Section 1

Duaa Raja

Chemistry Investigation

Table of Contents
Introduction........................................................................................................... 3
General information........................................................................................ 3
Natural processes and manufacture................................................................3
Uses................................................................................................................. 3
Collision theory...................................................................................................... 4
Pressure................................................................................................................. 4
Surface Area.......................................................................................................... 4
Catalysts................................................................................................................ 5
General info..................................................................................................... 5
Types of catalysts............................................................................................ 5
Enzymes (catalase)................................................................................................ 6
General function.............................................................................................. 6
Structure of catalase....................................................................................... 6
Function of catalase........................................................................................ 6
Possible Mechanisms....................................................................................... 7
Mechanism of catalase with H2o2.....................................................................7
Inhibition......................................................................................................... 8
pH.......................................................................................................................... 8
Temperature.......................................................................................................... 9
Arrhenius Equation.......................................................................................... 9
Example........................................................................................................ 10
Concentration...................................................................................................... 11
General.......................................................................................................... 11
Michaelis-Menten Kinetics.............................................................................11
Lineweaver-Burk Plot..................................................................................... 12
Rate equations..................................................................................................... 13
Aims..................................................................................................................... 14
Methods of making solutions...............................................................................15
Hydrogen Peroxide solution...........................................................................15
Catalase solution........................................................................................... 16
Potassium (IV) Manganate.............................................................................17
Methods of measuring rate..................................................................................18
Gas syringe................................................................................................... 19
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Suba seal gas syringe.................................................................................... 20

Inverted burette............................................................................................ 21
...................................................................................................................... 22
Titration......................................................................................................... 23
Risk Assessment.................................................................................................. 25
Bibliography......................................................................................................... 31
Introduction................................................................................................... 31
List of Figures................................................................................................ 32
List of Equations............................................................................................ 33
Methods......................................................................................................... 33
Risk Assessment............................................................................................ 34

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Introduction
General aims: I will investigate the kinetics of the decomposition of hydrogen
peroxide with the enzyme catalase experimentally by varying the concentration
and temperature.
General information
The equation for the decomposition of hydrogen peroxide with catalase:

2H2O2(aq)

catalase
2H2O(l) + O2(g)

equation 1

As illustrated in figure 1, hydrogen peroxide is a

compound of hydrogen and oxygen (it can be thought of
as water with an extra oxygen atom). Pure anhydrous
hydrogen peroxide is a colourless liquid that rapidly
decomposes into oxygen and water. The bond between
two oxygen atoms of the molecule is very weak, so the
molecule can very easily break down, making it reactive
and a powerful oxidising agent. [1]

the

Figure 1
Natural processes and manufacture
The human body produces hydrogen peroxide in the Shape of hydrogen peroxide
immune system. It is produced naturally as by-product of oxygen metabolism.
Most living beings have peroxidases enzymes that decompose small amounts of
hydrogen peroxide present into water and oxygen to prevent tissue damage
because it poisonous. Hydrogen Peroxide can
be produced indirectly by hydrogenation and
oxidation of ananthraquinone.[2] A direct
method
of
hydrogenation
not
only
hydrogenates O2 in H2O2 but also converts Figure 2
H2O2 into water.
Production of Hydrogen Peroxide by
Anthraquinone Process

Uses
Hydrogen peroxide is used to treat waste water and sewage from industrial and
domestic sources and for detoxifying organic pollutants in the environment. It is
also used as a commercial disinfectant and antimicrobial agent. Since it is
extremely reactive it is used as a component in some types of rocket fuel. As a
disinfectant, hydrogen peroxide has been shown to be generally effective with
very safe by-products. [3] At low concentrations (around 3-6%) it is used in
peroxide-based hair dyes. [4]
The rate of decomposition is dependent on the temperature and concentration of
the peroxide, as well as the presence of impurities and stabilizers. The use of a
catalyst (such as manganese dioxide, silver, or the enzyme catalase) increases
the rate of decomposition. [5]

Page | 3

Collision theory
Molecules need to collide for a reaction to take
place. But not all collisions will result in a reaction.
Figure 3 demonstrates the orientation in which the
molecules must be in order for them to react for
that particular reaction. [6] Activation energy is the
energy the reactants need to overcome the
repulsive electron forces between approaching
molecules and to break the existing bonds in the
reacting molecules.
A decomposition is an example of a unimolecular
reaction. This is where a single reactant molecule
transforms into one or more products. The rate at which a substance
decomposes depends on its concentration and it is Figure 3
often first order. The reaction I am investigating is
exothermic as heat is released and the reactants have more energy than the
products as shown in figure 4.
The rate of a chemical reaction can be changed by:

the pressure of the system (if some

reacting molecules are a gas)
pH
surface area (if some reactants are in solid
phase)
adding a catalyst
the temperature
the concentration of catalase (catalyst) and
hydrogen peroxide (substrate)

Pressure

Figure 4
The higher the Ea of a reaction
the smaller the amount of
energetic collisions present and
the slower the reaction

Increasing the pressure on a reaction increases the rate of

reaction only when the reactants are gaseous. Pressure is
usually increased by reducing the volume. If pressure is
increased then more particles are present in the same volume and there will be a
higher collision frequency leading to an increased rate of reaction. I will not need
to take the effect of pressure into account as the reactants are not gases but I
could represent the rate as the change in pressure as oxygen is produced.

