Pilot Experiment: Before writing the complete method, I did a pilot experiment to discover what potential problems I may come across. These problems included lack of suitable equipment, the problem of how often to take recordings, timing, accuracy and reliability and the technicalities of the method. I shall write about how the outcomes of the pilot experiment affected my choices. I was interested in how much gas was produced over 5 minutes when the Celery Extract concentration was at its highest, so I recorded this value. I found that in one minute no more than 10 cm3 of oxygen was produced and once 5 minutes had passed, the 50 cm3 burette was close to empty. This shows that 5 minutes is the maximum time one could take recordings of the volume of oxygen produced.
Basic Safety measures include no eating, or drinking, and long hair should be tied back. Solutions should be labelled so that people understand the risk involved when dealing with certain solutions. Chemical spillages can be wiped with a damp cloth and dried with a paper towel. Safety Goggles should be worn to prevent eye-contact with the solutions and apparatus. Any accident concerning the eye should be replied with flooding the eye with water and immediately reported to the supervisor. In this experiment, safety goggles must be worn and possibly lab coats/gloves (depends on availability) to protect your clothes and skin. From the pilot experiment I learnt that Hydrogen peroxide can burn and make your skin go white, so due care and attention must be given when handling the beaker containing this particular solution. Enzymes, such as Catalase in the celery extract, are irritants so one must wash their skin if skin contact is made.
Equipment
80 cm3 Hydrogen Peroxide. I calculated the volumes of reactants I would need, by working out how many tests I would be carrying out from the pilot experiment. The volume of Hydrogen Peroxide will never be changed as we are only testing one variable: how the concentration of Catalase affects the rate of reaction with Hydrogen Peroxide. As explained in the Variables section, I intend to use larger volumes as this will decrease the percentage error in measurement. The concentration of the hydrogen peroxide we are being given is 20 vol., so the concentration will remain the same.
50 cm3 100% Celery extract, undiluted and liquidised. The celery tissue has to be liquidised to ensure that the surface area of the celery tissue does not affect the rate of enzyme reaction, and so that the amount of celery can be easily measured.
50 cm3 Distilled Water. The water used to dilute the celery extract must be distilled so that there are no impurities in the water. The water used to dilute the celery extract in all the boiling tubes must be the same throughout as any contaminants will disrupt the reaction between the catalase in the celery extract and the hydrogen peroxide solution.
Distilled water is an appropriate solvent to use; it is used as a transport medium in the blood (83% water), the lymphatic system, the excretory and digestive system of animals, and more importantly in the vascular tissues of plants (xylem). It has this solvent property due to its unequal charge distribution between the Oxygen and 2 Hydrogen atoms (dipolar molecule). The use of water as a solvent in the body, amongst a plethora of other things, animals and in plants shows that water is unlikely to damage the catalase molecules. Water is also highly un-reactive - it will not act as an oxidising agent (it won’t bond with or take any electrons from the catalase molecule). Also, concern for the effect of water on cells’ membranes (osmosis, turgidity and flaccidity) should be wavered; the membranes have already been broken by the liquidising process.
From the pilot experiment I realised that I needed a 1 dm3 plastic beaker for a water bath, as what we had was too small, and a tap water supply to fill it. I found that the water bath is needed so that the direction of the gas can be seen and the position of the burette changed in order to collect the oxygen.
16 Boiling Tubes are needed to do all the experiments and the repeat experiments, so that an average amount of gas can be obtained.
A Thermometer is needed to make sure that the temperature of the reactants is the same, throughout the experiment, before they are mixed to bring about a reaction.
Again from the pilot experiment, I learnt that a 50 cm3 Burette, instead of an up-turned boiling tube, is needed to collect the gas because the amount of gas collected can be recorded easily/accurately and it has a larger capacity to collect more gas. Accurate observation (to within a millimetre) is imperative when filling up the burette to the marking points and when taking a measurement, measurements must be read from the deepest point of the meniscus at the top of the water in the burette.
Digital Stop Watch accurate to within a second is required in order to time the duration of the tests and to indicate when a reading of how much gas collected needs to be observed. To make a reading to within a millisecond or ten milliseconds would be hard to do with just my observation, so to be accurate to within a second is all we can possibly do. However, it can be done with specialist equipment.
A Connecting/Delivery Tube, with rubber adaptor for boiling tubes, prevents gas escaping whilst it flows down the tube to the water bath, where it is collected by the burette. Their integrity needs to be checked before hand by letting water flow through it to see if there are any leaks.
3 syringes with a 5 cm3 capacity, for each experiment, are vital so that the volumes of distilled water, hydrogen peroxide and celery extract can be accurately measured to within a millimetre. Clean and dry syringes must be used for each experiment as solutions from the past experiments will still be inside, which would contaminate the new solutions being drawn up. Also, each syringe should be used for one solution as using the same syringe for two solutions would lead to contamination, making the resulting solution inaccurate. To avoid this simple mistake the syringes must be labelled to. It is important for no bubbles to be taken up when filling the syringe and that no bubbles are created when squeezing out the solution into the boiling tubes. This can be helped by applying pressure to the syringe valve slowly and with a consistent speed.
A Retort Stand and Clamp helps to hold the burette perpendicular to the work bench, so that readings can be accurately read.
The pilot experiment showed that a small amount of Vaseline, or any suitable waxy lubricant, is required. Gas can escape from joints and seals, by applying Vaseline the joint can still fit and gas is less likely to escape.
