4. Method
4.1 Method
Set up water bath to 291K.
Measure 10mL of 5% Hydrogen Peroxide using a 10mL graduated glass pippette(士0.1)
Pour the 10mL solution into a 100cm3 Buchner Flask
Attach Buchner flask to a 50 cm3 gas syringe (士0.5)with rubber tubing
Place Buchner Flask with 10mL of %% hydrogen peroxide water bath for 5 minutes then check temperature with thermometer (士0.05)that it had reached 291K degrees.
Weigh 0.2000g of Yeast using an electronic balance
Pour yeast into buchner flask and close with stopper. At this time start stopwatch and recording of gas syringe and check how much oxygen has been produced every 2 seconds over a 30 second time period.
Repeat this for 295K, 301K, 308K, 310K.
4.2 Apparatus
Water Bath
10ml Graduated glass pipette (士 0.1)
50cm3 glass gas syringe with rubber tubing (士0.5)
Retort Stand
Buchner Flask 100cm3
150 ml of 5% H2O2
3g of yeast
Thermometer(士0.05)
Electronic Balance(士0.0001)
4.3 Table 3:Risk Assesment
Material
Risk Assemsent
Hydrogen Peroxide
As this is a very dilute hydrogen peroxide, it does not pose any significant risk. Avoid Eye contact as it may be irritant but pressure may build up in the buchner flask so one needs to take care when opening it.
Catalase (Yeast)
Catalase extracted from yeast has a low hazard and is unlikely to cause any significant risk. Still avoid eye contact and do not swallow.
Ethical Considerations
There are no ethical issues identified for this experiment
Environmental Considerations
When disposing hydrogen peroxide dilute the solution with water and flush it down a sink
18.0
19.0
18.3
士1.0
0.47
16
19.0
21.0
20.0
20.0
士1.5
0.82
18
20.0
22.0
21.0
20.8
士1.5
0.62
20
21.0
22.0
22.0
21.5
士1.0
0.41
22
22.0
23.0
22.0
22.1
士1.0
0.62
24
22.0
24.0
23.0
22.8
士1.5
0.85
26
23.0
25.0
23.0
23.5
士1.5
0.71
28
24.0
25.0
24.0
24.2
士1.0
0.62
30
24.0
26.0
25.0
25.0
士1.5
0.82
Table 7: Volume of Oxygen collected (in cm3)over a 30 seond time period at 308 Kelvin (35℃)
Time (seconds)
Trial 1 O2 Collected 士 0.5 cm3
Trial 2 O2 Collected 士 0.5
cm3
Trial 3 O2 Collected 士 0.5
cm3
O2 collected mean 士0.5
Absolute Uncertainty Mean
Standard Deviation
2
3.0
4.0
3.0
3.3
士1.0
0.47
4
6.0
7.0
7.0
6.7
士1.0
0.47
6
9.0
9.0
8.0
8.7
士1.0
0.47
8
13.0
14.0
12.0
13.0
士1.5
0.82
10
15.0
16.0
14.0
15.0
士1.5
0.82
12
17.0
18.0
17.0
17.3
士1.0
0.47
14
19.0
21.0
20.0
20.0
士1.5
0.82
16
20.0
22.0
21.0
21.0
士1.5
0.82
18
21.0
22.0
22.0
21.7
士1.0
0.47
20
22.0
23.0
22.0
22.5
士1.0
0.41
22
22.0
24.0
23.0
23.2
士1.5
0.62
24
23.0
24.0
24.0
23.8
士1.0
0.62
26
25.0
25.0
24.0
24.8
士1.0
0.24
28
25.0
26.0
25.0
25.5
士1.0
0.41
30
26.0
27.0
26.0
26.3
士1.0
0.47
Table 8: Volume of Oxygen collected (in cm3) over a 30 seond time period at 310 Kelvin (37℃)
Time (seconds)
Trial 1 O2 Collected 士 0.5 cm3
Trial 2 O2 Collected 士 0.5
cm3
Trial 3 O2 Collected 士 0.5
cm3
O2 collected mean
Absolute Uncertainty of Mean
Standard Deviation
2
5.0
4.0
3.0
4.00
士1.5
0.82
4
10.0
8.0
7.0
8.33
士2.0
1.25
6
12.0
14.0
10.0
12.00
士2.5
1.63
8
20.0
17.0
19.0
18.67
士3.0
1.25
10
24.0
19.0
22.0
21.67
士3.0
2.05
12
25.0
21.0
24.0
23.33
士2.5
1.70
14
26.5
22.0
24.5
24.33
士2.8
1.84
16
26.5
23.0
25.0
24.83
士1.0
1.43
18
25.0
24.0
25.0
24.67
士1.0
0.47
20
25.0
24.5
25.5
25.00
士1.0
0.41
22
26.0
25.5
25.0
25.50
士1.3
0.41
24
26.0
25.5
26.5
26.00
士1.5
0.41
26
26.0
26.5
27.0
26.50
士1.0
0.41
28
26.0
26.5
27.0
26.50
士1.0
0.41
30