Surface Area
Increasing the surface area by cutting it into smaller pieces or grinding it into a
powder will increase the rate of reaction. This is because more particles are
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exposed to reactant, therefore there are more frequent collisions and rate
increases. Surfaces area is not something I need to consider in my experiments
as I will be working with liquids.

Catalysts
General info
Catalysts are compounds that increase the reaction rate by providing an
alternative pathway for the reaction with lower activation energy. They will have
no effect on the energetic characteristics of the hydrogen peroxide and catalase
and the barriers between them.
The amount of a catalyst does not change during a reaction, it is not consumed
as part of the reaction process. Catalysts do not affect the equilibrium state. [7]
This is because it speeds up both the forward and backward reaction by the
same amount. If both reaction rates are increased to the same extent, only the
rate at which equilibrium is reached is increased.
Types of catalysts
Catalysts can be categorized into two types: homogeneous and heterogeneous.
Homogeneous catalysts are those which exist in the same phase as the
reactants, while heterogeneous catalysts are not in the same phase as the
reactants.
Normally, heterogeneous catalysis involves the use of solid catalysts placed in a
liquid reaction mixture. Homogeneous catalysis means more effective mixing of
the catalyst with the reaction mixture; heterogeneous catalysis allows recovery
of expensive catalysts, which is useful for manufacturing processes. [8]

Homogeneous
Figure 5

A two stage reaction profile for a

catalytic cycle (Ea = activation energy)

Ea1:activation energy thats leads

to an intermediate complex
forming.
Ea2:activation energy for the
changing of the intermediate into
products.
Ea3:activation energy of
uncatalysed reaction. [9]

the

Heterogeneous
Figure 6

The reactants are adsorbed on to

the surface of the catalyst. This is
a chemical reaction as there is an
interaction between the electrons
of the reactants and the atoms on
the catalyst
The adsorbed reactants are free to
migrate over the surface of the
catalyst
When the reactants meet they are
free to react but still attached to
the surface
Finally the products of the reaction
are desorbed from the surface
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allowing them to move away and
freeing up area for more reactions

Enzymes (catalase)
General function
Catalase is an example of a biological catalyst; it increases the rate of reaction
without being changed or depleted itself. It is a protein; it has a very precise
three-dimensional shape, which forms one specific active site on the enzyme
where molecules. Each enzyme can only convert one kind of substrate molecule
into one kind of product molecules.
The basic mechanism by which catalase catalyse the reaction begins with the
binding of the hydrogen peroxide to the active site on catalase. The active site is
the specific region of the catalase which combines with the H2O2 molecule. The
binding of hydrogen peroxide to catalase will cause changes in the distribution of
electrons in the chemical bonds of the catalase and cause the reaction that leads
to the formation of oxygen and water. These are released from the catalase
surface to regenerate the enzyme for another reaction cycle.
The enzyme is the lock and so hydrogen peroxide
is the key. Only the correct key/substrate fits into
the keyhole or the lock/active site. The active
site of the enzyme has a unique shape that is
complementary to the shape of the hydrogen peroxide
molecule. Enzymes specifically react with only one or a
very few similar compounds as shown by the lock and
key model in figure 7.
Figure 7

Structure of catalase
Catalase is a tetramer (a protein with four subunits) of four polypeptide chains,
each over 500 amino acids long [11] as shown in figure 8.

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Function of catalase
The catalyst I will be using for this investigation
is catalase which is a homogeneous catalyst. It is
made naturally in almost all living organisms and
are important to life as it helps the body break
down hydrogen peroxide (a powerful and harmful
oxidizing agent) and prevents the accumulation
of carbon dioxide bubbles in the blood. Catalase
is very effective; one molecule of catalase can Figure 8
decompose millions of hydrogen peroxide
molecules. It also uses hydrogen peroxide to
oxidize
potentially
harmful
toxins
like
formaldehyde, formic acid, alcohol, and phenol [10] from occurring in the body.
The optimum pH for catalase is roughly 7 as this usually the pH of the cells
where catalase carries out its functions. While pH can vary by species, the
catalase I will be using is derived from bovine liver which is also roughly neutral.

Possible Mechanisms
Enzymes can catalyse reactions through a range of mechanisms. Some of these
include:

Bond strain: induced structural rearrangement that take place with the
binding of substrate and enzyme produce strained substrate bonds that
can easily reach the transition state
Proximity and orientation: causes the enzyme-substrate interaction to
orient reactive groups in a way that bring them into close proximity with
one another
Proton donors and acceptors: the presence of acidic or basic groups can
affect bond polarization and reaction speed (the acid is often a donor
whereas the base is an acceptor)
Electrostatic catalysis: side chains or cofactors (non-protein part) can
stabilize the charge on the transition state
Covalent catalysis: the substrate is oriented to the active site in such a
way that a covalent intermediate forms between the enzyme and
substrate. The covalent complex is more reactive than the substrate itself
originally was. [13]

Mechanism of catalase with H2o2

The protein has a precise 3D structure when it is active, which contains a
channel into which the hydrogen peroxide can diffuse. In the channel is a heme
group which is an iron molecule bound to the centre of a ring-like structure called
Page | 7

a porphyrin ring. The heme group in catalase is important to the reaction,

because it can be oxidized from Fe(III) to the less common Fe(IV) form. This is the
first part of the reaction:

H2O2 + Fe(III) H2O +O=Fe(IV) equation 2

Hydrogen peroxide has bound to the heme group and oxidized it to Fe(IV).
The enzyme goes back to the Fe (III) form by reducing the second molecule of
hydrogen peroxide to water.

