A2: Plan a procedure
- Using suitable equipment and apparatus, list the steps to be followed (there is no need to list apparatus unless unusable items are suggested)
- Using a 5mm corer, core enough beetroot to make 45 specimens all 5mm in length.
- Rinse all of the beetroot to remove any excess betalain.
- Place 5ml of water into 3 test tubes measuring the water using a pipette.
- Prepare the waterbath.
- Place all three of the test tubes into the waterbath and regulate the temperature using a thermometer.
- Place the beetroot discs into the test tubes.
- Leave for 15 minutes, constantly monitoring the temperature during this time to ensure stability.
- Remove the beetroot discs from the test tubes leaving the water solution in the test tube.
- Repeat this experiment for all the other temperatures.
- Select a suitable range for the independent variable or suggest conditions for the experimental treatment
I will be carrying out this experiment at 40°C, 50°C, 60°C, 70°C and 80°C at will be carrying out the experiment 3 times for each temperature.
- Communicate an ordered sequence to follow procedure (noting any safety issues)
- Cut the beetroot specimens using a size 5 corer
- Measure the length of the cut beetroot cutting them at 5mm
- Place 3 of the cut specimens into each of the three test tubes and carry out the experiment
- Which provides a fair test?
Keeping the temperature constant using a thermometer and constantly regulating the temperature during the 15 minute period.
Using the same size of beetroot discs 5mm in diameter by 5mm in length.
Using the same amount (5ml) of distilled water in each of the test tubes.
- State clearly what must be recorded
The percentage absorbance of pigment release, this figure will be generated by the colorimeter, and basically tells me how dark the solution. Therefore the darker the solution the more permeable the beetroot membrane so more betalain is released.
A3: Planning for analysis
- State the level of accuracy or appropriate units of measurement
A constant temperature will be maintained in the water bath using a thermometer. The sizes of the beetroot discs are ensured the same with the help of the corer to create the 5mm diameter and a ruler to measure the 5mm length.
A pipette will be used to measure the amount of distilled water (5ml) placed into each of the test tubes. The 15 minute period will be monitored using a digital stop clock, and a blue filter will be used on the colorimeter to absorb the maximum amount of the red light.
- What format will you use to present your results
I will use the table shown overleaf to record and present the results. I will calculate the mean, standard deviation and standard error. Standard deviation measures all the variation in a sample, so the higher the standard deviation the higher the variation. So the lower the standard deviation the more accurate the results are.
(BLANK TABLE OVERLEAF)
- Decide which methods of analysis will be the most appropriate
I will be using 95% confidence limits which tell me if I was to repeat this experiment again there would be a 95% chance or the results falling within the upper and lower limits. The confidence limits also show if the results are significant or insignificant.
I will also be using standard deviation which shows me the variation between the results. The lower the standard deviation the more accurate the results.
- Decide how much replication you will need for successful analysis
I will be repeating this experiment three times for each of the temperatures and calculating the mean based on these results. I will also be using the class averages to create a second graph displaying the class results for this experiment.
If I had more time I would use more replicates and may try this experiment at different temperatures.
- Consider how much replication is feasible considering the time and materials available
I am going to repeat this experiment three times for each of the temperatures. If I had more time I would carry out more replicates. I also do not have a digital waterbath so it is vital that I repeat this experiment at least three times to develop an accurate mean.
(Results Overleaf)
C2: Interpretation
- Assessment of the reliability of the data
Having now carried out the experiment, tabulated the results and created graphs I can now confirm that the experiment followed my hypothesis. As the temperature increased the percentage absorption of the pigment also increased. For instance at 40°C the percentage absorption of the pigment was 0.41 compare this with 50°C and the absorption was 0.46. This trend continues throughout the temperatures but between 50°C and 60°C there is the biggest increase in the % absorption. At 50°C the percentage absorption was 0.46 whereas this was 1.62 at 60%. That’s a 1.16 difference, such a huge difference like this clearly show that at temperatures above 50°C the membrane has become seriously damaged releasing lots of the pigment.
The confidence limits for my results are quite wide which would indicate that there was some variation in my results. For instance at 60°C the upper limit was 2.024 and the lower limit was 1.216, this basically means that based on my results if this experiment was repeated there is a 95% chance of the results falling within these brackets. The upper limit for 70°C is 1.945 and the lower limit is 1.815. When compared with the confidence limits for the class results I can see that there is less variation as the limits are not so spread. For instance in the class results at 60°C the upper limit was 1.64 and the lower limit was 1.12. This only leaves a difference of 0.52, whereas the in my results there is a difference of nearly 0.8.
- Comment on the reliability of the data
I think the data I have collected is reliable due to the equipment and steps I have taken to ensure reliability. For instance I used a corer to create the 5mm diameter beetroot discs and cut them to 5mm using a ruler, therefore all the beetroot discs were the same size.
When I look at my graph I can immediately see that some of the bars are quite large, for instance at 60°C. This variation I feel is due to my inability to sustain an exact 60°C environment for the 15 minute period on three occasions.
I also feel that I was able to control the waterbath efficiently with the help of a thermometer and close inspection throughout the 15 minute period. However if I had a digital waterbath I would have been able to control this environment temperature even more accurately. I also used a digital stop clock to monitor the time and ensured the beetroot discs were removed after the 15 minute period.
I measured the distilled water in each of the test tubes using a pipette and ensured that exactly 5ml was placed into each.
