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IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Candidate Name Candidate Number Date of Practical

:Yoojin Lee :002213-067 :August 23, 2010

Internal Assessment – Rate of Reaction Research Question How will changing hydrogen peroxide (H2O2) concentration affect the rate of reaction, represented by the increase in pressure over time, measured using gas pressure sensor?

Introduction Hydrogen peroxide (H2O2)1 is a by-product of biochemical metabolism. An accumulation of hydrogen peroxide can be deadly, so it has to be decomposed. One of the decomposing factors is an enzyme called Catalase. Catalase breaks hydrogen peroxide into water and oxygen.

The chemical formula for the reaction is,

Since this is a decomposition reaction, it is exothermic. Although hydrogen peroxide can gradually degenerate itself, it decomposes much faster with the help of Catalase, because Catalase lowers the activation energy, the minimum energy barrier that hydrogen peroxide molecules have to overcome to decompose.2

1

“Hydrogen,” Wikipedia, the free encyclopedia, http://en.wikipedia.org/wiki/Hydrogen_peroxide (accessed October 22, 2010). 2

“The Hydrogen Peroxide Breakdown Examining Factors That Affect the Reaction Rate of Enzymes,” Alief Independent School Districts Vanguard,http://www4.alief.isd.tenet.edu/cahowe/biology/pak%202/The%20Hydrogen%20Peroxide%20Breakd own.htm(accessed October 22, 2010). 1


IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Figure 1 shows the reaction trend when enzyme is present.3 Since the enzyme lowers the activation energy, the rate of reaction increases without consuming enzyme. In this experiment, the substrate is hydrogen peroxide. The purpose of this investigation is to find out the relationship between the substrate concentration and the rate of reaction, which is measured by the change of pressure overtime, measured using gas pressure sensor.

The gas pressure sensor is used because the reaction produces oxygen gas. The faster the reaction is, the faster the pressure will increase. Thus, by examining the change of pressure overtime, the rate will be calculated and analyzed. For this investigation, the initial rate of the reaction is examined.

3

Ibid 2


IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Hypothesis When the amount of enzyme stays constant, the substrate concentration will determine the rate of reaction. However, when the number of substrate molecules exceeds the available number of enzyme, the rate of reaction will no longer increase, but stay constant. Since the gas pressure sensor can take up to 180 atm, it is very difficult to calculate the initial rate of a highly concentrated solution. Thus, this experiment will only consider low concentrations. The rate of reaction will increase as the hydrogen peroxide concentration increases. However, from a certain concentration, the rate will stay the same, even if the concentration increases, because the amount of enzyme used is fixed.

Figure 2 shows the relationship between the rate of reaction and the hydrogen peroxide concentration.

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IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Variables Variables Independent

Description Hydrogen Peroxide (Substrate) Concentration, c/%

Dependant

Rate of Reaction of hydrogen peroxide decomposition

Controlled

Recording initial rate

Amount of enzyme

Temperature

Method of Measuring Hydrogen peroxide solution was diluted to 50% using distilled water. Then the diluted hydrogen peroxide was further diluted to prepare 20%, 40%, 60%, 80%, and 100%. Distilled water was used for control (0% ethanol). Triplicate trials were performed on each concentration to obtain the mean. Rate of reaction is represented by the change of pressure over time. Pressure was measured by using the gas pressure sensor. The same sensor was used throughout the experiment. Also the shortest tube was used to reduce systematic errors. As soon as the hydrogen peroxide is put inside the test tube, it is immediately capped with the gas pressure sensor to record data. The amount of enzyme is set to 10 microliters. Micropipette was used for accurate measurement, because an extra drop of enzyme can alter the rate of reaction significantly. Since temperature is directly related to the rate of reaction, the entire experiment was conducted in the lab at a constant room temperature, which is

approximately 25℃. Volume of hydrogen peroxide Even though the solutions differ in solution concentration, the volume for all of the solutions stayed the same, which is 1.5 cm3 for all trials. For accurate measurement, micropipette was used throughout. Size and type of test tubes The size and type of test tubes were constant, because they can alter the pressure. The same size and type of test tubes were used throughout. Hydrogen peroxide A new hydrogen peroxide was used because hydrogen peroxide can degenerate naturally. All of the trials used hydrogen peroxide from the same container. Table 1 shows the independent, dependent, and controlled variables and the methods of measuring 4


IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Apparatus

Materials  Hydrogen peroxide  Catalase  Distilled water

 Gas pressure sensor  Micropipette (± 0.006cm3)  Magnetic Stirrer  25cm3 Pipette (± 0.03cm3)  Test tubes  Beaker

Procedure 1. Serial dilution was performed following the diagram below.

Figure 3 shows the diagram for serial dilution method 2. 10µl of Catalase is added to the testing tube and the pressure build up was measured instantly using the gas pressure sensor. -

While collecting data magnetic stirrer was used to release oxygen gas trapped inside the solution with constant stirring.

-

Top part of the test tube was manually held to minimize the temperature rise.