H2O2 +O=Fe(IV)H2O+Fe(III)

equation 3

The highly-oxidizing Fe(IV) reacts with the second peroxide molecule, releasing
water and an oxygen molecule. [12]

Figure 9

Summary of equations 2 and 3 to show how the

overall equation of H202 decomposition is
derived

Most enzyme reactions have optimum conditions due to irreversible damage

(denaturation) done by pH and temperatures as shown by the graphs below:

Figure 10

Figure 11

This means there is a limited range of temperatures I can use because the
reaction may be too slow to measure at lower or higher temperatures. Lower
temperatures mean the molecules will not have enough kinetic energy to result
in successful collisions and then reactions. However, approaching high
temperatures, the reaction rate will start to decrease as the enzymes denature
and cannot break down hydrogen peroxide to produce oxygen and water.
Similarly, changes in the pH can limit the reaction so I need to ensure the pH is
Page | 8

kept constant at the optimum value in my experiment. Any deviations around the
optimum will cause the catalase to denature and rate of reaction to decrease.
Inhibition
Enzyme inhibitors can reduce or completely inhibit the enzyme catalytic activity
either reversibly or permanently. Inhibitors can block or modify the active site
which means that the substrate is prevented from being broken down. Those that
have similar structure to the substrate and occupy the active site are called
competitive inhibitors and those that attach to other parts of the enzyme
molecule and distort the shape (and therefore active site) are non-competitive.
For catalase, copper sulphate is a non-competitive inhibitor. Cyanide is a
competitive inhibitor because it binds to the active site and prevents an enzymesubstrate complex forming with catalase and hydrogen peroxide. Since cyanide
is very toxic, another way the reaction can be stopped is by the use of
concentrated sulfuric acid which lowers the pH so much that all of the catalase
molecules become denatured and further decomposition is halted. I will use
copper sulphate, sulphuric acid and ethanol [14] to stop the reaction at certain
points so the concentration of hydrogen peroxide at that point can be measured.

pH
This is the measure of the acidity or concentration of hydrogen ions in a solution.
As catalase is an enzyme (and therefore a protein made up of amino acids with
-NH2 and -COOH groups on side chains) the balance of OH- and H+ ions influences
the shape of the active site so that it is most complementary to the shape of
hydrogen peroxide. If the ionization state of these amino acids is altered, the
bonds that create the shape of the enzyme will be altered and the substrate will
not bind to the active site and be catalysed. Different enzymes have different
optimum pH. In this investigation I will not be measuring pH because there will
be too many variables to consider and there will be too many calculations.
However it can be kept constant using pH buffer solution. A pH buffer

Temperature
An increase in temperature causes a rise in the energy of the molecules involved
in the reaction. As a result the rate of the reaction increases and conversely the
rate of reaction will decrease with a decrease in temperature.
Figure 12

Where temperature of T2 > T1

Increasing the temperature will make
the peak move lower and to the right.
The area under the curves should be
equal as the number of particles are
the same. Increasing the temperature
will increase the rate as a greater
proportion of particles will have the
minimum activation energy.

Page | 9

In enzyme controlled reactions when the temperature is too high the bonds
(especially hydrogen bonds) break and this causes the shape of the enzyme to
change. This causes the active site to change shape and the enzyme can no
longer accept substrate molecules. I cannot carry out the experiments at higher
temperatures as the enzyme denatures the rate may be too small to measure.
Arrhenius Equation
The Arrhenius equation can be used to show the effect of a change of
temperature on the rate constant and therefore the rate of the reaction.

Figure 13

k is the rate constant

A is known as the pre-exponential factor.

It is the fraction of molecules that would
react if the activation energy was above
0, or if the kinetic energy of all
molecules are above Ea

Ea is the activation energy

This is the minimum energy needed for

the reaction to occur. To fit this into the
equation, it has to be expressed in

The Arrhenius equation can be written in a non-exponential form which will be

more convenient to use and interpret graphically. This equation is of a straight
line (y=mx+c).

ln k =

Ea 1
+ ln A
R T

equation 4

I will need to determine the rate constant (page 13 shows how) while changing
the temperature and plot a graph of

1
T

against ln k. Measuring the gradient

Page | 10

will then give

E a
, which will give me the activation energy for the reaction.
R

[15]
Alternatively, the activation energy can be found algebraically by using two rate
constants and two corresponding reaction temperatures but it is less accurate.

ln

k 1 Ea 1
1
=

k2
R T1 T2

equation 5

Example
The rate constant for the reaction H2(g) + I2(g) 2HI(g) is 5.4 x 10-4 M-1s-1 at 326 oC.
At 410 oC the rate constant was found to be 2.8 x 10-2 M-1s-1.
We can calculate the activation energy using the integrated form of the
Arrhenius equation as we know the rate constant for the reaction at two different
temperatures. 326oC is 599K and 410oC is 683K.