3 x 100 cm3 beakers to contain solutions of H2O2, distilled water and celery extract, for transfer to a syringe.
It was hard to organise the solutions made, so a Boiling Tube stand is an essential piece of equipment. This holds the boiling tubes steady during the experiment, so that (mental) concentration can be maintained when making timed observations.
A plastic Tray was needed during the pilot experiment to help organise one’s own experiment and to make sure that spillage doesn’t spread too far. Also, Sticky labels are needed to identify the composition of solutions in the boiling tubes/beakers, so that a mistake can not be made.
Procedure
Below is a step-by-step process to ascertain values for how the concentration of the enzyme catalase in celery tissue affects the rate of reaction with hydrogen peroxide. The table below shows the concentration proportions for all of the experiments. The diagram after the numbered list illustrates how the apparatus should be prepared.
- Wash and dry your hands. Put safety goggles on. If you a lab coat and/or gloves are available then wear them, as they
- Collect all equipment and lay out on table and tray, and use sticky labels to identify solutions of celery extract, hydrogen peroxide and distilled water.
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Fill the 1dm3 beaker with tap water up to near the edge, allowing for the fact that the burette and delivery tubing will be put in making the water level higher.
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Take a beaker and fill it with water. Then pour this water into the burette till it is full. It is easier to use a beaker to fill the burette, because the burette is obstructed by the sink when using the tap to fill it, as I learnt from the pilot experiment. Make sure that the burette is full to the 50cm3 mark, close to overfilling; otherwise air bubbles will change volume of water in the burette when it is inverted.
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With your thumb tightly closed over the aperture of the burette, turn it upside-down, and lower it into the 1dm3 beaker full of water. Whilst holding the burette upside down in the water, take your thumb off the aperture, and use the clamp to secure the burette to the Retort stand.
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Adjust the burette so it is perpendicular to the bench surface (see the diagram). Assure yourself that the burette is still in the 1 dm3 beaker, and water isn’t spilling over the lip of the beaker.
- Get a boiling tube rack to hold a set of 6 boiling tubes. Using the sticky labels, label the boiling tubes from 100, 80, 60, 40, 20 and 0, which corresponds to celery extract concentration (see table). Rearrange and replace the boiling tubes as the experiment continues, so that they are close to you to reduce the risk of solution being lost as drips when transferring it from one place to another (however unlikely).
- Record the temperature of the distilled water, making sure that the beaker is not touching the actual beaker, otherwise the temperature of the beaker will be measured rather than of the water.
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Fill the 5 cm3 syringe with 5 cm3 water from the 100 cm3 distilled water beaker, the sticky label should help you identify which beaker you need. Whenever you fill the syringe, make sure that the syringe is sufficiently immersed in the solution, to prevent bubbles being taken up in to the vessel. Empty and refill the syringe until the contained solution is clear of gas.
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Label the syringe you have just utilised ‘H2O’, referring to its use as the distilled water syringe, with a sticky label.
- Then to the boiling tube labelled 0, referring to celery extract concentration (see table above), add the distilled water from the syringe, slowly squeezing it out to prevent bubbles from being created. Gently shake the boiling tube to get rid of any bubbles.
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Sticky label a 5 cm3 syringe ‘H2O2’.
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Record the temperature of the H2O2 in the beaker.
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Immediately after, take the ‘H2O2’ syringe and use it to collect 5 cm3 of hydrogen peroxide using the same technique as outlined in step 7.
- Now test the integrity of the delivery tube by letting water flow through it, and check for water leaks. If there are any then replace it with a new one and check the new one too. Also check that the rubber seal can be securely fit into the boiling tube. Take the Vaseline and apply enough to create a thin layer around the inner lip of the tube. Make sure that the delivery tube is empty of water before continuing with the next step.
- Rest the delivery tube, the rubber seal end, on top of the 0 boiling tube and make sure that the other end is in the water and near the opening of the burette. This is in preparation for a quick transition from adding the hydrogen peroxide and sealing the apparatus.
- Record the temperature of the distilled water in the boiling tube.
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Take the digital timer, and as soon as you have slowly pushed out all of the hydrogen peroxide solution into the ‘0% catalase extract concentration’ boiling tube start the timer, seal the boiling tube with the rubber seal and make sure that the delivery tube in the water is directly under the burette. Make sure that the delivery tube to the burette is lined up as the first gas bubbles start to flow through it. If gas escapes being collected in the burette then start this experiment again.
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Exactly when thirty seconds has passed record how much gas has been collected. Record the volume of gas collected every thirty seconds for 2 minutes so that you have four results for “0 cm3 of Celery Extract/5 cm3 Water” in the table below. This is the ‘control’ for the experiment.
- Repeat this experiment 2 more times (steps 4 to 17), leaving out labelling and other things which do not need to be repeated. Sticky label the boiling tubes correctly.
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Label a third 5 cm3 syringe ‘CE’, representing ‘celery extract’.
- Calibrate (refill and reset) the apparatus, as stated in steps 4, 5 and 6.
- Record the temperature of the celery extract.
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To the boiling tube labelled 20, add the corresponding amount of celery extract using the syringe technique as outlined in step 8. The label ‘20’ corresponds to the left column of the table above, “20% celery extract/catalase concentration”. This row shows that this experiment requires 1 cm3 of celery extract, 4 cm3 of distilled water and the mandatory 5 cm3 of hydrogen peroxide.
