Prediction
The reaction for the decomposition of hydrogen peroxide is:
2H2O2(aq) + catalase(aq) → 2H2O(l) + O2(g)
I predict that the higher the concentration and the temperature, the faster the reaction will be because of the points that I have mentioned above. I also predict that as I increase the temperature and concentration the initial rate of the reaction will increase. This is because if the concentration is higher there are more active sites available for the hydrogen peroxide to bind with at one time so more products will be formed more quickly. More products will also be formed, more quickly, when we increase the temperature because the molecules have more kinetic energy and more collisions between molecules occur and more of these collisions result in a reaction because more of the molecules have the activation energy.
Towards the end of the reaction less product will be formed because the catalase enzymes will have broken down most of the hydrogen peroxide and therefore the rate of reaction will be lower, and less oxygen gas will be produced per second.
Plan:
From the equation we know that the reaction forms oxygen gas as one of the products. This will be the substance that we will try to measure to give us our results; hopefully we will also be able to determine the initial rates of the different reactions, and the orders of reaction for this reaction
During my experiment I am going to alter the temperature of my reactants and the concentration of the hydrogen peroxide to see how varying each affects the rates of the reactions. Before carrying out my final experiment and the method and what volumes and concentrations of hydrogen peroxide and yeast solution I am going to use I am going to do some preliminary work to see which work best and also what is the easiest and most accurate method for me to collect my oxygen gas.
Preliminary Work
The first problem to overcome is how to mix both of the solutions in a suitable vessel, without letting any gas escape. This rules out the possibility of pouring one solution into a test tube containing the other substance and then putting the seal on top because some gas would be lost in this way. After my preliminary work I have decided to use a resealable bung for my seal and then push a syringe with a hypodermic needle through this. This method ensures that no gas is lost during mixing. I am also going to use a sidearm conical flask as my vessel as the sidearm provides me with an outlet to join my delivery tube onto and transport my oxygen gas through.
There are many different methods that can be used to collect my gas, I could use a measuring cylinder or burette to collect my gas (see fig 3 below), however this isn’t very accurate and gas could be lost when I turn the cylinder upside down in the water.
From my preliminary work I have decided that the best method for collecting my oxygen gas would be to use a gas syringe, these are accurate to 0.1cm3 and no gas would be lost during any stage. These gas syringes are very low friction as they are made from smooth glass; this will be joined to the conical flask via a rubbery delivery tube (see fig 4 below):
When we were carrying out our preliminary study we were given a 5% stock yeast solution and 20-vol. hydrogen peroxide. When altering concentration in my final experiments I will alter the concentration of the hydrogen peroxide with distilled water until I have the concentration that I want. The hydrogen peroxide will also be the solution I am adding through the syringe, as it is thinner than the yeast solution. When altering the concentration of hydrogen peroxide, I will use five different concentrations made up by diluting the 20-vol stock solution. This number of concentrations will enable me to draw a reliable graph to compare how a change in concentration affects the rate of reaction.
Also when altering my temperature I will also use at least five different temperatures for the same reason. When I carry out this specific investigation I will heat up my reactants in a water bath and also carry them out in the water bath so that the temperature stays constant. I found that it took around two minutes to warm up my reactants to warm my reactants to the right temperature, so I will leave them in the water bath for at least this amount of time before I carry out the experiment.
The final volumes of reactants I have decided to use are 2 ml of yeast solution and 4 ml of hydrogen peroxide. The volumes produce enough oxygen to read well but they do not do this so quickly that I cannot read the values accurately or that they react so much that froth rises up the conical flask and through the delivery tube. Another reason for not using the measuring cylinder to collect my gas is that water is required and oxygen dissolves in water so this could affect my results.
The time gap that I am going to take between measurements on the gas syringe will be 10 seconds and I will measure for a total of 90 seconds. This time scale gives me enough time between measurements to write down the reading and be ready for the next. Also once 90 seconds have elapsed I found that most of the reactions had finished and that all of the hydrogen peroxide had been broken down.