ln

5.4 x 104 mol1 s1 E a 1

1
=
K
K
4
1 1
R
599
683
2.8 x 10 mol s

3.9484=

E a
( 2.053 x 104 K 1)
R

Ea =1.923 x 104 K x 8.314 J /mol k

Ea =1.60 x 105 J / mol

Concentration
General
More collisions taking place means there will be more successful collisions and
this will lead to a faster reaction rate. When more hydrogen peroxide molecules
are added, more enzyme-substrate complexes can be formed and the rate of
reaction increases. Eventually, increasing this concentration further will have no
Page | 11

effect because the active sites will be full and so no more enzyme-substrate
complexes can form. As the catalase concentration increases, there will be more
active sites available and the reaction can happen faster. However there will
come a point where increasing its concentration beyond a certain point will have
no effect as the hydrogen peroxide concentration will become the limiting factor.
Michaelis-Menten Kinetics
The rate of the catalase reaction follows one of the general equations for
enzyme-catalysed reactions, the Michaelis-Menten equation. The rate of the
catalase reaction with hydrogen peroxide is:

reactionrate V 0 =

V max ( concentration of hydrogen peroxide )

K m + ( concentrationof hydrogen peroxide )

equation 6

V0 is the inital velocity of the reaction

Vmax is the maximum rate of the reaction
Km, the Michaelis-Menten constant is the concentration of the substrate at which
the rate of the reaction is one half the Vmax. It can also be thought of as a
measure of how well the substrate complexes with an enzyme (known as its
binding affinity). An equation with a low K m value shows a large binding affinity,
as the reaction will reach Vmax quicker. [16]
Km is also the concentration of the substrate in mol dm -3 which produces a
reaction rate half of Vmax.
The Michaelis-Menten equation shows that the reaction rates for enzyme
reactions saturate - they reach a maximum reaction rate (V max), and stay at that
maximum reaction rate regardless of how much more hydrogen peroxide is
added beyond that point.
Figure 14

Vmax is approached asymptotically as

concentration of substrate increases.
It is the maximum rate at which an
enzyme catalyses a reaction and
expressed as the amount of product
formed per minute.
A large Km requires high substrate
concentrations to achieve maximum
reaction velocity. This means the
enzyme isnt as effective

Lineweaver-Burk Plot
Simply graphing rate and substrate concentration would not give V max (therefore
Km) as it is an asymptote. Its value can only be certain if the reaction takes place

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at an infinite concentration of substrate which is impossible to do to

experimentally.
The Lineweaver-Burk double reciprocal plot rearranges the Michaelis-Menten
equation into a linear form:

K
1
1
1
= m .
+
V V max [S] V max

equation 7

Figure 15

Plotting 1/v against 1/[S] gives a

straight line where:

y intercept is 1/ Vmax

gradient is Km / Vmax

x intercept is -1/ Km

Rate equations
The order of an equation is what the concentration of a substance is raised to in
the rate equation. The greater that number, the greater effect it will have on
rate. The order of a reaction must be determined experimentally.
Page | 13

Figure 16

The concentrations of A and B have to

be raised to some power to show how
they affect the rate of the reaction.
These powers are called the orders of
reaction with respect to A and B.

Chemical reactions are categorized on the basis of kinetics as zero-order, firstorder, second-order:

Zero-orderthere is no effect on the initial rate of reaction r=k (ms -1)

First-order the initial rate of reaction doubles r=k[A] (s-1)
Second order the initial rate of reaction quadruples or r=[A][B] or r=k[A]2
(m-1.s-1)

However these are the general units and will vary on what method is used to
measure A and B.
The order of a reaction can be established from a concentration-time graph but it
is sometimes difficult to
distinguish between first and
second orders. A rateconcentration graph like
those shown in figure 17 will
show the order with respect to
the reactant much more
Figure 17
clearly.
In rate laws, k is the rate constant of the reaction. The value of k can be
considered constant for that reaction under those conditions. The rate constant
is constant for a given reaction only if the concentration of reactants is being
changed. However, k can vary based on conditions such as temperature, ionic
strength and the presence of catalysts. [17]
k can be worked out by rearranging the rate equation when the rate orders with
respect to both catalase and hydrogen peroxide are known:

Page | 14

B
a
A

rate
k=

Figure 18 shows how straight line plots can be used to determine the rate
constant depending on the order.
Zeroth
Second Order

First Order

Figure 18

The general rate law for hydrogen peroxide decomposition and catalase is k
[H2O2] [catalase]. The reaction is first order with respect to H2O2 and catalase as
well. The overall order of a reaction is given by (a + b) so the reaction is second
order overall.
To show this I will need to change the concentration of hydrogen peroxide (whilst
keeping the concentration of catalase constant) and measure by how much the
initial rate increases or decreases. I can then determine the order with respect to
hydrogen peroxide. The same can be done for varying concentrations of catalase
to find the order and hence k.
Page | 15

Aims
From my research I have found how rate constants and orders can be
determined. By carrying out the experiment with an appropriate method and
finding the rate at various concentrations to find order and then k for each
temperature. This will allow me to use the Arrhenius equation which is
rearranged into a linear form where I can calculate the activation energy as the
gradient is (

E a
) where R is the gas constant.
R

To find Vmax, the velocity at different hydrogen peroxide concentration should be

found. I will be able to plot initial velocity against substrate concentration which
should give me the characteristic hyperbolic Michaelis-Menten curve.