Although I took all these steps to ensure reliability my results still displayed some variation. For instance at 40°C there was a 0.2 difference between the upper and lower confidence limit. Possible reasons for this may be that the beetroot discs were not exactly the same size, some of the beetroot discs came from different beetroots which may have contained more or less betalain. Another possible reason could be that the waterbath may have at times during the 15 minute period experienced some slight fluctuations above and below the desired temperature.
- Using statistical evidence, identify the trend
As temperature increase the membrane in the beetroot becomes damaged. Therefore the beetroot membrane becomes more permeable and cannot carry out its function. Therefore as a result the betalain inside the cell begins to seep out through.
There is no significant difference between the results at 40°C and 50°C as the confidence limits fall within each other. However there is a huge significant difference between 50°C and 60°C as the confidence limits don’t even touch. This indicates to me that between 50°C and 60°C the membrane becomes seriously damaged and as a result this damage causes the membrane to loss its role. As a result lots more betalain can escape through the membrane and into the solution. The class results also indicate that the significant difference is between 50°C and 60°C. At these higher temperatures the proteins which make up over 70% of the cell membrane become denatured and the phospholipid bilayer becomes fluid.
My results show that as the temperature increase the permeability of the beetroot membrane also increases. This trend is also apparent in the class results.
My results are relatively accurate as the confidence limits are quite small. However I also used the class results to design the same table and if I compare the two I can easily see that the confidence limits in the class graph are much smaller. This indicates that the class results are more accurate and that if I was to repeat this experiment again there is a 95% chance of the results falling within the upper and lower limit.
- Briefly explain the trend
At these higher temperatures the proteins which make up over 70% of the cell membrane become denatured and the phospholipid bilayer becomes fluid. Therefore as the temperature increase the permeability of the beetroot membrane also increases. Due to this the membrane cannot control what enters and leaves the cell so the betalain pigment escapes. At higher temperatures more of the betalain can escape as the membrane is more permeable.
- Using appropriate biological knowledge and understanding, give a detailed explanation of the trend
Temperature has a huge impact on the structure of the cell membrane in a beetroot. It is composed of proteins making up over 70% and a phospholipid bilayer. At high temperatures the proteins in the cell membrane start to uncoil and become denatured and the phospholipid bilayer turns to fluid. Therefore since the cell membrane is damaged it cannot prevent cell organelles from escaping. Therefore the betalain pigment found in the vacuoles of the beetroots escapes.
In my experiment, as the temperature increases more holes form in the cell membrane and so more of the betalain can escape into the solution. Therefore there is more pigmentation in the solutions at higher temperatures, so therefore there is an increase in the percentage absorbance of betalain.
C3: Evaluation of the practical procedures
- Comment on the appropriateness of the range of the independent variable or the conditions of the experimental treatment
The temperature was quite difficult to maintain as I was using a make shift waterbath with the help of a Bunsen burner and a thermometer. I found that it was very difficult to maintain a constant temperature and noticed slight fluctuations. If I had a digital waterbath I would have been able to set the exact temperature and not need to worry about regulating as the waterbath would do this.
- Comment on the procedures used to prevent variation of factors not being investigated
A constant temperature was maintained to prevent variation of the replicates. This was done manually with the help of a thermometer and I found this quite a difficult task.
I used distilled water in the test tubes to ensure there were no impurities in the water which may have affected the % absorbance of light.
I calibrated the colorimeter after every test using a test tube filled with distilled water. These helped me to ensure that the colorimeter gave accurate readings.
I used clean equipment as dirty test tubes for instance would have affected the ability of the colorimeter to give accurate readings, as the dirt would affect the amount of light passing through.
I also tried to use the same size of beetroots when making the discs as different sizes may contain different amounts of the betalain pigment.
- Comment on the appropriateness of the observations/measurements
There is some variation in my results which the confidence limits on the graph show. I think the main reasons for this variation is due to the temperature in the waterbath as I found this quite hard to regulate. Therefore at times the water bath may have been above or below the desired temperature by a couple of degrees. This variation could also be attributed to the actual size of the beetroot specimens as they may not have all been exactly 5mm as it is quite hard to cut exactly to this length.
Some of the beetroot discs were also cut from different size beetroots and therefore some may contain more of the betalain pigment. Also some of the discs were from the core of the beetroot and the core contains more betalain.
However the class results show less variation between the confidence limits thus indicating more reliable results.
Both sets of results show that as the temperature increases the permeability of the cell membrane increases. As a result more betalain escapes and the solution becomes darker due to the presence of betalain, therefore the solution absorbs more light.
- Assess the validity of the implementation of the procedure
I think this procedure went quite well however I feel that the regulation of the waterbath may have affected my results. If I had the time and the resources I would like to repeat this experiment using a digital waterbath which would allow me to control the temperature to 0.5°C.
I would also like to use more replicates at each temperature and repeat this experiment at different temperatures.
I would also like to find out at exactly which point between 50°C and 60°C does the cell membrane start to become denatured.
- Suggest another variable which could be investigated
If I had more time and greater resources I would like to investigate other factors. For instance I would like to investigate the effects of detergents. Detergents help bring fat and lipids into a solution so in theory should break down the phospholipid bilayer in the beetroot cell membrane. Higher detergent concentrations should break down the membrane further therefore releasing the betalain into the solution.
I would also like to investigate what would happen if I used pickled beetroot instead of fresh beetroot. Pickled beetroots are treated and are designed to last longer therefore it would be interesting to see what effects temperatures would have on the breakdown of their membrane.