APPARATUS: fresh beetroot, narrow cork borer, beaker, water, knife, tile, ice, 4 waterbaths, stopwatch, colorimeter (with blue filter), detergent, ethanol and 1M HCl.
METHOD:
- Use a narrow cork borer to cut 9 cores, in parallel, down the length of half a fresh beetroot.
- Wash in water in a beaker until there is no further “bleeding” of pigment from cells whose membranes you broke while cutting the cores. It is most important to take your time and do this thoroughly. Store these cores in water.
Investigating the effect of temperature
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Measure 5cm3 of water into 6 test tubes. Place in ice, room temperature, and water baths at 30,40,50 and 60oC and leave for 5 minutes to thermally equilibriate.
- Add a core and start a clock
- Swirl gently and record the appearance of the water after 2,5,10 and 15 minutes.
- Set up a colorimeter with a blue (470 n ) filter.
- After 15 minutes remove the core, fill a colorimeter curvette with the water from each tube, and record the percentage transmission of light. This will give a relative indication of the anthocyanin pigment concentration.
- Plot a graph of 1/percentage transmission (vertically) against temperature.
Investigating the effect of acids and fat solvents.
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Place 5cm3 of each of 5% detergent, ethanol, and 1M HCl into each of the 3 test tubes, and place on to the bench at room temperature.
- Add a core, start a clock, and repeat the last 4 steps above.
PREDICTION: I predict that an increase in temperature will cause the plasma membrane to denature and allow anthocyanin and other substances contained within the membrane to leak out. As it is the anthocyanin that gives the beetroot its purple colour it will be possible to see the water change colour and show that the anthocyanin has leaked from the cell.
RESULTS:
From investigating the effect of temperature
From investigating the effect of acids and fat solvents
ANALYSIS:
From the graph I can see that my prediction was correct except for one anomalous result which can be accounted for. The corvette containing the solution from the room temperature test tube was accidentally spilt so when it was put into the colorimeter it was not full and therefor did not give and accurate result. The rest of the results follow my prediction that as temperature increases the plasma membrane is denatures and therefor allows substances from inside the membrane to leak out, anthocyanin is one of the substances that is leaked and can be seen as the pink colour in the water.
From the results of acid and fat solvent investigation I can see that the detergent causes most denaturing, as this has the highest percentage light transmission, 27 compared with 17 and 16 from the HCl and Ethanol respectively.
EVALUATION:
I thought the investigation overall was successful. We discovered that there was a correlation between the temperature change and the percentage transmission as we had predicted. The results clearly showed this, with the exception of 1 anomaly, the room temperature tube was accidentally knocked so the little solution that was put into the colorimeter did not give an accurate result. It also made it unfair to compare it to the others, as it had not been as full as the other cuvettes. The other problem we had was that we had put the solutions into the cuvettes and colorimeter too soon after they had come out of the water baths. The high temperatures caused the cuvettes to condense and therefor when we put them in the colorimeter the transmission reading did not show the solution we had obtained but included the steamed up sides of the cuvette. We allowed the solutions to cool before putting them into the colorimeter again (this is when 1 tube was accidentally knocked over) and then recording the results we read.
It was quite difficult to try and get all 9 beetroot samples the same size, although we used the same borer and cut them all the same length they were all quite small so it would have been easy for them to all be slightly different. By having the beetroot the same width and length they should have all had the same surface area, which would therefor allow the same amount of anthocyanin to leak. If we had measured the beetroot to have equal masses then they may have all had different surface areas, which would have made the experiment unfair in recording the integrity of plasma membrane.