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Repeat step 8 and 9, to obtain the new requirement of distilled water (4 cm3). Slowly release the distilled water into the boiling tube of 1 cm3 celery extract.
- If you find the temperature varies, then collect a new sample, but if it still varies, leave the solution in a cool or warm area accordingly. This could involve running water over the boiling tube or warming it for a very short time in front of a radiator. This must be done carefully to get the required temperature, because the sample must have an initial temperature/energy level, which is equal to the samples of the same composition.
- Repeat steps 13, 26 and 14, in that order.
- Repeat step 16.
- Repeat steps 18, 19, and 20, recording the results next to the row “20% celery extract/catalase concentration” and under the corresponding columns.
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The next solution to be prepared is “40% celery extract/catalase concentration”, which means that solutions of 2 cm3 celery extract, 3 cm3 distilled water and 5 cm3 of hydrogen peroxide are needed.
- Repeat steps 4, 5 and 6.
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Repeat steps 23, 24 and 26 with the aim to obtain 2 cm3 of celery extract, using the ‘40’ boiling tube.
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Repeat steps 25 (step 8 should be followed by 26) and 26 to obtain 3 cm3 of distilled water.
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Repeat step 13, 26 and 14 to achieve a 5 cm3 sample of hydrogen peroxide.
- Repeat steps 16, 18, 19 and 20, recording the results next to the “40% celery extract/catalase concentration” row and the correct columns.
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The next solution to be prepared is “60% celery extract/catalase concentration”, which means that solutions of 3 cm3 celery extract, 2 cm3 distilled water and 5 cm3 of hydrogen peroxide are needed.
- Repeat steps 4, 5 and 6.
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Repeat steps 23, 24 and 26 with the aim to obtain 3 cm3 of celery extract, using the ‘60’ boiling tube.
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Repeat steps 25 (step 8 should be followed by 26) and 26 to obtain 2 cm3 of distilled water.
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Repeat step 13, 26 and 14 to achieve a 5 cm3 sample of hydrogen peroxide.
- Repeat steps 16, 18, 19 and 20, recording the results next to the “60% celery extract/catalase concentration” row and the parallel columns.
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The next solution to be prepared is “80% celery extract/catalase concentration”, which means that solutions of 4 cm3 celery extract, 1 cm3 distilled water and 5 cm3 of hydrogen peroxide are needed.
- Repeat steps 4, 5 and 6.
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Repeat steps 23, 24 and 26 with the aim to obtain 4 cm3 of celery extract, using the ‘80’ boiling tube.
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Repeat steps 25 (step 8 should be followed by 26) and 26 to obtain 1 cm3 of distilled water.
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Repeat step 13, 26 and 14 to achieve a 5 cm3 sample of hydrogen peroxide.
- Repeat steps 16, 18, 19 and 20, recording the results next to the “80% celery extract/catalase concentration” row and the resulting columns.
- Repeat steps 4, 5 and 6.
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Repeat steps 23, 24 and 26 with the aim to obtain 5 cm3 of celery extract, using the ‘100’ boiling tube.
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Repeat step 13, 26 and 14 to achieve a 5 cm3 sample of hydrogen peroxide.
- Repeat steps 16, 18, 19 and 20, recording the results next to the “100% celery extract/catalase concentration” row and the resulting columns.
- Finish the experiment by tidying the apparatus away for cleaning/decontamination and removing any safety clothing.
There would be a results table to follow this procedure, but I have only displayed the results table for the communal, agreed method we used.
This diagram below shows the apparatus in its correct format.
N.B: The burette is perpendicular to the table.
Fig. 5.1
Implementation and Interpretation of the Results of the
Hydrogen Peroxide - Catalase reaction.
The results shown in the following pages are from an ‘Agreed Method’ experiment, where the method was the same for each person being supervised. For the vast majority, only one set of results were determined, so data for calculating individual averages of results are scarce. The graphs provide a Graphical analysis of the results.
Agreed Method Commentary
As an agreed method used by everyone, the class results can be used for statistical analysis. Some peoples’ results will have anomalies, for a number of reasons, and a graph of the class’ average rate of reaction would show that these results are too overbearing when calculating an average. By using certain statistical techniques, like standard deviation, we can deduce which results are inconsistent with the rest of the class and use the results that are accurate.
The main differences between my experiment and this ‘Agreed Experiment’ include the concentrations used, the times when results were noted down, the use of a holed rubber bung with delivery tube and how solutions were prepared. The concentrations used in the Agreed Experiment ranged from 0% to 100%, with a factor of 25%, whilst my experiment was to utilise concentrations with the factor of 20%.
Results table showing my individual results for the volume of oxygen gas (cm3) collected in the time indicated
None Achieved indicates that the experiment was not repeated in order to get another set of results to calculate averages of my data.
An example of the use of a Statistical Technique
Below I have utilised standard deviation to obtain a graph of the class results, minimising the anomalies, which would otherwise affect the shape of the line graph and have plagued mine.
Standard deviation is a measure of how widely values are dispersed from the average value (the mean).
Equation for Standard Deviation:
Already I have used Microsoft® Excel to create a spreadsheet of the data and a graph. Furthermore, the spreadsheet program has the capability to calculate the standard deviation of a set of data, so that one doesn’t have to go through the lengthy calculation process required for such a huge set of data.
