Rate of Decomposition of Hydrogen Peroxide

Research Question
How does the source of the catalyst (potato, radish, carrot) and concentration of substrate (hydrogen
peroxide) affect the rate of reaction (measured by the % concentration of oxygen after 500 seconds) while
controlling temperature?
Introduction
One of the most important enzymatic reactions in the human body is the decomposition reaction of
Hydrogen Peroxide. The body makes catalase to break down Hydrogen Peroxide before it can form
hydroxyl radicals. One molecule of catalase breaks down close to hundred thousand molecules 𝐻2𝑂2 per

second. A build up of 𝐻2𝑂2 causes the cell membrane to disintegrate. Maintaining the level of hydrogen

peroxide in cells is also essential to cellular signaling processes. Catalase breaks down hydrogen peroxide
into 2 molecules of water and 1 molecule of oxygen. This reaction is shown in the image below.

The rate of reaction or decomposition of hydrogen peroxide depends on the number of collisions between
the catalase enzyme and substrate molecules. The speed and intensity of these collisions depend on
various factors, including temperature, PH, concentration of the enzyme or concentration of the substrate.
This paper will specifically look at the impact of substrate concentration on the rate of decomposition,
measured through the percentage concentration of oxygen released.
Background Information:
Enzymes are proteins that catalyze metabolic reactions in the body by lowering the activation energy
required for the reaction. They convert substrates into products using catabolic or anabolic processes. The
human proteome consists of 1300 enzymes that are used for processes ranging from DNA replication
using the polymerase enzyme to fat digestion using Lipase. Enzymes are globular proteins that have
specific active sites that make them well suited to their target substrate. The lock and key model and
induced fit model are both used to explain the relation between a substrate and an enzyme’s active site.
The first model states that both the enzyme and substrate geometrically fit into each other while the
second model proposes that chemical reactions between the substrate and active site result in a
conformational change in the structure of the active site.
 Table 1: Variables

Variable Type Variable How it will be Justification

manipulated/measured

Independent Variable Percentage Concentration 5 concentrations of H2O2 were Different concentrations of

of 𝐻2𝑂2 made by diluting hydrogen hydrogen peroxide show the
peroxide solution with distilled change in the rate of reaction with
water. Concentrations of an increase in substrate particles.
0%, 0.6%, 0.9%, 1.5%, 3% and
6% were made. This increment is acceptable
because they are short and
frequent

Independent Variable II Source of Catalase Catalase from potatoes, radish Different plant tissues have
and carrots was compared to varying levels of catalase. Further,
determine the most efficient this catalase may be more
organic catalyst. accessible based on the complexity
of the plant tissue. To find the
most effective source of catalase,
the 3 organic sources were
compared.

Dependent Variable Percentage Concentration Measured using the labquest The greater the percentage
of Oxygen software and oxygen sensor. concentration of oxygen, the
higher the rate of reaction after
500 seconds.

Derived Dependent 𝐻2𝑂2 decomposition rate Derived from the analysis of Oxygen concentration will be used
Variable (change in % the data from the oxygen as a measure of the rate of

concentration of oxygen in sensor decomposition. A high

Au/second) (Final % conc - Initial % conc) concentration of oxygen is

indicative of a high rate of
reaction.
 Table 2: Controlled Variables

Controlled Variable How it will be controlled Reason for variable to be constant

Temperature The experimental and controlled setup Temperature affects the dependent variable - rate of
were placed in a water bath heated to decomposition. High temperature may denature the
𝑜
37 𝐶 which is the optimum temperature catalase enzyme.

for both catalase and manganese dioxide.

Volume and 3
A fixed measure of 20 𝑐𝑚 of catalase A higher volume or concentration of the enzyme will
Concentration of the solution, extracted from 20 grams of increase the rate of reaction.
Enzyme potato, was added to each trial, keeping
the volume constant.

Volume of hydrogen 3
20 𝑐𝑚 solutions of each concentration Hydrogen Peroxide is the substrate. A higher
peroxide solution of Hydrogen Peroxide was used concentration of the substrate would account for a
higher rate of reaction because there are more
molecules to collide with and decompose.

Type of The potato extract solution will all be Differences in the species of the potato/carrot/radish
potato/carrot/radish used from Yukon Gold potatoes. used can cause a difference in the volume of the
to extract catalase enzyme catalase found in the plant tissue.
The carrot extract solution will be from
Touchon carrots.

The radish extract solution will be from

Daikon radishes.

