Pre-tests
In order to find the most effective method I decided to carry out some pre-tests. These helped me to discover the most accurate and safest way to carry out this experiment. The volumes and temperatures I tested helped me to find out the best procedure to take.
Pre-test 1
- 0.1g yeast used
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5cm3 H2O2 used
- This experiment was carried out at room temperature.
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A gas syringe was used to measure the volume of O2 given off.
There was a large amount of O2 given off in the first 30 seconds, but I couldn’t see how it rose between 0-30 seconds clearly enough. So after this trial I needed to take readings after shorter intervals to give me a better idea of the rate of reaction. I then tried to take readings every 10 seconds, but this made it difficult to get accurate results, because it gave me so little time to write results down and watch the timer and prepare to take the next reading. From this point onwards, I took a reading every 15 seconds, which was more efficient and practical.
Pre-test 2
- 0.1g yeast used
-
5cm3 H2O2
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This experiment was carried out at 66°C.
At this temperature, the yeast stopped decomposing after 15 seconds, suggesting that the Catalase was denatured.
Pre-test 3
- 0.1g yeast used
-
5cm3 H2O2
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The experiment was carried out at 10°C
From this test I wanted to find out how long it would take for the volume of H2O2 I used to completely decompose. At 10°C the reaction was very slow and showed that complete decomposition could take at least 4 minutes. If I took readings for every experiment for 4 minutes, it would be very time consuming, so I decided on 2 minutes as a suitable time period. So then I decided to try the experiment at 20°C and not include 10°C in my range.
Pre-test 4
- 0.1g yeast used
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10cm3 of H2O2
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This experiment was carried out at 20°C
I used double the volume of H2O2 this meant that the O2 was given off much fast. This made it difficult to take accurate readings because the gas was being given off so quickly. So I decided to stick to using 5cm3 of the H2O2 solution.
Method
The variables that affect the decomposition of H2O2 are shown in this table.
Apparatus
For this experiment I will need:
- Retort Stand
- Gas Syringe
- Delivery tube with bung
- Boiling tube
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A 10cm3 measuring cylinder
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At least 30cm3 of 5 vol H2O2
- About 1.5g of yeast (this amount needed for repeats)
- Petri dish
- Test tube rack
- Large Beaker
- Bunsen
- Thermometer
- Heatproof Mat
- Splints/matches
- Timer
- Balance accurate to 2 decimal places
- Goggles
Procedure
Firstly the equipment has to be set up as shown in the diagram above. The first temperature at which will carry out my experiment will be 20°C. As I discovered in my pretests the water from the tap was at approximately 20°C and I will use a thermometer to make sure it is the right temperature, then I will use the 10cm3-measuring cylinder to measure out 5cm3 of H2O2. The H2O2 is then placed in a boiling tube, which is placed in the water bath until it too has reached 20°C. This is done to acclimatize the H2O2 to the temperature, thus making the results more accurate, which in turn gives a clearer picture of the rate of decomposition at this temperature. While the H2O2 is reaching this temperature, 0.1g of yeast is measured out in a petri dish ready to be added to the H2O2. When the H2O2 has reached 20°C then the yeast is added. The bung is placed in boiling tube immediately and then timer must be started. A reading for the volume of O2 must then be taken every 15 seconds, and recorded. This experiment must be repeated three times at this temperature, in order to achieve a more accurate average.
This procedure is then carried out for the other temperatures of 30°C, 40°C, 60°C and 70° C. For these higher temperatures the water will need to be heated with a Bunsen on the blue flame. For 60°C and 70°C I will carry out the experiment with the water bath and boiling tube still being heated by the Bunsen on a low flame because this is a high temperature and this will be more accurate as the temperature will not drop which would affect the results. The experiment needs to be carried out at all the temperatures three times.
Safety
100 vols. of H2O2 is corrosive
20 vols. of H2O2 is an irritant – dangerous if swallowed
Less than 18 vols. H2O2 to produce minimal hazard – I am using 5 vols. of H2O2 which is well with in the minimal hazard range.
I will be wearing goggles and should any spillage on myself or on other surfaces occur, I would wash it off with lots of water immediately. Because H2O2 is flammable, I shall take special care around the Bunsen burner as oxygen-rich air promotes burning.
Obtaining
This table of results shows the raw data from the experiment.
This table shows the averages I obtained from my repeats.
Analysis
From the graph that I drew using the averages from the results I can see that the yeast and H2O2 at 30°C produced the most O2 in two minutes, 32cm3, but at 40°C there was more O2 given out more quickly, 11.3cm3 after 15 seconds, than at any other temperature. The least O2 was given out at 70°C, just 9.5cm3 in two minutes
From the best-fit lines I drew on my graph, I have interpolated some data shown in this table. This is shown on my graph.
From this data I am going to draw a graph to show the rate of decomposition.
