Repeats: I will do three experiments on each temperature reading and then calculate an average. If variations of readings for certain temperature points are big and I have time at the end of my experiment, I will do one more trial on each temperature and get a more accurate average reading.
Safety:
- Goggles must be worn at all times, whilst Bunsen burners and chemicals (buffer) are being used in the room.
- Any loose clothing should be removed, this will minimise spillages and accidents.
- Hair must be tied back, to give a clear view of the working area and away from open fire.
Equipment / per temperature points experiment
- 3 x Boiling tube
-
2 x 500 cm3 beaker
- 2 x Thermometers
- 9 x Beetroot disks
-
200 cm3 x distilled water
-
200 cm3 x ice cubes
- 1 x Tripod
- 1 X Bunsen burner
- 1 x Mat
- 1 x White tile
- 1 x Scalpel
- 1 x Ruler
- 1 x Cork borer
- 1 x Tongs
- 1 x Stop watch
- 3 x Sticky labels
- 4 x Test tubes
Diagram of set up apparatus / per temperature experiment
Method:
1. Prepare beetroot disks
- Place the beetroot on the white tile
- Push the cork borer through the beetroot
- Cut of the outer skin and place a ruler along side the cut out cylinder of beetroot and measure 5mm along -width of the beetroot disk (to a 0.5mm degree of accuracy)
- Using a scalpel cut the beetroot to form disks
- Repeat these steps to accumulate 9 beetroot disks (3 for each boiling tube)
NB. Do not hold the beetroot at the base (opposite side to cork borer) as the cork borer is sharp and may cut your hand.
2. Rinse beetroot with distilled water before putting into boiling tube
-
Place all beetroot disks into a beaker and add 15 cm3 of distilled water
- Swirl beaker gently – this will wash off all pigment which has been released from cutting the disks and damaging some membranes.
- Pour out and replace the distilled water, repeat this step until the beetroot water remains colourless.
3. Set apparatus as shown in the diagram
-
Pour 150cm3 of tap water into a beaker (not the one used for washing beetroot) using a measuring cylinder (to 1cm3 degree of accuracy)
-
Pour 10cm3 (to 0.1cm3 degree of accuracy) of distilled water into 3 boiling tubes using a measuring cylinder and place them into the beaker.
- Place one thermometer into the large beaker and one into one of the boiling tube.
4. Preheat the distilled water to the required temperature. The required temperature should be in the boiling tube as that is the location of the experiment, not in the large beaker.
-
Use a Bunsen burner if the temperature is too low (-3oC below the required temperature) and ice-cubes if the temperature is too high (+3oC above required temperature).
N.B. For the experiment with ice-cold water, fill the beaker quarter with tap water and add ice-cubes. Allow 5minutes for the water to cool down to the minimum temperature.
5. After the specified temperature is reached, place 3 beetroot disks into each boiling tube and leave for 3 minutes, time with a stop watch.
- Constantly monitor the temperature, maintaining it at the desired temperature by adding ice cubes or heat using a Bunsen burner.
6. After 3 minutes remove all boiling tubes from the beaker using a pair of tongs (as the boiling tubes might be hot and will burn your hand at certain temperatures)
7. Take 3 test tubes and separate the water from the beetroot slices by pouring gently the water from one boiling tube into a test tube, the beetroot disks will remain in the set of boiling tubes.
-
Make sure the set of test tubes, with the water, are the same volume of 10cm3 (to 1cm3 degree of accuracy) and leave to cool.
N.B Do not use filter paper to cover the boiling tube whilst pouring the water into the test tube as pigment may be retained by the filter paper as this may give inaccurate results.
8. Pour 10cm3 of pure distilled water into a test tube. This test tube will be used to calibrate the colorimeter (C0). Wipe the test tube with a paper towel and place into the colorimeter, secure the cap on top. Reset the colorimeter so that the pure distilled water test tube recorded 100% transmission.
9. Remove the Co test tube. Wipe a sample test tube and place into the colorimeter, secure the cap and record the result.
10. Re-calibrate the colorimeter using the C1 test tube adjusting the percentage of transmission to 100% and repeat Step 9 for each sample test tube.
- Record results for each test in the results table.
11. Clean apparatus and repeat the experiment changing the temperature to all specified temperatures - just above 0oC, 20oC, 40oC, 60oC or 80oC - and making sure the same volumes, equipment and method is used. At the end of the experiment there should be 18 light transmission percentage recordings (3 test tubes x 5 temperatures) and 5 average results.