Methods of making solutions

Hydrogen Peroxide solution
I will need to make accurate solutions of various concentrations of hydrogen
peroxide. The concentration of hydrogen peroxide is given as the volume of
oxygen it produces. 1cm3 of 100 vol should produce 100cm 3 of oxygen at STP.
A 20 vol concentration of hydrogen peroxide is equal to 1.7 mol dm-3. [1]
Various concentrations can be prepared as shown on the following table:
Concentrati
on (vol)
20
15
10
5
4
2
1

Volume of 20 vol
hydrogen
Peroxide (cm3)
250.0
187.5
125.0
62.5
50.0
25.0
12.5

Volume of distilled water

(cm3)
0.0
62.5
125.0
187.5
200.0
225.0
237.5
Page | 16

Equipment

20 vol hydrogen peroxide

Distilled water
Pipette filler
250cm3 volumetric flask and stopper
10cm3 volumetric pipette
25cm3 volumetric pipette
10cm3 measuring (mohr) pipette
Pasteur pipette
Funnel
Labels

Method
1. Measure required volume of 20 vol hydrogen peroxide with volumetric or
measuring pipettes
2. Using funnel make sure all of it is transferred into 250cm 3 volumetric flask
by washing out with distilled water if needed
3. Add appropriate volume of distilled water to volumetric flask so meniscus
is aligned. Use a pasteur pipette for the last few drops.
4. Replace stopper and turn bottle upside down so the solution is thoroughly
mixed
5. Label with appropriate concentration of hydrogen peroxide

Catalase solution
Concentration (%)
100
80
60
40
20
Equipment

Volume of 100% catalase

solution (cm3)
100
80
60
40
20

Volume of distilled water

(cm3)
0
20
40
60
80

100cm3 volumetric flask

Pipette filler
250cm3 beaker
25cm3 volumetric pipette
10cm3 volumetric pipette
Pasteur pipette
Stirring rod
Solid catalase granules
Distilled water
Scales
Funnel
Labels
Page | 17

Method [2]
1. Place beaker onto scale, zero the reading and measure out 100g of
catalase
2. Pour some water in beaker and dissolve as much as possible with
stirring rod
3. Transfer all of this into a volumetric flask, wash beaker with more water
to get all into flask
4. Fill with water so meniscus is on calibration line. Use a pasteur pipette
for the last few drops
5. Replace stopper and turn bottle upside down several times to mix
solution
This is the 100% solution so to make the others I will need to:
6. Measure out with pipette chosen volume of 100% solution into
volumetric flask
7. Add distilled water until line is on meniscus
8. Replace stopper and invert several times
9. Label with appropriate concentration of catalase

Potassium (IV) Manganate

Page | 18

Methods of measuring rate

The equation for decomposition of hydrogen peroxide with catalase is:

2 H 2 O2 2 H 2 O+O 2

equation 1

The rate of reaction is usually shown as the change in a property of reactant or

product per unit of time.

rate=

change product /reactant

time

equation 2

There are several ways to measure the rate of reaction:

Products

the volumes of gases (O2) produced

changes in pressure (oxygen produced in a closed system creates
increasing pressure as reaction happens)
Reactants
Page | 19

temperature change (as this decomposition is exothermic)

change in mass (as one of the product is oxygen gas)
pH (pH would drop if one of the reactants is an acid)
concentration of H202 (this would drop as it converted into water and
oxygen while reaction progresses)

Methods I can use for my investigation are:

Gas syringe
This is perhaps the easiest way to measure gas production. A gas syringe will
measure the volume of oxygen produced from the reaction after catalase is
added.
Equipment

Clamp stand
Water bath
100cm3 gas syringe
250cm3 Bchner flask
10cm3 measuring (Mohr) pipette
Rubber bung
Pipette filler
Rubber tubing
Stopwatch
Hydrogen peroxide solution
Catalase Solution

Method
Page | 20

1. Clamp gas syringe horizontally to stand

2. Measure with measuring pipette 5cm 3 of hydrogen peroxide solution into
Bchner flask
3. Connect tube to side arm and the other end to gas syringe
4. Carefully place flask into the water bath and leave for 1 minute
5. Pour catalase solution using measuring pipette into flask and quickly place
bung onto flask
6. Swirl mixture and start stop clock
7. Record the volume of gas at 10 second intervals for at least 3 minutes or
until reaction stops
8. Clean conical flask and repeat 2-6 three more times with same
concentrations of hydrogen peroxide and catalase
9. Repeat three times but with different hydrogen peroxide and catalase
concentrations

Diagram:

Bchner
flask
closed
with
bung
containi
ng H2O2

Clam

Gas

Rubber
tubing
Stopwatch

Wate
r
Suba seal gas syringe
Equipment

Clamp stand
Gas syringe
10cm3 syringe
Hypodermic needle
250cm3 Bchner flask
Suba seal
Stopwatch
Water bath
5cm3 volumetric pipette
Pipette filler
Hydrogen peroxide solution
Catalase solution