After calculating the standard deviation and average of each column of data for each minute, and thus each concentration, I will use the data to evaluate whose data is best to use in a graph “The Average Rate of Reaction between Hydrogen Peroxide and Catalase for the class”. As my graph showed that there were some anomalous results, the use of the class results will help me to come to a logical conclusion, hopefully reflecting my results in some way.
This table is similar to the table shown two pages back; it includes everybody’s results, but no repeat results obtained by anyone have been replicated here. Nobody obtained a complete set of replicate results, so including them would change the value of some deviation/average values, disrupting any pattern I hope to observe. This table, and the next two, shows the standard deviation calculated for each column of results for the volume of oxygen collected (cm3) and also the average volume of oxygen collected. These figures have been rounded to within one decimal point, as that is the degree of accuracy to which the recordings of observations were made and the plots on a graph can be plotted by me.
Evaluation of the Standard Deviation-Average Data:
- Input the Average volume of oxygen gas produced of a column into a calculator.
- Add the standard deviation of that same column.
- Add 1.
- Look at the values in that column, if any exceed the value on your calculator then this person’s results are not to be used in the graph “The Average Rate of Reaction between Hydrogen Peroxide and Catalase for the class”. Record this person’s name, by, for example, underlining their name in the spreadsheet program.
- Clear the display on the calculator.
- Input the ‘Average volume of oxygen gas produced’ of a column into a calculator.
- Subtract the standard deviation of that same column.
- Subtract 1.
- Look at the values in that column, if any are less than the value on your calculator then this person’s results are not to be used in the graph “The Average Rate of Reaction between Hydrogen Peroxide and Catalase for the class”. Record this person’s name.
Consequently, the tables from the past few pages display the people who have had their results pulled out of the table; the ‘offending’ results and the person’s name have been struck through (e.g. crossed out) and are in italics. The data in italics are just part of the data which has been exiled, but wasn’t actually outside of the average+/-standard deviation calculation.
Example calculation
- Column: 100% Concentration, 1 minute.
- Value on Calculator: 13.3875
+/- 3.792953994
+ 1
= 18.18045399
- People eliminated at this stage:
‘Weller’, his value was 20.8 cm3 for this column and far exceeds 18.18045399, so he must be taken out.
The remaining data, the people who have not been crossed out, are to be used for a graph with a title close to “The Average Rate of Reaction between Hydrogen Peroxide and Catalase for the class”. If we were to do a graph with all of the results included, then we would find the persons’ results outlined above would change the shape of the graph too much, invalidating the usefulness of it. The discarded data was more widely dispersed from the average value compared to other results, so those values would have the greatest effect on the shape of the line graph.
Now we have to calculate the average of the remaining results by adding up the amount of oxygen produced for each minute, dividing by the number of results, and plotting the new values on a graph against the concentration of Celery Extract/Catalase.
Analysis
The graphs on previous pages show a relationship between the time and the volume of oxygen produced within that time. My results show that at 1 minute the amount of gas produced at 100% and 25% Celery Extract concentration was 12.8 cm3 and 4.6 cm3 respectively, whilst at 3 minutes the amount of gas produced increased to 18.4 cm3 and 6.3 cm3. Over the three minutes the amount of gas produced increased by 5.6 cm3 and 1.7 cm3, again respectively. This shows that when celery extract was present in the solution, an increasing amount of oxygen gas was produced over the three minutes, and the volume recorded increased every minute. When the celery extract concentration was 0%, the amount of gas produced over the three minutes remained at 2.6 cm3. By changing the concentration of celery extract, we were changing the concentration of enzyme in the solution. Also the amount of oxygen produced is an indicator of the rate of reaction, and thus the faster the rate of reaction, the faster oxygen gas is evolved from the reaction mixture. Therefore, a change in enzyme concentration, and no other factors, affects the rate at which the reaction occurs. In addition to this, from the results it has also been confirmed, when an enzyme is present the rate of reaction increases.
From both graphs, we observe that in each line there is a steep gradient within the first minute, followed by a more level line. Calculating the gradient of the lines shows that this is indeed true,
Gradient = increase in y-axis-direction
increase in x-axis-direction
(i) So, Gradient of line between 0 and 1 minute for
100% Celery Extract concentration = 2 cm3
9 seconds
= 0.222222222
= 0.222 cm3 of oxygen produced per second (to 3 significant figures)
(ii) And, Gradient of line between 0 and 1 minute for
75% Celery Extract concentration = 1.8 cm3
12 seconds
= 0.15
= 0.150 cm3/sec (3 sig. fig)
(iii) Also, Gradient of line between 0 and 1 minute for
50% Celery Extract concentration = 1 cm3
9 seconds
= 0.111111111
= 0.111 cm3/sec (3 sig. fig)
(iv) Gradient of line between 0 and 1 minute for
25% Celery Extract concentration = 1.2 cm3
15 seconds
= 0.08
= 0.0800 cm3/sec (3 sig. fig)
For curved lines, the gradient has been calculated by simply drawing a straight line to make the triangle, instead of using parallelograms, as this is clearer than drawing parallelograms all over the graph paper.
