washed more than once and shaking the boiling tubes unevenly before putting in the colorimeter could produce not the best results.
Also the age of the beetroot needs to be controlled to prevent unreliable results, the older the beetroot the more betacyanin and anthocyanin pigment available therefore the experiment will be unfair, hence I will use a beetroot that is new and just grown.
So overall my results have supported my prediction, the results were fairly accurate and reliable and allowed me to give a firm conclusion.