Method
1. Clamp gas syringe horizontally
2. Connect rubber tubing to side arm
Page | 21

Use volumetric pipette to put 5cm3 of hydrogen peroxide in the flask

Place suba seal on Bchner flask
Carefully place flask in water bath and leave for 1 minute
Fill syringe with catalase solution
Place syringe through suba seal and push down so all of the catalase is
transferred to flask making sure it is thoroughly mixed by swirling
8. Start stop clock and record the volume of gas in gas syringe at 10
second intervals for at least 3 minutes or until reaction stops
9. Wash out, dry flask and repeat 3-8 two more times with same
concentrations of hydrogen peroxide and catalase
10.Repeat three times but with different hydrogen peroxide and catalase
concentrations
3.
4.
5.
6.
7.

Diagram:

Bchner
flask
containi
ng H2O2
closed
with
suba

10cm3
syringe
filled with
Rubber
tubing

Clam
Gas
Stopwatch

Inverted burette
Watewill measure the volume of O produced from the reaction in an inverted
This
2
r
burette
which will displace the water inside of it.
Equipment

Clamp stand
Burette
Bowl/plastic tub
Water bath
10cm3 measuring pipette
25cm3 volumetric pipette
Pipette filler
250cm3 Bchner flask
Rubber bung
Rubber tubing
Stopwatch
Hydrogen Peroxide solutions
Catalase solutions

Method
1. Fill burette with water and invert it into tub of water.

Page | 22

2. Secure with clamp and make sure there is space for water to displace.
Release tap to remove any air bubbles and lower water where initial
measurement can be made.
3. Measure out hydrogen peroxide solution (based on following table as
burette can only hold 50cm3) in volumetric/measuring pipette and pour in
Bchner flask
Concentration (vol)
Maximum volume (ml)
20
2
15
3
10
5
5
10
4
12
2
25
1
50
4. Attach tube to side arm and put the other end under water in burette,
making sure it is secure
5. Carefully place flask into water bath
6. Use measuring pipette to transfer catalase solution into Bchner flask and
quickly replace bung while swirling
7. Start stop clock when first bubble appears
10.Record the volume at 10 second intervals for at least 3 minutes or until
reaction stops
11.Wash and dry conical flask, refill burette and repeat 1-9 two more times for
same concentrations
12.Repeat three times but with different hydrogen peroxide and catalase
concentrations

Diagram:
Bchner
flask
closed
with
bung
containi
ng H2O2

Inverted
burette
filled with
water

Tap
is

Clam
Rubber
tubing

Stopwatch

Wate
r

Tub filled
with

Page | 23

Titration
I can find the exact concentration of hydrogen peroxide by a redox titration with
potassium manganate (VII) in presence of an acid. [3]

2MnO4- + 5H2O2 + 6H+ 2Mn2+ + 5O2 + 8H2O

equation 3

By adding an inhibitor at certain times to stop the catalase from catalysing

hydrogen peroxide, I can titrate that sample to determine the concentration of
H2O2 at different times and therefore the rate.
Equipment

Clamp stand
2 burettes
Water bath
Funnel
2 25cm3 volumetric pipette
10cm3 volumetric pipette
10cm3 measuring pipette
Conical flask
3 pipette fillers
500cm3 beaker
6 100cm3 beakers
Page | 24

Hydrogen peroxide solutions

Catalase solutions
Potassium permanganate solution 0.02M
Copper(II) sulphate solution
Ethanol
Concentrated sulfuric acid
Stop clock
White tile
Labels

Method for determining most effective inhibitor using gas syringe:

1.
2.
3.
4.
5.
6.
7.

Measure out 20cm3 of hydrogen peroxide into conical flask

Add catalase and quickly replace bung
Start timer and record volume of gas produced
At 10 seconds add the copper sulfate
Continue to record the volume for at least 2 minutes
Clean conical flask and dry
Repeat 1-6 for ethanol and sulphuric acid

The best inhibitor will quickly stop the reaction resulting in a smaller amount
of oxygen produced.

Method:
1. Measure 15cm3 of copper sulphate into 100cm3 beakers with measuring
pipette
2. Label beakers with times 10-60 seconds
3. Measure out 60cm3 of hydrogen peroxide solution into beaker using
burette
4. Add 2cm3 of catalase solution to beaker with measuring pipette and start
stop clock
5. Every 10 seconds using pipette transfer 10cm3 of reacting solution into a
beaker of the chosen inhibitor (time intervals can vary depending on how
fast reaction seems to be taking place)
Now I have samples, I need to titrate them with permanganate to find the
concentration of hydrogen peroxide at each time
6. Rinse burette with potassium manganate (IV) allowing it drain into a waste
beaker
7. Clamp burette to stand and fill with the permanganate
8. Use volumetric pipette with pump to transfer known volume of
quenched solution to conical flask
9. Add permanganate while swirling the flask
10.Slow down when pink colour starts to appear
11.Stop when pale pink colour persists for 30 seconds
12.Record the end point (volume of permanganate used)
13.Wash conical flask and repeat for other times
14.Repeat 1- 12 two more times
Page | 25

