Results: 3ml of O2 in 60 secs
=0.03ml/s
Method 2:
Used: 5 ml yeast
1ml Hydrogen Peroxide
Results: 5 ml O2 in 60 secs
= 0.08 ml/s
Conclusions
From these results you can see that method 2 collected 2ml more gas in 60 seconds than method 1 therefore I conclude that using a gas syringe to collect the oxygen from the reaction is more effective than method 1, using an up turned measuring cylinder. The reasons I think for the gas syringe being more effective than the measuring cylinder are as follows:
- Oxygen dissolves in water therefore using a water bath and bubbling the oxygen through the water in the measuring cylinder means that some gas must be lost due to it dissolving. When using a gas syringe no water is involved except that which is contained in the yeast mixture there is therefore not as high a risk of dissolving oxygen.
- When you start the reaction off using the measuring cylinder, you have to insert the hydrogen peroxide prior to putting the bung in the test tube, gas is therefore lost between the time when the hydrogen peroxide is put into the test tube and when the bung is placed in the top. When using a gas syringe a hypodermic needle pushed through the rubber bung and a syringe containing the hydrogen peroxide is used, so no gas can escape as the bung is already in the test tube.
- The measuring cylinder method requires that there be a long delivery tube making the risk of kinks in the tube greater and may limit the amount of the oxygen that gets to the measuring cylinder. From the diagram of method 2 you can see that there is no requirement for a long delivery tube.
From this preliminary work I decided that I would use method 2, the gas syringe to collect the oxygen produced from the reaction.
Now that I know how I am going to collect the gas I need to know what substance containing catalase I am going to use I have three choices potato, liver or yeast.
With each of the forms of catalase we performed three trials, in each we used 1 ml of hydrogen peroxide. The yeast was measured using a 1 ml syringe and the liver and potato were weighed using a balance measured to 2 dp. We did the trials using the gas syringe method and we collected gas for 60 secs.
Results: Oxygen produced by hydrogen peroxide when using three different sources of catalase enzyme
All three substances containing catalase gave the same rate of reaction; I, therefore, based my choice of which substance to use on accuracy of measuring out each substance. I found that it was very hard and also time consuming to measure out both the liver and potato, as each time I measured it out I had to use a balance. Measuring the yeast in a syringe was much easier and took less time and also more accurate.
Another reason why yeast is a better source of catalase is due to it being a liquid therefore each volume I measure out will have equal surface area unlike potato or liver which would have very uneven surface areas. In addition, due to yeast being made in the lab, there is a more even distribution of catalase throughout the samples than there would be found naturally in both potato and liver samples. I have therefore decided to use yeast as my source of catalase enzyme.
From the preliminary work I decided:
- The gas syringe method will be used to measure the amount of oxygen produced.
- Yeast will be used as a source of catalase
- I will collect gas for 30 seconds instead of 60 as we found from our preliminary experiments that after this space of time the gas collected was a negligible amount.
- I will run the experiment three times for each concentration of hydrogen peroxide to increase accuracy and to reduce the chance of error.
- I will use 1ml of hydrogen peroxide because if it is any more than that then More than one syringe will have to be used to insert it into the test tube.
I have decided to use six different concentrations of hydrogen peroxide; They will be made from a stock solution of 100% hydrogen peroxide(20vol) and mixed with water to provide different levels of concentration. I am going to measure them in percentages :
Quantities of hydrogen peroxide and water used to make up the different concentrations of hydrogen peroxide
I decided that I would measure out 10 ml of each concentration to minimise error. Error will be reduced due to the whole quantity for each concentration being measured out in one go. The amount of error will be less significant because as a percentage of the quantity measured the possible ± 0.5 ml error is small. I will use a syringe to measure out all of the hydrogen peroxide which has 1 ml graduations it is therefore accurate to 1 dp.