I am using yeast as my source of catalase because it provides me with a constant surface area for the substrate to be exposed to. With potato or yeast I would have to use small chunks of either, this would give me unequal amounts of surface area for each experiment I carried out, this would affect my results and make them less reliable. Yeast is also easier to measure out the yeast solution, I just have to use a graduated pipette and take the volume I want out of the stock solution. There is also a more even distribution of the enzyme in the solution, than in the natural liver or potato samples, because it is constantly having air blown through it to keep it well mixed and to ensure that the enzyme doesn’t sink to the bottom of the solution.
Standardisation and control of variables
To ensure that there are no extraneous variables affecting the results we have to ensure that as many variables that can influence the results are kept under control. We will control them as best as possible with the equipment that is available. These variables include temperature, concentration of reactants, pH levels, and surface area, I more detailed explanation of how these affect the rate of reaction is shown on page 3. The volume of the reactants can also affect the rate of reaction, if the volume of either the yeast or hydrogen peroxide gets increased in one of the experiments, the amount of oxygen produced will increase and this will affect my results.
I am going to make sure that I wash all equipment between experiments where I change the variables, such as the syringe, conical flask, graduated pipette etc. I will also make sure that they are dried thoroughly as any excess water could increase the dilution of my reactants and affect my results.
I must ensure that I used the syringes for the right substances; if they get mixed up they could contaminate the reactants and affect my results. I will also start the stop clock at the same time for each experiment, I will start it as soon as I start to press down the syringe containing the hydrogen peroxide.
Safety
There are various safety issues that I had to take into account before carrying out my preliminary work. Hydrogen peroxide can be corrosive, and can act as an irritant to the skin, eyes and clothing if spilled and not washed off thoroughly.
All enzymes are potential allergies and can irritate membranes found in the eyes or nose. They can also cause asthmatic attacks. Spills of either substance should be washed immediately.
Following is a risk assessment for both chemicals:
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Skin exposure: Rinse affected skin with plenty of water and remove
contaminated clothing, rinse for at least 15 minutes and seek medical attention if problems occur.
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Eye exposure: Vapours are corrosive and can be damaging to eyes.
Symptoms include pain, redness and blurred vision. Splashes may cause tissue destruction. Wash eyes for 15 minutes, lifting upper and lower eyelids. Seek medical attention immediately. Hydrogen peroxide may release oxygen and accelerate combustion; it may react with organic materials and cause fire.
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Small spills: Flood area with excess water to dilute solution. Use a
broad spectrum absorbent (cat litter) to clean up the spill. Use a large amount of water to wash down spills and reduce flammable vapours.
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Large spills: Notify others in area of spill. Turn off ignition sources in
area. Evacuate area and post doors to spill area. Restrict people from area of spill until cleanup is complete.
For safety reasons when carrying out my investigation I will wear a lab coat and safety goggles at all times. The goggles will prevent harmful substances from splashing into my eyes and the lab coat will prevent spillages from getting onto my clothing and soaking through to my skin. After the practical I will wash my hand with antibacterial soap, as yeast will have been handled.
Final method
- Set up apparatus and water bath.
- Measures out 2ml yeast with graduated pipette and put into conical flask, place resealable bung on top of flask and join up delivery tube to sidearm.
- Measure out 4ml hydrogen peroxide into syringe and push hypodermic needle through resealable bung.
- Inject in hydrogen peroxide and start stop clock.
- Time for 90 seconds, measuring every 10 seconds recording results in a table.
- Repeat 3 times for each variation of temperature and concentration.
I will also carry out a titration of hydrogen peroxide; this will enable me to find out the concentration of the stock hydrogen peroxide solution (20-vol) that I am using I will be carrying out five titrations to enable me to get a reliable average for my calculations. The method for this is shown below:
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Take 25ml of 20-vol H2O2 into a graduated flask and top up to 500 ml with distilled water.
- Remove 25 ml of this new solution and add 200 ml of distilled water and 25 ml of sulphuric acid (1 molar).
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Then take 25 ml of this solution into a beaker and titrate with 0.005 mol dm-3 potassium manganate from a burette, making a note of the value that the manganate solution goes up to.
- When the solution retains its pinkish colour the titration is complete. Now take the new reading from the burette and subtract the starting volume from the end and I now have my titre.