26.0
26.5
27.0
26.50
士1.0
0.41
Absolute Uncertainties for O2 collected:
highest value recorded with uncertainty(+0.5) – lowest value recorded with uncertainty(-0.5)2
Example for second 2 at 37℃
5.5-2.52 = 士1.5
5.2 Propagating Uncertainties
Percentage Uncertainties = absolute uncertaintyreading taken 100
Sample Calculations for 291 K
Percentage Reading of weighing Yeast
=士0.00010.2000 100 = 0.05%
Percentage Uncertainty of H2O2 collected with pipette
= 士0.10.10 x 100 = 1.00%
Percentage Uncertainty of Thermometer
= 士0.0518 = 0.278%
Percentage Uncertainty of Gas Collected
= absolute unertainty gas syringetotal O2 gas collected x100
ex. at 18℃
士.0.520.3 x100 = 2.46%
Percentage Uncertainty of Stop Watch
士0.5 + Time taken to start recording of Gas syringe (0.86s see appendix for calculations)
= 士1.3630= 4.53%
Table 9 Displaying The Percentage Uncertainty of each Apparatus at for each replicate for each temperature
Temperature (K)
Scale to measure Yeast
(%)
Pipette to measure H2O2 (%)
Thermometer (%)
Mean Uncertainty of Gas collected in 30 seconds (%)
Stop Watch
(%)
Total Error
(%)
Mean Error of Investigation
(%)
291
0.05
1.00
0.278
2.46
4.53
8.318
7.80
295
0.05
1.00
0.227
1.90
4.53
7.707
301
0.05
1.00
0.178
2.00
4.53
7.758
308
0.05
1.00
0.142
1.90
4.53
7.622
310
0.05
1.00
0.135
1.89
4.53
7.605
Rate of Reaction Graph
Graph 1: A rate of reaction graph, where O2 gas collected in cm3 was plotted against time over a 30 second time period
Time (seconds)
Legend for Graph:
In order to determine the actication energy, the initial rate of reaction was calculated from this graph. In order to do this a tanget was drawn at t=0.5 Then as k=ck, as the hydrogen peroxide and catalase concentration are assumed to remained unchanged and therefore are a constant. Thus, the natural logarithm (ln) of the initial rate was taken and plotted against 1/T where the temperature is displayed in Kelvin.
Table 10: Proccessed Data showing the values of 1/T againt ln(initial rate)
Temperature in Kelvin
1/T(K)
(10-3 K-1)
Initial Rate (gradient of tangent)
ln(initial rate)
291
3.45
1.09
0.09
295
3.39
1.19
0.17
301
3.32
1.42
0.35
308
3.25
1.74
0.55
310
3.23
1.92
0.65
Subsequently, these values where plotted in a graph with 1/T on the X-axis and ln(initial temperature) in the Y-axis.
Graph 2: Ahrrenius Graph in which the ln(initial rate) is plotted against 1/T in Kelvin showing the temperature dependance of the value of the initial rate
The slope of this graph is equal to =−Ea/RT. Therefore in order to determine the value of the of the acrivation enegry the slope (-3094.9) is multiplied by -R (-8.3145) in order to determine the Ea in Jmol-1.
= -2660.8 x -8.3145 = 21280 Jmol-1.
= 25.732 Jmol-1.1000= 21.28 KJmol-1.
As the proportion of molecules with the enrgy greater than or equal to Ea can be given by the exponential term e-Ea/RT. Thus at 25 ℃ this can be calculated as follows: e-21280/8.3145 X 298= 1.86 X 10-4. Compared to literature values for the uncatalysed decomposition of hydrogen peroxide this is equal to 6.9 × 10-14, the yeast-catalase catalysed decomposition has a relative rate of 2.70 × 1010.
In order to account for uncertanties, the final result is multilplied by the accumulated average uncertainty (see table 9. Therefore the ativation energy is equal to 25.75 x .2085 = 5.38, 25.73 KJmol-1 士4.91KJmol-1.