Hypothesis: As the concentration of Hydrogen Peroxide increases, the percentage concentration of

oxygen after 500 seconds increases until the saturation point, after which the percentage concentration of
oxygen will remain constant and hence the rate of reaction.
Justification: As the concentration of 𝐻2𝑂2 increases, the number of particles in the solution increases.

This allows for a greater number of collisions between the catalase and 𝐻2𝑂2 molecules, therefore

increasing the rate of reaction. However the rate of reaction will increase at a slower rate with each
 increment of increase in the Hydrogen Peroxide concentration. It will eventually stop increasing at a point
in which the concentration of catalase becomes the limiting factor.
Null Hypothesis: This is no correlation between the change in concentration of Hydrogen peroxide and
percentage concentration of oxygen.
Alternative Hypothesis: There is a correlation between the change in concentration of hydrogen
peroxide and the percentage concentration of oxygen.

Table 3: Materials

Apparatus Reagents and Materials

- 1 Nutribullet PRO 900 Blender - 6% Hydrogen Peroxide solution - 500 𝑐𝑚

3

- 4 beakers - 200 cm3 (±10) - Distilled Water - 10 liters

- LabQuest Oxygen Probe (±0.01) - 10 Yukon Gold Potatoes
3
- 5 Measuring cylinders - 50𝑐𝑚 (± 5) - 8 Touchon carrots
- 12 test tubes - 8 Daikon radishes
- 2 Test tube racks
3
- 10 conical flasks (250 𝑐𝑚 )
- 1 Stopwatch (± 0. 01 seconds)
𝑜
- 6 Lab Thermometers (± 0. 1 𝐶)
𝑜
- 1 Water Bath (6 test tube slots) (± 0. 1 𝐶)

Methodology
Preliminary Experimentation
The preliminary experimentation that was undertaken before the actual experiment involved various
procedures to determine the ideal volume of
- Hydrogen peroxide solution
- Catalase solution
3
It was found in order to control the height reached by the foam, 20𝑐𝑚 of each solution has to be added to
a 250 cm3 conical flask. This would allow readings to be recorded without the foam damaging or
interfering with the sensor.
Optimum Method of Extraction (of catalase)
To find the most efficient way of extracting catalase without damaging the enzyme in the process, 3
methods of extraction were identified and compared. While the final data collection compared carrot,
potato and radish, the methods of extraction were performed only on potatoes and generalized for the
other sources.
1. The first method involved cutting 20 grams of the potato into 1x1 cubes where the surface area of
volume ratio of the cube determined the rate of decomposition.
2. The second method cut 20 grams of the potato into pieces and added them to a Nutribullet blender
with 20cm3 of distilled water. The solution was blended for 10 seconds.
3. The third method followed the second one, but ran the solution through a filter paper to eliminate
any solid pieces of the potato.
4. The fourth method involved grinding 20 grams of potato in a mortar and pestle to extract the juice
of the catalate.
𝑜
All four methods of extraction were compared by first setting a temperature equilibrium of 37 𝐶 between
all the solutions using a water bath. Catalase enzyme from each method was placed / poured into a conical
3
flask to which 20 𝑐𝑚 of 3% 𝐻2𝑂2 solution was added. The oxygen sensor was placed into the conical

flask and the percentage concentration of oxygen after 500 seconds was found for each method used.
Through this experiment it was found that method 4, using the mortar and pestle, extracted the catalase
most efficiently, without damaging the enzyme. However it was later found that this method of extraction
was highly time consuming, which caused the potato to darken and the catalase to denature. Hence the
final method of extraction used was a blender (method 2).

Collection of Data
Part A - Preparing the Hydrogen Peroxide solutions
A 3% working standard solution of 𝐻2𝑂2 was made from 6% 𝐻2𝑂2 and the subsequent concentrations of

the substrate solution were made from the 3% working standard solution. The working standard was
diluted using appropriate volumes of distilled water, to make 1.5% ,0.9% and 0.6% 𝐻2𝑂2 solutions.

Distilled water was used as a control for the experiment.

Part B - Preparing the Catalate Solutions
1. 20 grams of potato were cut into large pieces and placed into a blender.
3
2. 20 𝑐𝑚 water was added and the solution was blended for 10 seconds.
3. The same was done with carrot and radish to prepare the catalase solutions.
Part C - Setting up the experiment to measure rate of reaction
 3
1. 20𝑐𝑚 of 6.0% hydrogen peroxide was poured into 5 conical flasks and placed in a water bath of
𝑜
37 𝐶.
3
2. 20 𝑐𝑚 of catalase from potato was poured into 5 test tubes and placed in the water bath.
3. Once both solutions reached equilibrium, one test tube of catalase was poured into one conical
flask; the oxygen sensor was placed inside and data collection began.
4. The same was repeated for the other 4 test tubes of catalase.
5. Steps 1 - 4 were repeated using catalase from carrot and radish (step 2).
6. Steps 1 - 5 were repeated using hydrogen peroxide concentrations of 0%, 0.6%, 0.9% and 1.5%
and 3.0%.