3. Steps 1 and 2 were repeated and performed with different concentrations to obtain valid triplicate trials for each concentration. 5


IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Data Collection – Qualitative Data The reaction started as soon as Catalase touched the surface of hydrogen peroxide. More concentrated hydrogen peroxide produced more oxygen bubbles and the reaction rate was faster, because it produced oxygen gas rapidly. On the other hand, more diluted hydrogen peroxide reacted slowly and the oxygen bubbles were released sporadically.

Data Collection – Quantitative Data Pressures of Different Hydrogen Peroxide Concentrations/ kPa Time, t/ sec

1.5

0.75

0.375

0.1875

0.09875

1st

2nd

3rd

1st

2nd

3rd

1st

2nd

3rd

1st

2nd

3rd

1st

2nd

3rd

0.00

100

100

103

100

100

103

100

103

103

100

100

103

100

100

103

60.0

100

100

103

100

100

103

100

103

103

100

100

103

100

100

103

120

105

100

100

104

100

103

100

103

103

100

102

103

100

100

103

180

114

107

100

106

103

105

102

105

105

102

103

104

100

103

104

240

119

117

107

108

105

108

105

108

107

104

104

105

100

103

105

300

124

123

116

110

108

113

108

110

109

105

105

106

100

104

106

360

127

129

125

112

110

117

110

113

111

106

106

106

100

104

106

420

130

134

132

114

112

120

112

115

112

107

106

108

103

105

108

480

134

138

136

115

114

123

114

116

113

107

107

105

103

105

107

540

137

141

140

117

115

125

115

117

114

108

107

106

103

105

108

Table 2 shows condensed raw data for the experiment extracted from Logger Pro.

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IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Graph 1 shows the raw data for pressure build up of different hydrogen peroxide concentrations over time 7


IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Data Processing The gradient of a graph represents the change in pressure over time. Thus, it represents the rate of reaction. Rate of Reaction, r/ kPa s-1 Hydrogen Peroxide Concentration, c/%

Trials Mean(a)

Mean ± SD(b)

1

3

3

1.50000

0.130

0.110

0.0994

0.113

0.015

0.75000

0.0527

0.0417

0.0437

0.0460

0.0059

0.37500

0.0389

0.0299

0.0306

0.0331

0.0050

0.18750

0.0174

0.0251

0.0128

0.0184

0.0062

0.09875

0.0156

0.0251

0.0150

0.0186

0.0057

Table 3 shows the rates of pressure increase for different hydrogen peroxide concentrations Mean: average of triplicate trials for each set. (b) SD: standard deviation for triplicate trials. (a)

Sample Calculations Rate of reaction =  Calculation of the mean rate of 1.5% hydrogen peroxide from the triplicate trials. Mean ( ) = 0.113 kPa s-1

=

 Calculation of the standard deviation of 1.5% hydrogen peroxide from the triplicate trials. Standard deviation =

= = 0.015 kPa s-1 8


IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Average Rate of Pressure Increase of Different Hydrogen Peroxide Concentration

y = 0.0678x + 0.0063 R² = 0.9695

Average Rate of Reaction, a/ kPa/sec

0.14

(a)

0.12

(b) 0.1 0.08 0.06 0.04 0.02 0

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Concentration of Hydrogen Peroxide, c/%

Graph 2 shows the processed data of average rate of pressure increase over the hydrogen peroxide concentration Vertical error bar shows the standard deviation of the triplicate trials for the rate of reaction (b) Horizontal error bar shows the uncertainty in H2O2 concentration. (a)

9

1.8


IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Uncertainties % uncertainty for volume use Volume of water using % uncertainty in % uncertainty in Micropipette volume/ % volume/ % (ΔV = ± 0.006) cm3 0 0 0 (0.012/1.5)x100 (0.012/1.5)x100 1.5 ± 0.012 = 0.8 = 0.8 (0.012/1.5)x100 (0.012/1.5)x100 1.5 ± 0.012 = 0.8 = 0.8 (0.012/1.5)x100 (0.012/1.5)x100 1.5 ± 0.012 = 0.8 = 0.8 (0.012/1.5)x100 (0.012/1.5)x100 1.5 ± 0.012 = 0.8 = 0.8 (0.012/1.5)x100 (0.012/1.5)x100 1.5 ± 0.012 = 0.8 = 0.8 Table 4 shows the percent uncertainty for volume use Absolute % uncertainty in % uncertainty in concentration, concentration, Δc/% Δc/%

3.00000

Volume of H2O2 using Micropipette (ΔV = ± 0.006) cm3 0

1.50000

1.5 ± 0.012

0.75000

1.5 ± 0.012

0.37500

1.5 ± 0.012

0.18750

1.5 ± 0.012

0.09875

1.5 ± 0.012

Concentration of H2O2, c/%

Concentration of H2O2, c/% 3.00000 1.50000 0.75000 0.37500 0.18750 0.09875

0.0 0.0 1.6 (1.6/100)x1.50000 = 0.02400 3.2 (3.2/100)x0.75000 = 0.02400 4.8 (4.8/100)x0.37500 = 0.01800 6.4 (6.4/100)x0.18750 = 0.01200 8.0 (8.0/100)x0.09875 = 0.00790 Table 5 shows the absolute uncertainties 10