15.Repeat for different concentrations of hydrogen peroxide and catalase

Diagram:

Inhibitor

10cm3
volumetric

Beaker
containin
g
hydrogen
peroxide
and

Stopwatch

Clamp
Burette
with

Conical flask with

sample of
quenched

White

Page | 26

Risk Assessment

Page | 27

Chemi
cal

Hydrog
en
peroxid
e
(20
volume
)
[1.1]

Hazard
symbol

Risks

Precautions

eye
protec
tion
gloves

Irritant

Hydrog
en
peroxid
e
(15
volume
and
less)
Catalas
e
(powde
r)
[1.2]

Avoid
contaminat
ion as this
may speed
up the
formation
of oxygen
and
pressure
build up

Ove
rall
Risk
Medi
um

Low

Harmful

Catalas
e
(all
solutio
ns
above
1%)

irritatin
g to
eyes
and
skin

Protectio
n

Irritant

harmful
as
powder,
irritatin
g to
eyes
Irritatin
g to
eyes
and
skin.
Risk of
serious
damage
to eyes

irritant
for
solution
s
greater
than
1%,
irritatin
g to
eyes
and
skin
Enzyme
s are
sensitiz

eye
protect
ion
use of
fume
cupbo
ard

To avoid
powdered
enzymes
escaping
into the air
switch off
the fume
cupboard
when carry
out the
weighing so
particles
arent
inhaled

Medi
um

eye
protect
ion
gloves

Avoid
contact
with skin
and eyes

Medi
um

Page | 28

General Precautions

Avoid contact with skin and eyes

Avoid inhalation of vapour or mist
Wear lab coat to avoid contamination of clothes
Spillages should be taken care of immediately especially on floor to avoid
slips
Move seats under bench to prevent tripping around working area
Be careful moving around and handling to prevent breaking glassware

Emergency Actions [6]

Contact with skin: wash with soap and plenty of water

Contact with eyes: flood eyes with tap water for at least 15 minutes
Swallowed: rinse mouth, do not encourage vomiting
Inhaled: move into fresh air

Page | 29

Bibliography
Introduction
[1]
Hydrogen Peroxide general
http://brownperiodfxhydrogenperoxide.blogspot.co.uk/2011/02/this-iswhat-hydrogen-peroxide-molecule.html
[2]
Why is hydrogen peroxide stored in dark bottles?
http://humantouchofchemistry.com/why-is-hydrogen-peroxide-stored-indark-bottles.htm
[3]
Toxicology data network, human health effects: hydrogen peroxide
http://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?
dbs+hsdb:@term+@DOCNO+547
[4]
Hydrogen Peroxide - Toxicological Overview
http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1246260031509
[5]
(Summary) The Decomposition of Hydrogen Peroxide by Liver Catalase,
John Williams, Muspratt Laboratory of Physical and Electrochemistry,
University of Liverpool, 1927 Journal of General Physiology
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140981/pdf/309.pdf
[6]
Collision Theory
http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Rate_Laws/Gas_
Phase_Kinetics/Collision_Theory/Collision_Theory_I
[7]
The Effect Of A Catalyst - Chapter 14, section 4 Chemistry by
Boundless
http://books.google.co.uk/books?
id=fa3pAAAAQBAJ&printsec=frontcover&dq=chemistry+by+boundless&
hl=en&sa=X&ei=Y8F6UsjDOMii0QW0ooGgAw&ved=0CDEQ6AEwAA#v=
onepage&q=chemistry%20by%20boundless&f=false
[8]
Homogeneous Catalysis - Chapter 13, section 5 Chemistry by
Boundless
http://books.google.co.uk/books?
id=fa3pAAAAQBAJ&printsec=frontcover&dq=chemistry+by+boundless&
hl=en&sa=X&ei=Y8F6UsjDOMii0QW0ooGgAw&ved=0CDEQ6AEwAA#v=
onepage&q=chemistry%20by%20boundless&f=false
[9]
Two stage reaction profile for a catalytic cycle
http://www.docbrown.info/page03/ASA2rates.htm
[10] Some biochemical properties of Catalase from Kohlrabi, H. TayefiNasrabadi, Department Of Biochemistry, University of Tabriz, 2008
Journal of Biological Sciences
http://198.170.104.138/jbs/0/317-JBS-ANSI.pdf
Page | 30

[11]
[12]

[13]

[14]
[15]

[16]
[17]

Catalase: Product Information

http://himedialabs.com/TD/TC037.pdf
Proposed Mechanism of Catalase
http://biology.kenyon.edu/BMB/Chime/catalase/frames/cattx.htm#Propos
ed Mechanism of Catalase
Enzyme Catalysis
https://www.boundless.com/chemistry/chemicalkinetics/catalysis/enzyme-catalysis/
Inhibition of Catalase Activity in Wines
http://ajevonline.org/content/26/2/92.abstract
Mathematics of Arrhenius Equation
http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Reaction_Rates/
Temperature_Dependence_of_Reaction_Rates/Arrhenius_Equation
http://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Michaelis_a
nd_Menten_Equation
http://www.chemguide.co.uk/physical/basicrates/orders.html#top