I worked out the rate by dividing the volume of O2 given off in 15 seconds by the time it
took, which was 15 seconds. This graph shows that the rate of reaction was highest at
40°C, at 0.87cm3/second, suggesting that the optimum temperature was close to 40°C, as
I predicted. The increase between 20°C and 30°C and then to 40°C was a steady one,
increasing by about 0.25cm3/secs between each 10°C interval. The rate almost doubled
between 20°C and 30°C, which again is something I predicted. As far as I can tell from my graph, the rate decreased after 40°C which agrees with the idea of it being near to the optimum temperature. The rate of reaction appears to decrease as quickly as it increased, in fact the line on the graph is almost symmetrical between 20°C and 60°C. It is impossible to say exactly at which the catalase was denatured without further investigation, but it is probably safe to say that that the catalase was denatured by the time the temperature reached 60 and 70°C.
As I have said in my prediction, catalase is an enzyme found in human cells. This
means that the optimum temperature should be body temperature, which is approximately
37°C. The closest temperature to body temperature that I tested at was 40°C, and this
was temperature when the rate of decomposition was the highest, suggesting that 40°C
was the optimum temperature out of the range I tested. This means that at 40°C, all the
available H2O2 molecules were combining with the active sites of all available catalase
molecules. In other words there were the most amount of successful collisions at 40°C
of all the temperatures in my range.
Theoretically, the rate of reaction should have been highest at 70°, and it would have been if this were not a reaction that involved enzymes. According kinetic theory, the higher the temperature, and the faster the rate of reaction. This is because as the temperature increases, the molecules in the reaction have more kinetic energy. This means that they move faster and they collide more frequently. The molecules also already have the activation energy required, so the bonds break on collision. So if the higher the temperature, the faster the rate of reaction, why wasn’t the rate of reaction fastest at 70°C? It is not fastest at 70°C, because catalase is an enzyme and being an enzyme means it is a protein. Which means that heat can change the structure, or that catalase can be denatured. As I said in my prediction, heat changes the structure of the catalase, so if we use the lock and key analogy, the lock has changed its shape, and key no longer fits. The catalase is no longer the right shape for H2O2 molecules to bind with the active sites of the catalase. Oxygen is still given off, because the decomposition of H2O2 does not need catalase, it merely speeds up the decomposition. This is why there is still oxygen given off at 60 and 70°C, but the rate of reaction is not highest at these temperatures. So from this experiment I can conclude that the optimum temperature for the enzyme catalase is around 40°C, I can also conclude the rate of reaction, or decomposition in this case increases as the temperature rises until the optimum temperature is reached. After 40°C the catalase molecules were denatured, and the rate of decomposition decreased, and using scientific theory as I have done, I can explain it.
Evaluation
My results agreed with my prediction and scientific theory, they also showed clear trends. I do not think there were any significantly anomalous results because the repeats meant that if one set of results was significantly different to the other two sets of results at that temperature, it did not affect the average very much, which was in the end what I used to plot my graphs and calculate the rate of reaction.
There were many faults to the procedure I used which could have affected the accuracy of my results. Oxygen escaped in the short time between adding the yeast to the H2O2 and placing the bung in the boiling tube. This was important because the rate of decomposition was fastest in those first few seconds, so this could mean that the results for the first 15 seconds could be inaccurate. To improve on this a boiling tube or conical flask with a side arm could be used and this would mean the delivery tube was already connected the reactants, so less oxygen would be lost. There was also the problem of the yeast sticking to the side of the boiling tube, due to it being wet from the H2O2 being poured in. This meant that not all the yeast and therefore not all the catalase could react with H2O2. A solution to this would be to add the H2O2 to the yeast rather than the other way round. The H2O2 could be acclimatised to the temperature required in a separate boiling, then be syringed into the boiling tube containing the yeast. Another problem with yeast was that the particles of yeast that we used varied greatly in size, meaning that not all the particles had the same surface area. If the yeast had been crushed first then it would have had a more uniform size and a larger surface area, and as the reaction between the catalase and the H2O2 occurs on the surface, a greater surface area means more collisions take place. So maybe the results would have been higher. The last two problems were to do with temperature. Under the circumstances of this experiment, the temperature at which the experiment was carried out could be controlled fairly well. It is however impossible to say if the temperature remained constant through out all the tests at one temperature, especially the higher temperatures of 60 and 70°C even though a water bath and Bunsen burner were used. This could make a big difference to the results, because if the temperature was supposed to be 30°C but it had dropped to for example 24°C it would be difficult to tell because water is a poor conductor and the thermometer might not measure the temperature drop. So this theoretical set of results could be closer to what they should be at 20°C and bring the average down. In an ideal situation, the best thing to use in this experiment would be an electrical thermostatically controlled water bath, then the results would almost certainly be more accurate because the temperature would be kept more constant. Finally, another reason that the results for the higher temperatures of 60 and 70°C could be inaccurate is the fact that gas expands at high temperatures, so the oxygen could have taken up a larger volume than there was oxygen actually produced.
If I was to carry out this experiment again I would definitely try the experiment at 50°C because it is unclear from my results what happens after the optimum of 40°C has been reached. I would also probably experiment with smaller intervals of temperature, maybe using 5° intervals, which would again give me a better idea of the curve on the rate of reaction against temperature graph, because I wasn’t actually able to draw a smooth curve. If I was going extend the experiment, I would maybe repeat the same experiment but with small pieces of liver instead of yeast, as it also contains catalase and it would be interesting to see if the results from the two experiments were similar. I might also use a narrower temperature range between 30, 40 and 50°C to find out exactly where then optimum temperature was.