Total % uncertainty/ %

Concentration with uncertainty

0

0

0.8+0.8 = 1.6

1.5±1.6%

0.8+0.8+1.6 = 3.2 0.8+0.8+3.2 = 4.8 0.8+0.8+4.8 = 6.4 0.8+0.8+6.4 = 8.0

0.75±3.2% 0.375±4.8% 0.1875±6.4% 0.09875±8.0%

Concentration, c/% 3.00000±0.00000 1.50000±0.02400 0.75000±0.02400 0.37500±0.01800 0.18750±0.01200 0.09875±0.00790


IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Conclusion The data suggests that as the hydrogen peroxide concentration increases, the rate of diffusion increases and that my hypothesis is valid. The linear regression and the high R 2 value show that there is a positive correlation between the rate of reaction and the hydrogen peroxide concentration. However, it cannot be proved that the correlation is always directly proportional. When observing the first two data on 0.09875% and 0.1875%, there is no obvious increase and rather seems overlapping. Perhaps, this might be due to the fact that the hydrogen peroxide concentration was too low. On the other hand, 1.5% has a huge error bar, representing that the triplicate data were not consistent. Thus, it will not be taken into account for examining the trend for this investigation. Otherwise, the graph seems to be smoothly increasing. All data except 0.09875% show a legitimate positive correlation, because the vertical error bars to not overlap at all. The results tell that when the hydrogen peroxide concentration is high, the reaction rate increases. Thus, the results lead to the conclusion that the rate of reaction is directly proportional when the hydrogen peroxide concentration is greater than 0.09875%.

Evaluation Although the trials for 1.5% had a wide range, the experiment is justifiable because reliable triplicate trials were obtained. This is also reflected by the small vertical deviation on the Graph 2. The uncertainty of the hydrogen peroxide, shown by the horizontal error bar, varies with the concentration; higher concentrations have wider uncertainty than lower concentrations. Although it seems likely that there is a positive correlation according to the Graph 2, the linear regression does not pass through the origin of the graph, because based on the linear regression equation, the y-intercept is 0.00063%. This shows that both systematic and random errors were present.

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IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Since this experiment dealt with small amount of Catalase as well as the substrate hydrogen peroxide, small systematic errors led to large uncertainties. For instance, in preparing different concentrations of hydrogen peroxide, one extra drop of a solution could have altered the concentration to a great extent. Major errors could have been reduced if the experiment was conducted under larger system.

Limitations and Improvements Limitations Since the amount of Catalase was limited and small in quantity, only 10Âľl of Catalase was used for each trial to ensure enough is left for multiple trials. Although a micropipette was used, there was small amount of the enzyme solution left on the wall of the tip after releasing. Because only small amount of Catalase was used, this could have resulted in huge uncertainties in measuring the rate. Additionally, the entire substrate amount was reduced to 1.5ml. Thus, small errors yielded huge uncertainties. Since the test tube had to be manually capped with the gas pressure sensor, it inevitably included human error because of human reaction time. Thus, the initial rate might not be accurate.

Improvements

The whole system needs to be enlarged to reduce uncertainties. Thus, a greater amount of Catalase and hydrogen peroxide is needed. In larger scale experiments, small errors will be negligible and the results will be more reliable and justifiable.

To reduce human errors, more advanced apparatus has to be used. To obtain accurate data, the pressure has to be measured as soon as Catalase hits the surface of hydrogen peroxide, because the reaction starts instantaneously, even if the concentration is low.

The solutions were prepared by performing only serial dilutions due to time constraints and limited amount of Catalse. Since the To prepare more solutions of different different concentrations were obtained by concentration, more amount of hydrogen performing serial dilution, the concentration peroxide has to be used and enough Catalase decreased by half, lacking variety in must be present. A variety of solutions could concentrations. The results would have been be prepared by altering the percentages better if more solutions of different manually and not perform serial dilutions. concentrations between 0.4% and 1.5% were tested. Table 5 shows the limitations and the improvements

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IB Chemistry HL Name: Yoojin Lee Candidate Number: 002213-067

Bibliography 1

“Hydrogen.” Wikipedia, the freeencyclopedia. http://en.wikipedia.org/wiki/Hydrogen_peroxide(accessed October 22, 2010).

2

“The Hydrogen Peroxide Breakdown Examining Factors That Affect the Reaction Rate of Enzymes.” Alief Independent School Districts Vanguard.http://www4.alief.isd.tenet.edu/cahowe/biology/pak%202/The%20Hydrogen%20 Peroxide%20Breakdown.htm(accessed October 22, 2010).

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Hydrogen peroxide experiment  

Hydrogen peroxide experiment

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