Hypothesis:
My prediction for this experiment is that as the temperature increases, more pigments will leak out, causing the transmission of the solution to decrease as the dye absorbs more light. This is because rise in temperature damages the cell membrane structure by denaturing the proteins, forming holes that cause the pigments to leak out.
Method:
Independent variable-
- Temperature of water baths (0˚C to 70˚C)
Dependent variable-
- Absorption or transmission measurement for each of the dye solution.
Variables that are kept constant-
- Size of each slice of beetroot (1 cm in length)
- The kind of beetroot (each beetroot might vary in concentration of dyes)
- Time in keeping beetroot sections in water baths (30 min)
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Volume of distilled water (5cm3)
- Cut using a size 4 cork borer, eight 1cm length slices from a single beetroot.
- Place the slices in a beaker of distilled water, leave for 10 min to wash off the excess dye.
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Place 8 labelled boiling tubes each containing 5cm3 distilled water into water baths at 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C and 70°C. Leave for 5 minutes till the water reach required temperature.
- Place 1 beetroot section into each boiling tubes and leave for 30 min in the water baths.
- Remove the beetroot sections from the boiling tubes; shake the solutions to disperse the dye.
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Switch on the colorimeter and set to read % transmission which is how much light goes through the sample, set the filter dial to the blue/green filter.
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Using a pipette, measure accurately 2 cm3 of distilled water into a cuvette. Place it into the colorimeter, without touching the bottom of the cuvettes so the bit light shines through is not smudged, making sure that the light is shining through the smooth side.
- Adjust the colorimeter to read 100% transmission for clear water as the control, keep the setting through out the experiment.
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Place 2 cm3 of dye solutions from each of the boiling tubes into cuvettes and take readings for transmission.
Safety:
- Use cork borer and scalpel carefully to prevent injuries by cutting
- Water in some of the water baths would be very hot so watch out
- Watch out that you don't break any glass apparatus to prevent cutting
Results:
To plot the graph, I put independent variable (temperature °C) on x-axis and dependent variable (% transmission) on the y-axis
Analysis:
From my results and graph, I can see that my prediction was correct, the transmission of the solutions decreases and the absorbance increases as the temperature increases.
From 0°C to 40°C, the decrease in % of transmission is quite small. But from 40°C onwards there is a sharp decrease in transmission with a huge difference between each reading, but the decrease level off after 60°C.
This is probably because temperature up to 40 °C does little damage to cell membrane structure; the proteins within the phospholipid bilayer can withstand the heat. But after optimum temperature has been reached, the proteins start to denature. These proteins are made up of polypeptide chains which are joined together by hydrogen and peptide bonds, once the temperature has increased above 40°C the molecules vibrate so energetically that these bonds break easily and therefore creating holes within the cell membrane, causing large amount of beetroot pigment to leak out. But after 60°C, nearly all the pigments has leaked out from the beetroot, absorbing almost all of the light, therefore the decrease in transmission levels off again.
Evaluation:
Nearly all of the points fit on the curve of best fit on my graph, but there are many things I could have done to make this experiment more reliable and accurate.
There were many stages in my procedures that could have led to errors. There were many random errors, for example, although the cross sections of the beetroot would be the same by using a cork borer, there could have been inaccurate measurements of length for each beetroot section. If the slice was cut slightly smaller it would have reduced the surface area therefore decreasing the amount of pigment leaked from the sample. Also, although I cut the sections from 1 single beetroot, the slices were cut from different parts, some near the center and other near the surface; this could have led to unreliable results as the pigment concentration might have varied. During the preparation, many of the cells could have been damaged causing pigment to leak out so reducing the amount of pigment leaked during the experiment.
If any errors were to occur from the equipment, i.e. systematic errors, the most likely would have been the temperatures of the water baths. The temperatures of these baths would not always have been constant and may have been slightly over or under the desired temperatures. This is because the heater on the water baths only start working once the temperature has dropped below what is required, once the heater has started working once again it causes the temperature to rise above what is required, then the bath once again cools to below the optimum temperature and the whole cycle starts once again. Also, for the 0°C, I used a beaker of ice cubes, but that didn’t reach right down to 0°C, and some of the ice must have melted during the 30 min waiting time, raising the temperature.
Also I have found that the colourmeter was particularly unreliable because during the testing numbers on screen changed often for each of the cuvettes, and it would be better to have a more accurate colourmeter too which measures light over a narrower range of wavelengths.
To achieve a fairer and more reliable result, the experiment should have been repeated two times, working out a clear average between the results, seeing any trends more clearly.
