Fair test: The variables, as stated above, are temperature and oxygen. We wil alter temperature, the oxygen level will alter as an effect of that. Everything else will be kept the same – we will have 10cm3 of hydrogen peroxide, and 2g of liver. The same people shall each do the same task so they are familiar with their task. The apparatus will be kept the same throughout and we will do three tries at each temperature to obtain a more accurate average. We will put the liver in the water bath/ice for a minimum of 20 minutes to try and make the temperature accurate.
We decided on 2g of liver during our preliminary experiments.
They were carried out in the same way this experiment will be, but instead of altering the temperature, we changed the mass of liver used to find the best amount to use in these experiments.
Preliminary experiment:
Set up as follows:
Method in short: With the apparatus set up as shown above, we weighed 3 amounts of liver 3 times, so we had 3 bits of 1g liver, 3 of 2g, and 3 of 3g. In turn, we put a piece of liver into the boiling tube, and added the 10cm3 of hydrogen peroxide, immediately putting the bung in the tube. The test was carried out at room temperature. We left the bung in for two minutes, and then recorded the amount of water displaced in the measuring cylinder, the results were as follows:
Amount of Oxygen Produced
We also decided, along with having 2g of liver, that we should have a stronger molar solution of hydrogen as the amount of oxygen wasn’t very much, and we would need a bigger range of results to see a pattern. We were using a diluted solution, so for the main experiment we decided to use a solution of concentration 20% (instead of 10%).
In the main experiment, I expect that the reaction at 40oC will produce the largest amount of oxygen, and the one at 60oC will produce the least – the enzymes should have totally denatured at this temperature, and should not be working at all (there should be no oxygen produced). The temperature at room temperature should be second highest, and the ice should be third. I think this due to my scientific knowledge explained previously about the optimum temperature for enzymes, and denaturing. I think that at 0oC I should get around 50cm3, at room temperature (about 24oC, I should get double that - 100cm3, and around 130cm3 at 40oC. If you imagine a graph of the oxygen produced at these temperatures, the curve should look something like an upside-down ‘U’ shape, though with flatter sides. It should climb steadily to 40oC, then drop off steeply as the enzymes denature.
Method:
Diagram: Set up as in preliminary experiment.
Other equipment also used - electronic scales, scalpel, cutting tile, water bath, measuring cylinder, stopwatch.
Using the scalpel, cutting tile and electronic scales, we cut and weighed 12 pieces of liver, all 2g each. We tried to make them roughly the same shape, so they had the same surface area and would react evenly.
We then put them all in boiling tubes, and put them into groups of three, each group would be heated or cooled to a different temperature - 2oC (in ice), 22oC (room temperature), 40oC (in water bath), 60oC (in water bath). For the ice and water bath, they will be stood in a test tube rack, with the liver submerged in the water in each case. They should be left in there for a minimum of 20 minutes, so the liver pieces are at the right temperature.
One at a time, we put a boiling tube into the set up above, and measured 10cm3 of hydrogen peroxide in a measuring cylinder. We then poured the hydrogen peroxide into the tube, and immediately put the bung in the top and started the stopwatch. We would time it for one minute.
Once the minute was up, we would record the level, then disconnect the equipment.
We repeated this for each piece of liver cut, at each temperature, refilling the measuring cylinder each time so the water level was full.
Results:
Amount of Oxygen Produced
Graph:
Analysis:
The results that I obtained were more or less as I predicted – the temperature the enzymes worked best at was 40oC, producing an average of 121cm3 of oxygen, and the enzymes worked least effectively at 60oC, with an average of 44cm3.
The enzymes worked best at 40oC because this was the optimum temperature for enzymes before they start to denature. Enzymes correspond to one particular substrate – in this case hydrogen peroxide (the enzyme was catalase). They do this by having a shape that carries an inverse version of the substrate, so it fits into the enzyme, which will break it down (in this case, into water and oxygen). The enzymes work best at an optimum temperature, after which it will start to denature, and lose its shape, which means that it will not work as effectively because its shape won’t correspond to the substrate properly anymore. If the shape is altered it may not fit the substrate, and not be able to break it down anymore:
The rate of reaction increases with temperature up until the optimum temperature of 40oC. This is because of the particle collision theory that states that the faster particles move, the more chance of a collision (think of cars on a road). The faster the particles are going, the more likely the collision with an enzyme, and because of this, the reaction will be faster overall.
The results go accordingly to the optimum conditions for the enzyme, proving my prediction.
However, there was one big anomalous result – the one at 60oC - 44cm3. As stated earlier – after 40oC, the enzymes should start to denature, and at 60oC, they should all have denatured, and not do their job at all, producing no oxygen at all. In this case, some oxygen was produced. This was probably because the liver wasn’t left in the water bath long enough – 20 minutes must not be enough.
There was one other slightly anomalous result at 22oC on the third try – I think it was a bit too high. I didn’t think this fitted in with the general trend, and was too high, bringing the average up with it. This could have been because of a few things – the test tube was heated by accident, and was used without realising, it could have been left in there for more than a minute, the measuring cylinder could have been less than full, or more hydrogen peroxide could have been added than necessary. Inaccurate measuring will probably account for the differences in readings that we obtained.
Evaluation:
In conclusion, our results were fairly far spread – there was a big range between results at certain temperatures. This was probably due to reasons already stated – most likely inaccurate measurement. For example - some liver pieces could have been bigger than others, or have a larger surface area, increasing the chance of collision, according to the collision theory, which states that the more points of contact for particles, the more likely a collision. The amount of hydrogen peroxide could be inaccurate – if there was more than needed, then there would be more substrate for the enzymes to work on, meaning more oxygen produced, or less if the volume was lower. This was due to the equipment more than anything – the equipment wasn’t accurate enough. It’s impossible to get exactly the same amounts using a measuring cylinder, and reading by eye, or cutting liver with a scalpel and weighing to 2 decimal points on a machine. This means that the measurements would not be exactly the same, but roughly accurate – enough to get the general trend of results. This was surprising since we’d had to change liver half way through the experiment. This was unfortunate, and meant that at room temperature and 40oC the liver was from the same animal, but at 2oC and 60oC, it wasn’t. This accounts for slight changes to the curve, but overall it wasn’t too bad – it showed the basics – the thing it lacked was like the rest of the experiment – accuracy.
To improve this experiment, more accurate equipment is required, and the liver must be kept the same (we had to change it because the original had gone off – so this means the experiment will have to be done in a few days). Further investigations could be into different temperatures, or trying to alter a different factor, like surface area. Will cutting the liver up into smaller pieces make it react better? – according to the particle collision theory it should, but does it in practice?
Another experiment could be trying to find the relation between the amount of liver, and the oxygen given off, so you could predict what results you would get with different amounts of liver and different temperatures – will it work in a ratio? You could also try the same experiment with potato or celery, which produces catalase, and may be easier to shape and control surface area. I could also do additional experiments with different enzymes, instead of amylase, for example, protease or maltase. I could then plot the results of those experiments with those of amylase to see if all enzymes work at the same optimum conditions.