Results
Below is a list of tables and graphs for each varied temperature, during these concentrations the hydrogen peroxide used always came from the stock solution (20-vol), it was not diluted:
Below is a list of tables and graphs for each varying concentration of hydrogen peroxide; all of the experiments were carried out at room temperature that was measured to be 17oC, the concentration is shown as a ratio with the first number being the amount of hydrogen peroxide in ml and the second being the amount of distilled water in ml to make up the diluted solution:
Below is a table of results for my titration:
Analysis
As we can see from both sets of results and graphs the experiments went as expected, when we increased either temperature or concentration the amount of oxygen gas produced increased. The rate of reaction also increased. From the graph we can work out the initial rate of reaction by taking a tangent and then use the formula: rate = rise / tread. All of the initial rates of reaction are marked on each of the graphs. Below are two graphs comparing the rates:
Because the second of these graphs has a straight line I can say that the reaction is first order with respect to hydrogen peroxide.
The reason why the rate increases as the concentration increases is because more oxygen gas is being produced per second (this can be seen on the graph, when the concentration is 2-3 the rate is lower than when the concentration of hydrogen peroxide is 4-1. This is due to there being more hydrogen peroxide molecules to bind with the enzyme. The enzyme joins with the substrate to form an enzyme substrate complex and then breaks down the hydrogen peroxide to form two products:
2H2O2(aq) → 2H2O(l) + O2(g)
The rate of reaction increases as temperature increases because the enzyme and substrate molecules have more kinetic more energy. This means that the molecules collide more often and more of these collisions result in a reaction, producing more oxygen gas. At 28oC, graph 4, the rate of reaction was 2.00, however at 51oC, graph 6, the rate of reaction was 5.56.
From my results of the titration I am also able to work out the concentration of the 20-vol hydrogen peroxide solution, see below:
We know that it takes two moles of MnO4- to react with five moles of H2o2
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Moles of MnO4- (average titre) = cv = 0.005 x (20.6/1000) = 1.0x10-4
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Moles of H2o2= 1.0x10-4 x (5/2) = 2.5x10-4
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In 1 dm3 = 2.5x10-4 x (1000/25) = 1x10-2 moles
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Dilution = 500/25 = 20 x 2x10-1 = 2x10-1
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250/25 = 10 x 2x10-1Concentration = 2 mol dm-3
The actual concentration of hydrogen 20-vol hydrogen peroxide is 1.6 mol dm-3
so the value that I obtained was very close to the actual value.
If we look at graph 7 we can see that the reaction only starts after 60 seconds, this can’t be due to the enzyme breaking down the hydrogen peroxide because the catalase enzymes will have been denatured by the high temperatures and will no longer be able to break down the hydrogen peroxide molecules. The reaction must begin because of the hydrogen peroxide decomposing on its own. The temperature of the substrate is high enough to allow this to happen, the temperature will be high enough to break the bonds in the molecule quickly and break the substrate down to oxygen gas and water.
Also from the tables of results we can see that the temperature of the reactants increases by the end of the reaction, and happens most at lower temperatures, if we look at table 1 there is a change of 12.3oC but looking at table 6 there is no temperature change. The change happens because the reaction is exothermic and energy is being given to the surroundings as the experiment advances, and the products end up with less energy than the reactants. To break bonds energy is required, this is endothermic, however the energy given out by the reaction must be higher than the energy needed to break the bonds present in the hydrogen peroxide, otherwise the temperature would go down.
Conclusion
My results agree with my prediction that the higher the concentration of hydrogen peroxide and the higher the temperature, the faster the rate of reaction because more oxygen gas was produced in less time.
If concentration increases the rate increases because there is more substrate molecules, therefore there is a higher possibility that a substrate molecule will bind with a catalase molecule, fit its active site and a reaction will occur, producing more product, and essentially for this experiment more oxygen gas. This increased oxygen production means that the gas syringe moves out more quickly so my measurements increase as we increase concentration.
If temperature increases the rate increases because the molecules have more kinetic energy. This means that they move around faster and collide more often. Because the kinetic energy is increased, more of the molecules have the activation energy, this means that there is a higher chance of reaction when they collide, producing more oxygen gas per second and increasing the rate.