Control of variables
To ensure that there are no extraneous variables affecting the results it must be insured that as many that would influence the results are kept under control we will therefore control as best we can with equipment available:
Temperature: changes in temperature can affect the rate of reaction of the breakdown of hydrogen peroxide; if the molecules get heated up then they will possess more kinetic energy, meaning an increase in collisions per second resulting in an increase in rate of reaction, we will therefore attempt to do the whole experiment on the same day in the space of 1 hour in which there will only be minor changes in temperature. Another reason for temperature to be kept constant is the fact that if the temperature got too high usually around 37°C the enzyme would be denatured, that is, the hydrogen bonds holding the tertiary structure together would be broken and the shape of the active sites would change meaning that it wouldn’t work correctly.
Volume of enzyme and substrate: An increase in either enzyme or substrate would lead to an increase in reaction rate we therefore need to make sure that we measure everything as accurately as possible.
Concentration of yeast: If the concentration of the yeast was increased then it would mean that there would be more catalase for the hydrogen peroxide to bind with as there would be an increase in the number of active sites available. We will therefore use yeast from the same sample and attempt to do the experiments all on the same day.
pH level: Enzymes are denatured at certain pH levels and work at different speeds depending on the level:
The effect of pH on rate
of reaction
We will have to use the same batches of yeast and of the different concentrations for each experiment.
Washing of equipment: When washing for example test tubes it is essential that they are dried thoroughly because any water left in them would dilute the solutions.
Syringes: we must make sure that when syringes are used, they are used for the same substance each time because if we mixed them up, it would contaminate either the yeast or the hydrogen peroxide.
Timing: we must make sure that we start the stop clock at the same point for each experiment. We will start the timer once the hydrogen peroxide starts being added to the yeast i.e. as soon as the syringe is starting to be pushed down.
Safety
There are various safety issues that need to be addressed before the experiment is carried out:
- safety goggles must be worn throughout the practical and whilst clearing up to prevent substances that may be harmful splashing into the eyes.
- Lab coats will be worn throughout the practical as hydrogen peroxide can damage skin and clothes if it is spilt, lab coats will help prevent and spillages reaching the skin.
- After the practical hands must be washed using anti-microbial soap as yeast will have been handled.
- All test tubes must be stored in test tube racks and the gas syringe must be held in a clamp stand to stop the glass items from rolling off the surfaces.
Final plan
- measure out the 6 different hydrogen peroxide concentrations putting them in 6 separate labelled beakers
- set up the equipment as shown in the diagram, The test tube is sitting in a test tube rack.
- add 5 ml of yeast to the test tube
- making sure all of the air is out of the gas syringe, put the bung securely in the test tube.
- measure out 1 ml of 0% hydrogen peroxide in a 1 ml syringe and attach it to the hypodermic needle.
- start the stop clock as soon as soon as the syringe is starting to be pushed down.
- wait until 30 seconds is gone and then take down the reading from the gas syringe of how much gas has been produced.
- Repeat the same steps again 3 times for each concentration to make sure that the results are reliable
When we collect the data we will put it in a table that looks like this:
The rate will then be worked out using this formula: gas collected = oxygen/ sec
time taken
This data will then be put onto a graph:
Method
The experiment was carried out exactly as planned, see ‘final plan’ for method on pg 9.
Results
Oxygen produced by different concentrations of hydrogen peroxide
Analysis
My graph showing the rate of reaction shows that as the concentration of the hydrogen peroxide increases the rate of reaction does also. At the beginning of the graph from 0-80%concentration, the rate of reaction increases uniformly this is shown by a straight line from 0.1ml/s through to 0.68 ml/s but you can see from 80% to 100% concentration the rate of reaction starts to plateau.
The reason why the rate of reaction increases as the concentration is increased is due to there being more substrate, which for this experiment is hydrogen peroxide, to join with the enzyme. The enzyme joins with the substrate to form an enzyme substrate complex the enzyme then speeds up the break down of hydrogen peroxide and the two products are released:
2H2O2 →2H2O + O2
At (a) on the graph there are more and more substrate particles in the mixture so there is an increase in number of collisions per second therefore more enzyme substrate complexes will be formed.