List of Figures
1
Four different models used to represent a H2O2 molecule
http://www.windows2universe.org/physical_science/chemistry/h
ydrogen_peroxide.html
2
Anthraquinone Process for Production of Hydrogen Peroxide
http://www.lookchem.com/Chempedia/Chemical-Technology/InorganicChemical-Technology/2863.html
3
The Orientation of Collisions
http://www.chemguide.co.uk/physical/basicrates/introduction.html
4
Activation Energy
http://www.chem.fsu.edu/chemlab/chm1046course/activation.html
5
Catalysts or making it happen- heterogeneous
http://www.spaceflight.esa.int/impress/text/education/Catalysis/index.ht
ml
6
Catalysis theory and practice
http://www.docbrown.info/page07/appendixtrans06.htm
7
Enzymes - 7.6.2 Induced-fit model
http://www.click4biology.info/c4b/7/pro7.6.htm
8
Catalase Structure
http://en.wikipedia.org/wiki/File:Catalase_Structure.png
9
On the Mechanism of the Decomposition of Hydrogen Peroxide by
Catalase, D Keilin EF Hartree, University of Cambridge, 1937
http://rspb.royalsocietypublishing.org/content/124/837/397.full.pdf
10
Factors affecting Enzyme action
11
http://www.biologymad.com/resources/EnzymesRevision.pdf
12
Factors that affect rate of reaction - temperature
http://www.webchem.net/notes/how_far/kinetics/rate_factors.htm
13
The Arrhenius Equation
http://www.chem.fsu.edu/chemlab/chm1046course/arrhenius.html
14
The Michaelis-Menten model
http://depts.washington.edu/wmatkins/kinetics/michaelis-menten.html
Page | 31

15
16
17
18

Lineweaver-Burk plot
http://en.wikipedia.org/wiki/Lineweaver%E2%80%93Burk_plot
Orders of reactions and rate equations
http://www.chemguide.co.uk/physical/basicrates/orders.html
Rate Laws
http://www.personal.kent.edu/~cearley/gen50/review/kinetics.htm
Figure 14.16, Properties of Reactions That Obey Zeroth-, First-, and
Second-Order Rate Laws
http://2012books.lardbucket.org/books/principles-of-general-chemistryv1.0m/s18-04-using-graphs-to-determine-rate.html

List of Equations
1 September 1995 issue of Chemistry Review
http://www.york.ac.uk/chemistry/schools/chemrev/projects/peroxide/
2 Evolution of Catalases from Bacteria to Humans Marcel Zamocky, Paul G.
3 Furtmller, Christian Obinger, University of Natural Resources and Applied Life
Sciences, Vienna, 2008
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2959186/
4 Arrhenius equation definition
http://chemistry.tutorvista.com/inorganic-chemistry/arrhenius-equation.html
5 Two Point Form of the Arrhenius equation
http://www.chem.fsu.edu/chemlab/chm1046course/activation.html
6 Structural Biochemistry/Enzyme/Michaelis and Menten Equation
http://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Michaelis_and_M
enten_Equation
7 Vmax and Km can be determined by double-reciprocal plots, Biochemistry 5 th
Edition
http://www.ncbi.nlm.nih.gov/books/NBK22557/
Methods
[1 Hydrogen Peroxide vol
]
http://en.wikipedia.org/wiki/Hydrogen_peroxide#Decomposition
[2 Catalase Solution
]
http://www.sigmaaldrich.com/technicaldocuments/protocols/biology/enzymatic-assay-of-catalase.html
[3 Titration
]
http://www.titrations.info/permanganate-titration-hydrogen-peroxide

Page | 32

Risk Assessment
[1]
[1.1]

[1.2]

[1.3]

[2]

[3]

[4]

http://www.jmloveridge.com/cosh/hydrogen
%20peroxide%206.pdf
Hazcard 50
http://www.cleapss.org.uk/attachments/article/0/Co
mpleteSet-2013-08-20.pdf?
Secondary/Science/Hazcards/
Hazcard 33
http://www.cleapss.org.uk/attachments/article/0/Co
mpleteSet-2013-08-20.pdf?
Secondary/Science/Hazcards/
Hazcard 81
http://www.cleapss.org.uk/attachments/article/0/Co
mpleteSet-2013-08-20.pdf?
Secondary/Science/Hazcards/

Copper Sulfate
http://www3.icb.usp.br/corpoeditorial/ARQUIVOS/residuos_quimicos/n
ovos/COPPER%20SULFATE%20SOLUTION%20(Cupric%20sulfate
%20standard).pdf
Ethanol
http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?
country=GB&language=en&productNumber=02877&brand=FLUKA&P
ageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog
%2Fproduct%2Ffluka%2F02877%3Flang%3Den
http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?
Page | 33

[5]
[6]

Hazard

country=GB&language=en&productNumber=435589&brand=SIAL&P
ageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog
%2Fproduct%2Fsial%2F435589%3Flang%3Den
https://wiki.bath.ac.uk/download/attachments/61374982/LAB2_4hypodermics.pdf
Emergency actions
http://www.chem.ufl.edu/facilities/safety-dealing-with-anemergency.shtml
http://chemistry.about.com/od/healthsafety/ig/Laboratory-SafetySigns/index.01.htm

Page | 34