Figure 1: Catalase extracted using different methods Figure 2: Foam seen after 500 seconds

Safety, Ethics and Environmental considerations:

A risk assessment was conducted for all the materials used. This included possible dangers of
setup/materials as well as methods of dealing with a compromised setup. Hydrogen peroxide is a known
irritant and hazardous substance. Any 𝐻2𝑂2 spill was cleaned with excess amounts of water. To decrease

risk, safety goggles, a lab coat, and gloves were used throughout the experiment. The environment was
considered in the minimal use of materials. Further, most of the materials used were organic and
biodegradable. Labware was reused wherever possible. Lastly, animal safety was protected by sourcing
catalase only from plants. Sources such as liver were not used due to the risk posed to animals in a
slaughterhouse, from where they are typically obtained. Research about the materials found the following:
Wash immediately, for around 15 mins, if it comes in contact with skin or eyes. [ORCBS – Hydrogen
Peroxide]. Catalase can cause “allergy or asthma symptoms or breathing difficulties if inhaled. Should
any be inhaled, remove yourself to fresh air.” In case of skin / eye contact; wash with water for at least 20
minutes and seek medical attention if any symptoms occur. [Safety Data Sheet Catalase].
 Qualitative Observations
The height of the foam after 500 seconds was higher for higher concentrations of Hydrogen Peroxide. The
height of the foam was highest when the carrot was used as a catalyst. When the potato solution was left
for an extended period of time, it decolourised. A decolourised solution led to the formation of small
amounts of foam. This decolorization was not seen in carrot or radish solutions.

Quantitative Observations
Raw Data Table (Table 6) - Initial and Final % concentrations of oxygen

Percentage concentration of oxygen (±0.01)

Concentration of
Hydrogen
Peroxide (%) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Initial Final Initial Final Initial Final Initial Final Initial Final

Source of Catalase : Radish

0 14.21 14.21 14.21 14.21 14.21 14.21 14.21 14.21 14.21 14.21
0.6 14.85 16.15 14.61 16.52 14.74 15.76 14.88 15.66 14.87 15.5
0.9 15 15.9 15.15 16.62 14.86 15.88 14.91 16.43 14.88 17.44
1.5 14.49 17.18 14.5 16.65 14.53 18.9 14.65 16.05 14.59 17.74
3 15 17.03 14.92 17.07 14.95 17.14 14.92 17.23 14.88 18.95
6 14.99 19.85 14.93 20.32 14.88 17.36 15.06 19.08 14.95 18.25
Source of Catalase : Potato
0 14.21 14.21 14.21 14.21 14.21 14.21 14.21 14.21 14.21 14.21
0.6 14.62 15.69 14.04 15.38 15.01 15.18 15.12 15.39 14.99 15.25
0.9 14.83 16.44 14.92 16.22 15.09 16.36 14.67 15.89 14.95 16.35
1.5 15.12 19.07 15.06 17.67 15.12 19.02 14.94 20.26 14.9 16.79
3 14.92 19.12 15.04 19.21 15.09 18.8 15 18.37 14.99 17.57
6 14.99 19.84 14.93 20.9 14.88 21.41 15.06 20.25 14.95 25.05
Source of Catalase : Carrot
0 14.21 14.21 14.21 14.21 14.21 14.21 14.21 14.21 14.21 14.21
0.6 15 28.35 14.89 31.18 16.17 29.93 14.8 23.74 15.33 32.21
0.9 18.25 28.38 14.36 26.93 16.83 29.17 14.46 28.41 14.7 31.87
1.5 14.95 32.21 18.66 32.21 15.06 32.21 15.12 32.21 15.16 32.21
3 14.86 30.8 15.25 32.21 15.12 32.21 16.24 32.1 14.86 32.21
6 15.87 32.21 17.8 32.21 16.34 32.21 15.23 32.21 15.32 32.21
 Data Processing:

Table 7 - Calculations

Calculation Formula Sample calculation

Change in percentage (Final % concentration) - (Initial % concentration) Carrot; 0.6 concentration; trial 1
concentration oxygen 32.21 - 15.87 = 16.34