At (b) on the graph a plateau is beginning to form due to there not being enough catalase enzymes to provide active sites for all the extra substrate particles. Therefore adding more substrate after the plateau effect would be pointless because it would make no difference unless more catalase enzyme was added to provide more active sites for the substrate particles
For 0% hydrogen peroxide, a reading of 3 ml was recorded. It seems that there is an error here because if there is no H2O2 then there shouldn’t be any products. The explanation is that when the hydrogen peroxide is inserted into the test tube, air from inside the test tube must be displaced, air displacement would also have occurred when the rubber bung was put into the test tube, the air displaced would have travelled into the gas syringe. It was an equal amount each time meaning that 3ml of air must be displaced for every experiment.
Conclusion: The results from my experiment support my prediction there was an increase in rate of reaction due to an increase of concentration of substrate and there was also a plateau effect due to the active sites of the catalase being occupied.
Evaluation
To try to minimise the chance of inaccuracy and unreliability, I repeated the experiment for each concentration three times and then worked out an average of the three experiments, the graph was then plotted of these averages.
The first source of error I encountered was the fact that some of the equipment was not easy to get accurate volumes measured. An example of this is the syringe used for measuring out the yeast. The syringe measured to the nearest ml, therefore there was room for error as each measurement could have been 0.5 ml either larger or smaller than that amount. Another problem with the syringes was that when you ejected the yeast from it, some was still left I the end. There fact that some yeast stayed in the end of the syringe would not significantly affect the results due to approximately the same amount staying in the end each time. However, the syringes not measuring correctly could affect the rate of reaction, if there was more than 5mls it would increase the rate of reaction due to an increase of active sites and if there was less than 5mls it would decrease the rate of reaction due to less active sites.
Another aspect of the experiment which may have produced inaccurate results is the fact that yeast is a living organism. The fact that it is living means that throughout the course of the experiments which took 1 hour, the yeast cells could have reproduced, this would mean that the rate of reaction of the experiments done at the end would have an increased rate of reaction compared to those at the beginning this would be due to the fact that more yeast cells would have been produced meaning that more catalase enzyme would be contained in the mixture at the end of the experiment than at the beginning, an increase in number of enzymes would mean an increase in number of active sites available meaning that more substrate molecules could form enzyme substrate complexes hence increasing the amount of oxygen produced in 30 seconds. This would affect the results because it would mean that not all of the experiments would have been done in the same conditions. We did our experiments as fast as possible which reduced the possible affect this may have had.
The yeast used in this experiment contains water; oxygen can dissolve in water. This means that not all of the oxygen produced by the reaction will have made it to the gas syringe. This would mean that the results are lower than they should but would not affect the shape of the graph due to all the experiments being affected equally.
Another way in which oxygen dissolving in water could have affected the results is the fact that different amounts of water were used to dilute the hydrogen peroxide to make the different concentrations. Highest volumes of water were used for lowest concentrations this would mean that it appears that they have a lower rate of reaction than they should compared with the higher concentrations when less water was in the solution meaning less gas was being dissolved.
When the bung is put into the test tube when setting up the experiment and when the hydrogen peroxide is inserted into the test tube via a hypodermic needle, air will have been displaced this would make the readings higher than they should be, but because the same amount of air was displaced for each experiment it would not affect the shape of the graph. This is demonstrated by the fact that the experiment for 0% H2O2 produced 3ml of gas when if there is no substrate for the enzyme to act on, no products can be formed. It can therefore be said that the 3ml of gas in the gas syringe was due to air displacement. In order to rectify this 3ml could be subtracted from the results of each experiment but as it makes no difference to the shape of the graph and therefore the conclusion there is no point.
One last confounding variable that was beyond my control was the temperature of the room. If the room heated up at any time in the experiment it would have speeded up the rate of reaction due to the molecules possessing more energy, this would result in more movement meaning more collisions per second resulting in successful reactions. If there were a decrease in temperature, the opposite would happen. If there were any temperature changes throughout the experiment it would make the results unreliable, as the solutions would have had different levels of energy. E.g. If there was a temperature increase at the beginning (a) and then a decrease back to normal in the middle(b) of the experiment, then a further decrease in temperature at the end(c), the graph that would be produced is shown alongside what it would be like it temperature remained constant