Evaluation
As I said earlier to try and minimize the chance of inaccuracy and unreliability, I repeated the experiment for each temperature and concentration change three times and then worked out an average of all three. I then used this average to plot my graphs. However I did notice an anomaly on graph 13 the reading taken after 10 seconds seems to be lower than the rest. We can also see anomalies on table 4, experiment 1 after 50 seconds, these results are about 10 cm3 lower than the other two experiments for that temperature.
These anomalous results could be due to the glass syringe sticking, not moving as freely as it should and projecting a value lower than it should. Even though I tried to make my experiment as accurate as possible there were still some areas that I couldn’t control fully. One being the timing, I only used a stop clock and the timing relied upon my reactions, if I sometimes measured at the wrong time then the reading would be wrong. Also I had to decide when to start timing, this could provide another slight inaccuracy.
I don’t think that the measuring equipment was inaccurate, all of the equipment that I used to measure volumes had an accuracy of at least 0.1 ml so the error for measurement could have only been 0.05 ml either side of that, which isn’t really a very large or significant amount to have made much, if any difference on my results. However even though the syringe measured accurately, there may have been times when I didn’t press down fully and expel all of the hydrogen peroxide inside it, this would mean that less oxygen would be produced and could account for some of my measurements being lower than expected.
Another aspect of the experiment, which may have produced inaccuracies, is the fact that yeast is a living organism. This means that as the day went on the yeast cells could have reproduced, this would mean that the experiments carried out at the beginning would have a lower rate than those carried out at the end, because there would be less active sites available to break down the substrate molecules at one time. This would affect the results and would mean that not all of the experiments would have been carried out under the same conditions. I carried out the experiment as quickly as possible and looking at my graphs I don’t think that it had an effect on my results.
The yeast used in this experiment contains water; oxygen can dissolve in water and this means that not all of the oxygen produced by the reaction will end up in the gas syringe. This would mean that the results would be lower than expected but would not affect the shape of the graph as all of the experiments would be affected equally.
Another way in which oxygen dissolving in water could affect my results would be in varying the concentrations of hydrogen peroxide, distilled water was used to dilute the hydrogen peroxide. Therefore the concentrations that were diluted the most would be most affected because they contain more water. This would mean that it appears that they have a lower rate of reaction than they should compared with the higher concentrations where less water is present meaning less gas gets dissolved in the water.
When the concentration of hydrogen peroxide is zero, we can see from graph 14 that 4 ml of oxygen was produced, however this value doesn’t increase as the time goes on. This release of gas can’t because of the breakdown of the hydrogen peroxide because there is no enzyme present and the temperature of the hydrogen peroxide isn’t high enough for it to breakdown on its own. The gas is due to air displacement when we inject the hydrogen peroxide into the conical flask, we inject 4ml of hydrogen peroxide so an equal volume of air is displaced into the gas syringe. This happens for every experiment and the volume displaced is the same each time because we always add the same amount of hydrogen peroxide, therefore it doesn’t alter my results or affect my graphs.
One last variable that I was unable to control was the temperature of the room. As the experiments where I varied the temperature were carried out in a water bath fluctuations in room temperature wouldn’t really affect them. However, the experiments where I varied concentration were carried out on the desk so these may get affected. If the room had heated up during the experiment it would have speeded up the rate of reaction due to the molecules possessing more kinetic energy, colliding more often and more of these collisions resulting in a reaction. If the temperature had of decreased then the opposite would have happened, the molecules would have less kinetic energy, less of them would possess the activation energy, so they would collide less often and less of the collisions would result in a reaction. If there were any temperature changes during my results it would make them unreliable, as the solutions would have different levels of energy. For example if there was a temperature increase at the beginning of the experiments, as I carried out the higher concentrations first the rate of these experiments would be increased. Then if the temperature went to normal the middle set of experiments would have the expected rate of reaction, and if it decreased further the last experiments I carried out would have a lower rate of reaction than expected. However I don’t think that this happened because my tables of results and graphs seem to be accurate, and follow my prediction.