Mean 𝑠𝑢𝑚 𝑜𝑓 𝑎𝑙𝑙 𝑣𝑎𝑙𝑢𝑒𝑠 𝑜𝑓 𝑐ℎ𝑎𝑛𝑔𝑒 𝑖𝑛 % 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 Carrot; 0.6 concentration;

𝑛𝑜. 𝑜𝑓 𝑡𝑟𝑖𝑎𝑙𝑠 13.35 + 16.29 + 13.76 + 8.94 + 16.88
No. of trials = 5 5
= 13.844

Standard Deviation Standard deviation was

2
∑(𝑥𝑖−𝑥) calculated using the STDEV
σ= 𝑁 function on excel.
Where σ is the standard deviation, 𝑥 is the mean
change in % concentration of oxygen, 𝑥𝑖 is each
value from data and N is the number of
samples/trials.

Processed Data (Table 8)

Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
Mean Change
% Concentration Change in % Change in % Change in % Change in % Change in % in %
of Hydrogen Concentration Concentratio Concentration Concentration Concentration Concentration Standard
Peroxide of Oxygen n of Oxygen of Oxygen of Oxygen of Oxygen of Oxygen Deviation
Source of Catalase : Radish
0 0 0 0 0 0 0
0.6 1.3 1.91 1.02 0.78 0.63 1.128 0.51
0.9 0.9 1.47 1.02 1.52 2.56 1.494 0.59
1.5 2.69 2.15 4.37 1.4 3.15 2.752 1.11
3 2.03 2.15 2.19 2.31 4.07 2.55 0.77
6 4.86 5.39 2.48 4.02 3.3 4.01 1.04
Source of Catalase : Potato
0 0 0 0 0 0 0 0
0.6 1.07 1.34 0.17 0.27 0.26 0.622 0.54
0.9 1.61 1.3 1.27 1.22 1.4 1.36 0.14
1.5 3.95 2.61 3.9 5.32 1.89 3.534 1.19
3 4.2 4.17 3.71 3.37 2.58 3.606 0.6
6 4.85 5.97 6.53 5.19 10.1 6.528 1.88
Source of Catalase : Carrot
0 0 0 0 0 0 0 0
 0.6 13.35 16.29 13.76 8.94 16.88 13.844 2.8
0.9 10.13 12.57 12.34 13.95 17.17 13.232 2.32
1.5 17.26 13.55 17.15 17.09 17.05 16.42 1.4
3 15.94 16.96 17.09 15.86 17.35 16.64 0.62
6 16.34 14.41 15.87 16.98 16.89 16.098 0.93

Analysis and Evaluation

The graph shows the relation between concentration of Hydrogen Peroxide and rate of decomposition
when catalyzed by catalase from potato, radish and carrot. The trend in this graph follows the general
pattern that the percentage concentration of oxygen increases with an increase in the concentration of
Hydrogen Peroxide. This shows a direct relationship. An increase in substrate concentration has led to an
increase in the enzymatic activity of catalase. An increase in the concentration of the substrate, increases
the number of molecules of 𝐻2𝑂2 present. This increases the probability of successful collisions between

the enzyme’s active site and the substrate molecule, increasing the rate of reaction.
In the graph for Potato, the mean % concentration of oxygen (for the 𝐻2𝑂2 concentration 1.5 %) deviates

from the general trend of values. This can be attributed to the anomalies found in trial 4 (% concentration
of oxygen = 5.32) and trial 5 (% concentration of oxygen = 1.89). Similarly, anomalies are noticed in the
graph for radish in trial 3 (% concentration of oxygen = 4.37) and trial 4 (% concentration of oxygen =
1.4) for the same concentration of 𝐻2𝑂2. This leads to a standard deviation of 1.11. While the graph for

carrot follows a similar trend initially, the enzyme active sites become saturated at the 𝐻2𝑂2 concentration

0.9. After this point, an increase in the concentration of Hydrogen Peroxide has little effect on the %
concentration of oxygen because the volume and concentration of the catalase enzyme becomes the
limiting factor.