(v) Gradient of line between 1 and 2 minutes for
25% Celery Extract concentration = 0.8 cm3
22.5 seconds
= 0.035555555
= 0.0360 cm3/sec (3 sig. fig)
(vi) Gradient of line between 1 and 2 minutes for
50% Celery Extract concentration = 1 cm3
10 seconds
= 0.1
= 0.100 cm3/sec (3 sig. fig)
(vii) Gradient of line between 1 and 2 minutes for
75% Celery Extract concentration = 0.8 cm3
15 seconds
= 0.053333333
= 0.0533 cm3/sec (3 sig. fig)
(viii) Gradient of line between 1 and 2 minutes for
100% Celery Extract concentration = 0.8 cm3
9.75 seconds
= 0.082051282
= 0.0821 cm3/sec (3 sig. fig)
For example, compare (i) to (viii), the rate of the reaction decreases to 0.0821 cm3/sec from 0.222cm3/sec. The same is repeated throughout the other results; (ii) and (vii) dropping from 0.150 cm3/sec to 0.0533 cm3/sec; and (iii) 0.111 cm3/sec makes the small drop to 0.100 cm3/sec; finally 0.0800 cm3/sec for 25% celery extract plummets to 0.0360 cm3/sec. The common feature of these rates is that the velocity of the reaction is faster in the first minute than it is in the second. This illustrates the fact that the initial rate of reaction, the first minute, is rapid, followed by a slower minute of velocity, when the amount of oxygen gas being produced per second decreases.
From the hand-written line graph, we can see various lines indicating gradient calculations in progress; they still need to be calculated in order to obtain a rate of reaction for the last minute. I would like you to take notice of the bracket, indicating a patch of anomalous results from the 50% and 75% Celery Extract concentration results. These are inconsistent results, but by calculating the gradient of the last section of these two lines, I hope to show that the anomalous results are not useless.
Gradient = increase in y-axis-direction
increase in x-axis-direction
So, Gradient of line between 2 and 3 minutes for
75% Celery Extract concentration = 1.8 cm3
33 seconds
= 0.054545454
= 0.0545 cm3of oxygen gas produced per second (to 3 significant figures)
So, Gradient of line between 2 and 3 minutes for
50% Celery Extract concentration = 1.9 cm3
39 seconds
= 0.048717948
= 0.0487 cm3/sec (3 sig. fig)
The rate of reaction at (a) (see graph) 75% Celery Extract Concentration is 0.0545 cm3/sec, and for 50% it is 0.0487 cm3/sec. Although the 75% Concentration line ends up lower than the 50% line, due to unforeseeable errors, the rate of reaction is higher at this point. We can have an educated guess that if the rate of reaction were to drop as predicted, the rate of reaction for the 75% concentration solution would decrease more slowly than the 50%, as the initial rate of reaction is faster. For the 25% concentration solution the rate of reaction in the third minute is slowest,
(v) Gradient of line between 2 and 3 minutes for
25% Celery Extract concentration = 0.4 cm3
22.5 seconds
= 0.017777777
= 0.0178 cm3/sec (3 sig. fig)
The curve decreases in steepness, and thus gradient, reflecting the ever slowing rate of reaction, 0.0800 cm3/sec (in the first minute)… 0.0360 cm3/sec (in the second minute)… 0.0178 cm3/sec (in the last minute). However, this levelling off of reaction velocity is not as obvious in my results. We have seen how the rate of reaction decreases after the first minute, and that the velocity never goes higher than this initial rate of reaction. We have also observed the drop in rate of reaction in between the first and second minutes, and, to an extent, the third (as shown by the comment above).
The graph on page 18 shows “The Class Average Rate of Reaction for the Hydrogen Peroxide-Catalase reaction". The lines on the graph are relatively straight, showing that the amount of oxygen gas produced per second remained relatively steady. However a slight curvature can be seen, getting less steep in every line, apart from the 75% concentration line which slightly bends upwards. For the other concentrations, the line gradient showed that the rate of reaction slowed down slightly over the three minutes, but it didn’t decrease and level off as predicted. The 75% Catalase concentration line had a slight increase in rate of reaction at the third minute; this line does not follow the pattern so the standard deviation calculation did not get rid of every anomalous result.
I predicted that “at any given enzyme concentration, whilst the substrate amount and concentration is limited (not infinite), the initial rate of reaction will be relatively quick and eventually reach a maximum velocity, before which the rate of reaction appears to level off and decrease”. The initial rate of reaction was quick in all of the results because there was a large amount of substrate available for the Catalase enzyme to bind with. The overall rate of reaction decreased after the first minute in all of my results, but they did not level off as predicted, they fluctuated, which indicated that there might have been problems in the experiment.
In the “The Class Average Rate of Reaction…” graph, the majority of the lines showed that the rate of reaction decreased after a rapid initial rate of reaction, appearing as if the rate of reaction would have reached a maximum velocity after a longer period of time. The decrease in line gradient/rate of reaction was due to the amount of the substrate molecules being reduced, as more substrate was being converted to product helped by the enzyme Catalase. Consequently there was less chance of collisions, ones that resulted in the binding of the enzyme with the substrate, because there was simply less substrate left for the enzyme to react with. In some of the experiments, more time was needed for more of the enzyme to be engaged in enzyme-substrate reactions, resulting in the maximum reaction velocity not being achieved.
In my results, the celery extract 25% concentration line gradient decreased more steadily, showing that the rate of reaction was decreasing at a stable rate, compared to the 100% which has a very steep line gradient. The volume and concentration of Hydrogen Peroxide never changed, so when the catalase was at 25% concentration there were many more hydrogen peroxide molecules per catalase molecule. When the concentration of enzyme is low the enzyme becomes saturated with substrate molecule; the rate of reaction reaches the maximum velocity more quickly because at any two points in time there are less and less active sites available to bind with the substrate. At 100% concentration, the catalase molecules were in larger number, there were more catalase molecules relative to hydrogen peroxide molecules, so enzyme active sites were more readily available, keeping the rate of reaction high.