Analysis:
Graph 1 shows my initial results and their averages for temperatures 10oC, 20oC, 40oC and 60oC. The general trend of the graph looks like a linear negative correlation between temperature and light transmission as a percentage.
The results for 10oC (64%, 72%, 38%) were the first results to be obtained. These results do not fit this pattern and have a very large variation, another problem with this result is that ideally the temperature should have been above zero, however this was very hard to achieve using purely ice. This makes me come to the conclusion that the results are inaccurate and the experiment should be retaken. The readings for 60oC (3%, 2%, 2%) were extremely low. If a result was reading 2% the substance would practically be opaque allowing limited amount of light through. However, by visual observation this was not the case, hence it might be a false reading.
Having two incorrect results and a discrepancy in the temperature scale, these tests were retaken. I also adjusted the scale by added intermediate temperature points, 30oC and 50oC, to see how this would affect my linear graph.
Graph 2 shows results for temperatures 10oC, 20oC, 30oC, 40oC, 50oC and 60oC.
The new results for 10oC (84%, 82%, 87%) are much more compliant and have a very small variation, making the average figure 84.3%. This confirms that my previous result was erroneous indeed, possibly due to calibration error and lack of experience.
The results for 30oC (70%, 78%, 82%) with an average of 82.6% clearly show that the graph in fact is not linear but a curve, shallow at low end and becoming steep at 30oC. The result for 40oC does not conform to the rest of the results to form a smooth curve. It also overlaps with the results of 50oC. As variation of readings at 40oC and 50oC is big enough to overlap, statistically this difference is not significant and more trials would be needed at 40oC and 50oC to see how the points actually line up. Results for 60oC look much more realistic than at the initial test (shown in Graph 1).
Graph 3 shows the average light transmission percentage against the temperature. The general trend is a negative relationship between temperature and percentage of light transmission due to pigment release. The lower the temperature is associated with the higher percentage of light transmission, therefore, there is less pigment release at low temperature. As temperature increases, the percentage of light transmission decreases due to increasing pigment release.
There are at least two possible explanations to why pigment is released: i) change in membrane proteins that make the membrane more permeable, and ii) increased diffusion through pores in the intact membrane.
All living cells have a cell membrane. The cell membrane consists of a phospholipid bilayer (due to hydrophobic tails and the hydrophilic heads), studded proteins, polysaccharides and other sorts of lipids. All of these components are in a fluid mosaic structure meaning the molecules move about within their respective layer meaning the membrane behaves like a fluid. Carrier proteins in the membrane are responsible for control of pigment release.
Proteins have a primary, secondary, tertiary and quaternary structure. Primary structure is the sequence of amino acids help together with peptide bonds. Secondary structure is the formation of alpha helix’s or beta-sheets, held together with hydrogen bonds. Tertiary structure of the protein is the configuration of the polypeptide bond, hydrogen, ionic and disulphide bridges. The quaternary structure is when multi polypeptide chains interact and have hydrogen, ionic and disulphide bonds between them.
Proteins denature under extreme conditions – high temperature, a process in which a protein unravels and loses its native conformations, thereby becoming biologically inactive. With extreme heat conditions the hydrogen bonds within the secondary structure are broken and the proteins open allowing pigment to be released.
Another reason for pigment leakage is due to diffusion. As the temperature increases the molecules kinetic energy increases. This increase of energy allows diffusion to occur quicker and be more sufficient.
When the temperature was increased from 10oC to 20oC the average light transmission percentage decreased from 84.3% to 82%, a decrease of 2.3%. This is the lowest decrease (shallowest gradient) because beetroot’s surviving conditions range from 5oC to 20oC – English growing climate.
As the temperature increased from 20oC to 30oC the average transmission fell from 82% to 76.6%, a decrease of 5.4%. A slight increase in the amount of pigment released. 30oC is equivalent to an extreme summer’s day. The hydrogen bonds are weakening allowing pigment movement.
Once the temperature increased from 30oC to 40oC there was a decrease of 32% from 76.6% to 44.6% transmission of light. This big decrease shows that something has happened in the membrane, the pores have opened allowing beetroot pigment to leak out into the surrounding water.