6. Analysis of Results
6.1 Conclusion
To conclude it could be said that the effect of catalase on Ea for the decomposition of H2O2 was successfully found, although there was a slight divergence in the literature values and found values. This was found to be 21.28 KJmol-1. Although these values are above literature values of 18KJmoles-1 (Derek) this can be accounted for regarding the fact that different sources of catalase may lead to different acitvation energies as well as the fact that the source from which it was derived (yeast) is highly sensitive to temperature changes as it operates at an optimum temperature at 37℃. Nevertheless, one must also consider the Systematic error in this investigation which can be determined as follows:
Total Error = |accepted value – experimental value| accepted value x 100%
Total Error = |18.00 – 21.28| / 18.00 x 100
Total Error = 18.22%
Systematic Error = Total error – Random Error = 18.22% − 7.80% = 10.42%
This indicates that there has been a high systematic error leading to the discreoancies between literature vales and the obtained value. Nevertheless, although te results did not prove to be accurate they were precise as could be seen by the similar values observed in the repeated trials.In a larger context of the scientific community this investigation can be interpreted as relevant, as it shows the substantial increase in molecules with the activation energy greater than or equal to the one needed for the decomposition of hydrogen peroxide into oxygen and water. The relative rate compared to the non-catalysed decomposition increased by 2.70 × 1010. When further examining the role of catalase in biological systems such as the human body the increased rate is of great importance as hydrogen peroxide may be damaging to tissue cells from oxidative damage. In addition, it can be seen that catalase is an extremely efficient enzyme in particular at temperatures similar to body temperature. It is also significantly more efficient in comparison to other enzymes such as most activation energies for reactions with biological catalysts range from 25- 63 kJ mol-1. (Derek)
6.2 Evaluation: Strengths and Weaknesses
The low standard deviation amongst replicates can be seen as a strength of this experiment. This indicates that eventhough there was a large systemic error, the results remained reproducable. In addition, when looking at the ahrrenius plot it can be deduced that the high R2value of (0.99). The regression coeffcient serves as a statistical measure of how close the data are fitted on a regression line (Minitab Blog) and hence suggests a strong correlation between the data points. Ultimately, this reinforces the idea that there is a correlation bewteen ln(initial rate) and the temperature, which is what is originally poposed byt he ahrrenius plot. The gathered data therefore reiterates scientific findings between the relation of the rate constant (k) of a reaction and its temperature dependance, despite its high systematic error.
Nevertheless, there were many weaknesses regarding the investigation; one of them being the small range of independant variables. Although, five different temperatures can be seen as sufficient, and clearly indicated a trend line with a strong regression coefficient, increasing the range of independant variables allow for more data to be plotted on the ahrrenius plot. This is of large significance as the trend line and its gradient are the basis for the calculation of the activation energy. By recording the rate of reaction at more temperatures, a gradient with a higher reflection of the true activation energy could be used to calculate the imapact of catalase on the decomposition of hydrogen peroxide. Additioanlly, temperatures closer to the optimum operating temperature of catalase could be used as these would serve as the most accurate reflection of its ability to provide an alternate reaction pathway. Low temperatures should also be avoided as these allow for mote oxygen to dissolve in water.Furthermore, when extending the range of temperatures it may not be disregarded that the enzyme is denatured at temperatures above 55℃. (Eyester)
Another Weakness, which may have contributed to the systematic error of this investigation could be the control variable of the concentration of catalase. Although the grams of yeast added to the reaction at each given temperature remained the same, the exact concentration within the yeast itself could not be determined and therefore poses a significant source of error in the investigation. The low standard deviation suggests that the concentration of catalase did not diverge to an extreme extent however there may still be a difference which may ahve impacted the rate of reaction.
Regarding the method by which the ahrrenius equation was manipulated, there were negative as well as positive aspects associated with it. A strength of using the method of initial rate of reaction was that it allowed for extraneous factors which may alter the function of catalase (such as pH or the presence of side reactions) to be reduced. Additionally, as the rate of reaction is definded as the change in concentration of an infinitely small time interval, the tangent used in order to detertmine the rate of reaction serves as a more accurate representation of the true rate of reaction as opposed to an average rate of reaction, as it allows for the assumption that the conentration of both catalase and hydrogen peroxide remained unchanged. Eventhough activation energy itself is independant of the temperature and concentration of reactants, these factors contribute to the initial rate of a reaction which wer eultimately employed in order to determine the activation energy. Nevertheless, this method gave rise to an increased random error of investigation, as the volume of gas collected over a given time period allowed for many difficulties. The exact initial rate of reaction may not be accurately determined due to the fact that the buchner flask must be closed and the delay in reaction time when the volume of oxygen collected is recorded. Although these errors were accounted for (by the calculation of time needed to start the recording of gas syringe), it allowed for large random error.Thus, the first two data points for the rate of reaction graph may be seen as unreliable and thus have a significant impact on the slope/value of the instantaneous rate of reaction. Nevertheless, researchers have suggested that the error embedded in the investigation by approciamting the gradient is within the normal limits of kinetic studies (Casado).
6.4 Extensions
One may consider analyzing the impact of different catalysts on the decomposition of hydrogen peroxide. By examining the activation energy of inorganic catalysts such as Manganese(IV)Oxide or iodide ions the magnitude of difference betwen the non-catalysed and catalysed decomposition may be further examined. This could be done using the same method as suggested above, and altering temperatures at which data is collected. By establishing the most efficient catalyst one may furhter look into their purpose and ideal environments in which they are used such as for industrial purposes etc.