If you look at the ‘Class Average Graph’, as the concentration of catalase molecules decreased the overall amount of oxygen gas produced decreased. Increasing the enzyme concentration increased the amount of gas produced because this increased the probability of successful collisions with Hydrogen Peroxide molecule producing more water and oxygen gas at a quicker rate. There is not an unlimited supply of substrate molecules so all reactions would have reached a maximum velocity.
Summary of Findings:
2H2O2 → 2H2O + O2
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An enzyme decreases the activation energy so that the substrate molecules have energy greater than, or can easily acquire enough energy to reach, the new activation energy, and thus the rate of reaction increases. For this to occur the catalase molecule must collide with the Hydrogen Peroxide molecule at the correct orientation to each other and the reacting molecules must haven enough energy to bring about the breakage of chemical bonds. Enzymes overcome the need for larger amounts of energy because it decreases the amount of energy needed. Totally random movement from the particles within the reacting solution resulted in collisions, which happened to be at the correct orientation. The ‘correct orientation’ for an enzyme and the substrate is one at which the active site of an enzyme can attach itself to a location on the substrate molecule. The lock and key theory dictated that the substrate molecule must be an exact complementary shape to the active site on the enzyme for the orientation factor to be satisfied. However, the induced fit theory showed that the substrate molecule can induce the enzyme to become flexible so that the two molecules closely fit. The outcome is the same either way; the enzyme binds to the substrate for a fraction of a second, before detaching and binding to another substrate. The nature of the enzyme never changes, so if all the factors affecting the experiment are controlled, the amount of catalase molecules never changes.
- At higher concentrations the initial rate of reaction was fastest, showing that as the substrate is being converted to product at a high velocity, the amount of substrate molecules are being reduced at a high velocity too. The initial rate of reaction is quick because there is a large amount of substrate (Hydrogen Peroxide) available for the Catalase enzyme to bind with.
- At lower concentrations the initial rate of reaction was slower, showing that the substrate is being converted to product at a slower velocity. As there is less enzyme molecules, the probability of substrate molecules coming into contact and binding successfully with the active site on the enzyme decreases.
- Increasing the enzyme concentration will increase the rate of reaction as this will increase the probability of successful collisions, but it will reach a maximum velocity as there is not an unlimited supply of substrate molecules. The amount of enzyme molecules remains the same whilst the substrate molecules decrease in number, until all have decomposed to water and oxygen, in the case of Hydrogen Peroxide. If the amount of substrate molecule decreases more and more, the enzyme molecules are less likely to collide with substrate molecules to form a product and so the rate of reaction will slow down. Eventually the reaction will stop and no more products produced, because there is no substrate left to catalyse.
Evaluation
In this evaluation I will consider the Agreed Method, any differences between that and my method, anomalous results and their sources, limits of procedures, strategy and equipment, any improvements and their justifications, and the effect of unreliable evidence on my conclusion.
My method and the ‘Agreed Method’ had many differences, some were positive whilst others were negative. I used concentrations of 100%, 80%, 60%, 40%, 20% and the mandatory 0%, but the Agreed Method dictated that we used variations with the factor of 25. My planned method would have supplied a wider range of results, which could have helped in the conclusion by providing back-up data if one or two concentrations had errors or anomalies, as they did do.
In addition to this, I planned to do three replicate results instead of the two as stated by the Agreed Method, which would have helped by offering even more data, which in turn could have been used to calculate more accurate averages. We could haven even afforded to discard an experiment if a replicated set deviated from the other two replicates too much. However, most of the group did not have the time to do a repeat for the Agreed Method, so to do three repeats of my experiment would have been a hard task.
Another difference between my method and the ‘Agreed Method’ was what time the results were noted down. They would have been noted down every thirty seconds for 2 minutes so that I had four results, but they were recorded every minute for three minutes, to get three results. Again my method would have provided a wider range of results, which would have made a smoother curve on a graph, showing where anomalous results are more easily.
For both methods a ‘control’ recording was made; the control was when no celery extract was used, just distilled water. This expressed the amount of gas displaced from the apparatus when the solution was liberated from the syringe into the conical flask closed system. The liquid changed the pressure inside the system, forcing air through the delivery tube and to the burette where the amount of gas produced was recorded. The amount of gas collected for the other concentrations (25%, 50%, 75% and 100%) should have the control amount of gas deducted from their values, because this is air which wasn’t produced from the reaction but from the nature of the apparatus. I would have liked more time to get replicate results for the control (and other concentrations obviously) to find out if the calibration of the apparatus changed over time and to obtain a more accurate control value.
The Agreed Method used a double-holed rubber bung and syringe to introduce the Hydrogen Peroxide solution into the conical flask, and the syringe remained in the bung throughout the three minutes of the experiment. It did not exactly specify when the stopwatch must be started but I started it once the syringe had been emptied completely, and before swirling the flask gently. This ensured that I had the exact same technique for each experiment and that any gas collected was recorded. By using a double-holed rubber bung and leaving the syringe inside, no gas could escape, as it might have done in my experiment, where I would have had to move quickly to close the rubber bung after opening it to let in the hydrogen peroxide from the syringe. This was a good idea as it prevented any loss of oxygen gas which would affect the results and hence my conclusion.