At low concentrations of hydrogen peroxide with excess of enzyme and inadequate substrate, the reaction
is limited from substrate availability, resulting in a low percentage concentration of oxygen produced after
500 seconds. For carrot as a source of catalase, the percentage concentration of oxygen after 500 seconds
increases, until the substrate is completely used up. This point is the saturation point where the graph
plateaus and will not show a positive increase without an increase in catalase volume or concentration.
At low concentrations (0.6%, 0.9% and 1.5%) of hydrogen peroxide the graphs for potato and radish
show an increase at the beginning of the experiment. However, a positive trend continues for higher
substrate concentrations and the graph doesn’t plateau as it does for the catalase from carrots. This can be
attributed to the free active sites that slow the efficiency of the reaction. However, the graph would
eventually plateau if the experiment was conducted for a longer duration. Error bars are a graphical
representation of the variability of data. The short error bars on the graph show that data is both precise
and accurate.
This experiment supports the hypothesis. There is indeed a positive relationship between the
concentration of Hydrogen Peroxide and the percentage concentration of oxygen. However graph 1 and 2
don’t plateau as predicted in the second part of the hypothesis. It is certain that they would plateau if the
experiment was conducted for a longer duration of time. Due to this, the alternative hypothesis is
confirmed and the null hypothesis is refuted.
This conclusion is supported by Sam Adam Day, who stated that “Increasing Substrate Concentration
increases the rate of reaction. This is because more substrate molecules will be colliding with enzyme
molecules, so more product will be formed.” (Day, 2019). This increases the rate of decomposition at
higher concentrations of Hydrogen Peroxide is seen through the graph.
Strengths
The apparatus used for this experiment was precise and reduced the chance of human error. An oxygen
probe was used as opposed to a gas syringe or water displacement setup, which has greater scope for
human error. Measuring cylinders (instead of beakers), graduated pipettes (instead of droppers), a high
precision laboratory scale and a stopwatch was used to make the apparatus accurate and appropriate. All
equipment was thoroughly washed before trials and different measuring equipment were used for
hydrogen peroxide and different sources of catalase. All possible controlled variables (Table 2) were
considered before conducting the experiment, in order to minimize chances of error and ensure that a
change in the dependent variable was due to the independent variable. In terms of reliability, the data is
reliable because 5 trials were conducted for each concentration of hydrogen peroxide. A mean was taken
for each concentration to increase reliability of the data. Temperature was strictly regulated using an
electrostatic water bath. Thermal equilibrium was established by ensuring the contents in the test tube
were the same temperature as the water bath (digital thermometer).
Limitations and Improvements
Some volume of catalase was lost during transfer from the measuring cylinder to the test tube. This
affected the volume of the substance used in each trial. This could have been improved by using different
measuring equipment for each trial or thoroughly washing and drying the equipment between trials.
There was also a time lag in pouring in the catalase and placing the oxygen sensor to begin data
collection. Given the rapid nature of the decomposition reaction, the measurement of the change in %
concentration of oxygen could have been affected. This could be improved by placing the sensor in
𝑜
immediately after mixing the solutions. Further, while all the solutions were at 37 𝐶 when combined to
measure the rate, the time they were removed from the water bath varied. This is because all the solutions
were placed in the water bath at roughly the same time. However some stayed longer while the
experiment was performed on others. This prolonged exposure could have reduced the activity of the
catalase enzyme, accounting for the difference in readings for the trials. This error can be minimized by
placing each test tube in the water bath for a fixed duration of incubation, after which, the next test tube
can be placed for the same amount of time.
Another limitation takes form in the starting oxygen concentration for each trial. When the oxygen probe
was placed from the conical flask of one reaction to the other, it wasn’t given enough time to equilibrate
to the environmental oxygen concentration. This could have been improved by allowing a gap of 5
minutes before each trial. This fluctuation in initial concentration of oxygen leads to high values for
standard deviation.
Lastly, the catalase was extracted from each source using a ‘Nutribullet’ blender. However, the rapid
motion of this blender released heat which could have damaged some of the catalase enzymes. This could
have been avoided by using a cold press machine.
Extensions
All the sources of catalase in this experiment come from root vegetables, other groups could be explored
to extend the research. Further, the range of concentrations of H2O2 could be increased to incorporate a
larger number of increments and better model the curve shown in the graph. To further this research from
modeling bodily reactions, the findings can be applied to larger industries. The rate of decomposition of
hydrogen peroxide from using an organic catalyst (catalase from carrot) can be compared with that of an
inorganic catalyst (manganese dioxide). This will explore the feasibility of replacing a polluting inorganic
catalyst with a more sustainable alternative. A large difference between the rates of reaction would be a
deterrent in the replacement of inorganic catalysts.

Conclusion
After comparing the three sources of catalase, it can be concluded that the percentage concentration of
oxygen after 500 seconds is positively correlated with an increase in the concentration of hydrogen
peroxide. Further, it was found that catalase extracted from carrots has the highest rate of reaction. This is
shown by the rapid increase in the percentage concentration of oxygen with a small increase in the
concentration of the substrate. This is because carrot tissue contains a higher concentration of catalase
enzymes.

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