When temperature was increased from 40oC to 50oC the percentage transmission increased from 44.6% to 54%, an increase of 9.4%. If these two averages are significant then an increase in average light transmission, a decrease in amount of pigment released could mean that at this certain temperature the pores shut close again. However, the result at 40oC seems to be an anomaly (discussed in the next section) and, therefore, I will discard a biological explanation for the decrease in amount of pigment released.
As the temperature was increased from 50oC to 60oC the average transmission decreased from 54% to 28%, a decrease of 26%. This decrease is due to the amount of kinetic energy present in the molecules of the cell membrane. As the membrane is in a fluid mosaic structure with kinetic energy generated from the increased heat of the surrounding environment, the gaps in formed are large enough to allow beetroot pigment out of the cells.
Evaluation
I have one anomaly in my results at 40oC with the average transmission percentage of 44.6%. This is an anomaly because it does not fit the smooth negative curve shape, I would have predicted an average value of 66% transmission. The reason for this anomaly may be due to sources of error, these are discussed below. This is a very large discrepancy which alters the value of my average result.
Whilst doing my experiment I realised that there were a few unavoidable sources of error. To cut the beetroot to the required dimensions was difficult because the equipment used to cut the width of the beetroot disks was a scalpel and ruler. This means that every disk will be slightly different in dimension and hence have a slightly different surface area.
Due to the circumstances of the experiment the reliability of the results has not had a huge impact in this experiment. The results have not been affected. To overcome this source of error would be difficult, especially in the working environment available, therefore, this error should be taken into consideration with the knowledge that perfect disks and equal surface area is very difficult to achieve.
Another source of error is glassware: whilst doing the experiment when pouring substances from boiling tubes into test tubes some water and traces of pigment were on my hands which I transferred onto the test tube. Using a rough paper towel simply smudged the watermarks.
This might have affected the readings on the colorimeter and have given me slightly skewed results. However, the procedure was exactly the same for all test tubes and therefore, I have minimised the chances and the degree of the skew. For this error not to occur in future experiments I should wash my hand and dry them thoroughly before handling test tubes. However, still clean the test tube with a paper towel before placing into the colorimeter.
Distilled water is a possible source of error too. I presume that as there is no pH buffering, the pH might not remain the same in the boiling tube after the beetroot has been added. This should be observed by using indicator paper and recorded.
Temperature is a major source of error. It was extremely difficult to maintain a constant temperature using a Bunsen burner and ice-cubes, the temperature fluctuated +/-7oC, a range of 14oC, thereby decreasing the degree of accuracy.
This has subsequently affected my results and made them less reliable. To overcome this problem, a thermostatically controlled waterbath should be used. This would also increase the number of trials that can be done at one constant temperature.
Another problem with the temperature points was the scale. I initially planned to do temperature points with an interval of 20oC, starting from just above zero. This proved to be nearly impossible to achieve with purely using ice, the lowest value recorded was 9oC, however, as seen on the first graph, those results do not give me accurate information and the trend was unclear, hence I did intervals of 10oC.
This concludes that the smaller the intervals between each temperature point the move accurate the results are. Ideally a result should be taken for a temperature point with the interval at 1oC, this will show the exact temperature the proteins denature and pores open to release beetroot pigment.
The volume of liquid may have been a source of error. The volume of water in the beaker which is acting as a water bath does not have to be a specific volume. However, it is important that the volume of water within the boiling tube is constant throughout. This was easily achieved by using a small 10cm3 measuring cylinder.
This possible error did not affect my results as my accuracy was to 0.1cm3. A nearly 100% accurate measurement can be achieved if a precision pipette is used.
Another source of error is the colorimeter. This is a piece of electronic equipment that may malfunction, as possibly happened with the initial result for 10oC. It is vitally important to calibrate the colorimeter before taking a measurement.
This did affect my results, once I obtained results for 20oC and saw they were higher in percentage light transmission than for 10oC. The 10oC temperature point was retaken and the results complied. This error is very hard to avoid because it is an electronic piece of apparatus, therefore several results should be taken for every test tube.
The number of repeats is an additional source of error. The more repeats done on temperature points the average values calculated will be more reliable. This is the issue with the anomaly at 40oC, if more results were taken for temperature points 40oC and 50oC then it would be clear to see how the averages for these two points truly relate to each other.
This did affect my results however, this error was consistent throughout the whole experiment which decreases the errors influence on my results. To overcome this error, more trials should be taken for each temperature point.