Another important and negative point, about the double-holed rubber bungs, and tube which went through it to connect the outside to the inside of the flask, is that once I disconnected the syringe and rubber bung from the conical flask, solution was left on my hands. I knew that this solution was Hydrogen Peroxide because it made my skin go white soon after and I had to wash it off in response to safety guidelines. This meant that not all of the solution from the syringe went to the bottom of the conical flask, so not all of it reacted, and was left on the underside of the rubber bung and inside the hole. The hole through the rubber bung had no air/liquid pressure applied to it as the last of the syringe solution went through, so it wasn’t all ‘squeezed’ through. By lengthening the rubber bung hole tubing so that it reaches far inside the vessel, one can see whether or not solution is not going through and take action by tapping the syringe, or some similar action. Also the rubber seal wasn’t always tight enough and some people in the group had to repeat experiments because they realised that gas was escaping. The use of a lubricant such as Vaseline would have made sure that any results were valid.
I would have used boiling tubes rather than a conical flask as outlined in the Agreed method, because of the loss of solution from transfer. As you empty the syringe into a vessel, it is probable that some of the Hydrogen Peroxide will drip down the inside of the vessel and flow to the bottom. If the inner surface area of the reaction vessel, sometimes termed as the system, is larger, then more solution will be left on the inside of that vessel, reducing the volume originally measured. A 100cm3 conical flask has a larger inner surface area than a boiling tube, so more solution may have been lost in the Agreed Method when transferring the syringe solution.
As identified on the graph paper, there are five possible anomalous results, they are possibly anomalous because they might, however unlikely, actually be the truth. These anomalous results are situated in the upper right quadrant of the graph and are highlighted in bold below:
These are identified as anomalous results because the lines cross on the graph and the line does not create a smooth curve, the line for 100% for example, instead it is erratic and unnatural. Sources of these errors include the holed rubber bung delivery strategy, which I pointed out as inaccurate earlier on due to the loss of solution and the use of a conical flask (as outlined earlier). When timing it is hard to pinpoint the exact volume of oxygen gas produced because bubbles of gas are evolving and rising through the burette continuously, especially at higher concentrations of enzyme where more oxygen is being produced, so one has to remember where the meniscus was on the minute. This could have been a problem when observing and measuring the amount of oxygen produced on the burette.
A major source of error is sample preparation and maintenance of the solution throughout the experiment. We could not prepare our own samples or store them before hand so very little is known about this, but basic precautions can be made to reduce this significant source of error. Samples of Hydrogen Peroxide, Celery Extract and Distilled water should be put on ice throughout the experiment in the same ice vessel or a thermostatically controlled water bath should be used if it can be made available. Using Ice to keep the solutions cool is less advisable, as this would affect the performance of the catalase enzyme, which is used to operating in temperatures ranging from 20oC to 40oC. A thermostatically controlled water bath is much more accurate and reliable as it is independent of our observation, unlike a thermometer and does not require as much attention. If this was used then a factor affecting the experiment could be effectively controlled reducing a significant source of error and cause of anomalous results. “Book of Data Nuffield Advanced Science” states that the enzyme catalysed reaction Hydrogen Peroxide (aq) + Catalase has an activation energy Ea/kJ mol-1 of 36.4, so this would be the optimum temperature for the experiment.
The effectiveness of liquidising celery tissue may be questionable. When celery extract is liquidised there could still be impurities from the source (the celery) and there could still be larger solid pieces or gelatinous solution in the solution which would change the surface area of the reactants, another factor affecting the experiment. A possible improvement could be timing the liquidising of the celery extract, so that we have some control over it to make sure that it is conducted in a fair way. By liquidising celery extract, the membrane-bound catalase is released as cell membranes are physically broken, “A native, membrane-bound catalase molecule can detoxify nearly 100 million H2O2 molecules per second! (Isolated "purified" catalase is only about 10% as fast.)”, but the use of actual celery is hard to implement because more factors are introduced. We could use isolated purified catalase as an alternative to celery extract, which would be a much more accurate practice, as there are no impurities. The availability of a centrifuge would greatly increase the accuracy of the experiment, after taking into account possibly errors using this equipment, as it would separate contaminations from the pure solution we want.
These impurities, which change the concentration of the celery extract, also introduce different enzyme concentrations within the celery in the beaker. The celery extract can also settle whilst in a beaker, and the different concentrations separate out slightly, although we may not be aware of it. The syringe position at which one takes celery extract from the beaker could result in taking celery extract of slightly different concentrations, and therefore catalase of slightly different concentrations. This unnoticeable error means that the solutions of celery extract measured where even more inaccurate as it was assumed that the solution within the beaker was of consistent concentration. The last paragraph divulged some stratagems to counter this.
Another (minor) error includes the measurement of solutions using the syringes. It is possible that I could have been slightly out on some measurements and not noticed it, or that some bubbles had entered the syringe, but this is unlikely as it is easy to accurately use a syringe and to see any bubbles in the solution. I realised after the experiment that by tapping the syringe, or flicking it, the bubbles would rise to the top, so bubbles can be pushed out. However, a syringe is only accurate to within a cubic centimetre, or millimetre, and the rest depends on my observation so an error could be from the limits of the apparatus and observation. The percentage error could be anything up to 10% per cubic centimetre of solution measured in the syringe, as my observations could only be accurate to within a very small margin of 0.1 cm3. An improvement here could be the use of high-quality syringes, accurate to a smaller unit that a millimetre, so that the likeliness of an error occurring could be reduced furthermore. Bubbles are also a problem when they are created in the reaction mixture by the addition of a solution from a syringe as the action of squirting from a syringe can create bubbles. Bubbles change the surface area of the reactants (see Variables). The use of a hypodermic needle would dramatically reduce the creation of bubbles but extra safety precautions we need to be made. ‘Gently swirling’ (step 4 of Agreed Method) the reaction mixture is not very exact and gives the reactants more energy, making the experiment unfair, but can be countered by using specialist equipment which shakes solutions (name unknown).
A problem I found with the conclusion is that the rate of reaction never reached a maximum velocity, and it couldn’t be proved that a maximum velocity of reaction could be reached. This depended on the amount of time we had. If we could have done each experiment for something close to 5 or 6 minutes, and still do three replicate results, we might have been able to observe the slowing down of the reactions and a more obvious curve, rather than a borderline straight-line. The pilot experiment I did showed that 100% concentration of 5 cm3 Celery extract, reacted with 5 cm3 of Hydrogen Peroxide to produce a little less than 50 cm3 of Oxygen gas, so it is possible to implement.
An improved strategy for timing could involve the use of a partner to start the timer once one had added the Hydrogen Peroxide to the reaction mixture. The other partner could concentrate on the strategy of adding the syringe solution to the reaction mixture, whilst the other waits for the signal to start the timing. The extra person could also be ‘used’ to tell one when to take a measurement from the burette, and other procedures which require doing two things at once. This would increase timing accuracy to within a fifth of second, making measurements more accurate, but this could be deemed inappropriate for this level of work, and there must be apparatus available that can take accurate measurements for you. An increase in timing accuracy from half a second to within a fifth of a second is a big reduction in error, but even then a fifth of a second is still too long; the availability of equipment is the most significant adversity.
The burette used to measure the gas was only accurate to within a cm3 but by observation one could record the amount of gas produced to within a tenth of a cubic centimetre. This is a good degree of accuracy but the degree of error could be slightly higher still. The difficulties in paying attention to the timing and the recording of the amount of oxygen gas produced, could have resulted in a loss of time and thus an increase in the amount of gas recorded or vice versa. Taking this into account, the percentage error in timing was probably much higher than previously though; the effect of this on the results is not negligible. It is now easy to see how my anomalous results came about. However the trend in the gradient of my graph line, and consequently the results, was similar to across the whole of the class’, so decisions made in the conclusion still stand.
A possibly significant source of error in the experiment is stemmed from the nature of Hydrogen Peroxide, and I found out about this only till after writing half of this investigation. Hydrogen Peroxide can be easily decomposed by UV light (‘Hydrogen Peroxide’ by Iman Ahmad, University of Iowa). UV light is a component of sunlight so sunlight was a simple factor which I had overlooked. UV radiation energy, or light, provides 400kJ mol-1 (a mole of photons). To overcome this factor, the implementation of a different light source and a dark room (one devoid of sunlight) would be the most effective improvements. A simpler, but less effective, solution is to paint the vessels, which will contain the Hydrogen Peroxide black and to cover them with black lids in order to reduce sunlight exposure.
After looking at my own results and plotting them on a graph, I became aware of the inadequacies of the data I had. To counter this I used the class’ data and standard deviation and average/mean calculations, to create a graph which would have less anomalous results. I used a combination of these to analyse the data to find out what had happened, and I tried to estimate what should have happened as I had anomalous results. The ‘Class Average Graph’ did have some slightly linear lines but still they had a slight curvature, indicating a slow down in reaction velocity over the three minutes. The processing of the results was in no way biased in order to show the reaction slowed down, for example, the standard deviation was added and subtracted from the mean. Even though my data was error-ridden, it did show a similar pattern. Certain factors will have a greater effect on the validity of the conclusion I made earlier on, but these are not major sources of error repeated over time that could have totally changed the outcome of the experiment. The amount of sunlight would remain relatively unchanged, bubbles would have a very minimal effect on the rate of reaction and the liquidising of the celery extract would certainly not have a major impact on the experiment (unless something was obviously wrong it). Although, the temperature of the samples and transfer of solutions remain as dubious aspects of this experiment and I would have liked to use a thermostatically controlled water bath and a different strategy for the latter. The temperature could account for the drop in rate of reaction after an initial rate of reaction: the temperature of the reactants increased as the reaction progressed changing the rate of reaction. A different strategy for the transfer of solutions would include modifying the equipment, or finding new equipment; the limitation of the apparatus can hinder the effectiveness of the strategy, or the strategy needs to be modified using suggestions made earlier.
Bibliography
Cambridge Advanced Sciences; Biology 1; Cambridge University Press 2000; ISBN 0 521 78719 X.
Hydrogen Peroxide by Iman Ahmad, Free Radical and Radiation Biology Program, University of Iowa, Spring 2001.
P.119, Book of Data Nuffield Advanced Science; Longman Group Limited;
ISBN 0 582 35448 X.
Undergraduate Biological Sciences Textbook.
New Scientist Archive.
http://medlib.med.utah.edu/NetBiochem/hi1a.htm
http://www.google.co.uk/search?q=Hydrogen+Peroxide+Catalase&hl=en
http://www.catalase.com/catalinks.htm
http://esg-www.mit.edu:8001/esgbio/cb/cbdir.html
http://www.sbu.ac.uk/water/index.html
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