Hydrogen peroxide is a toxic by-product of metabolism in organisms and must be decomposed into its products water and oxygen (in the form of effervescence) in order not to be harmful to the cells of the organism.

Biology Method – Skill AB

BACKGROUND KNOWLEDGE

Hydrogen peroxide is a toxic by-product of metabolism in organisms and must be decomposed into its products water and oxygen (in the form of effervescence) in order not to be harmful to the cells of the organism. The enzyme catalase speeds up the decomposition.

Catalase catalyses the following reaction: 2H2O2 2H2O + O2

INDEPENDENT VARIABLE: in this experiment the independent variable i.e. the one that is controlled, is the pH level of the solution in which the potato chip is immersed.

The independent variable in this particular experiment is not the only factor that affects the rate of the decomposition reaction of H2O2, there are many other factors which are confounding in this experiment:

CONFOUNDING VARIABLES:

Temperature of the substrate, buffer solution and the enzyme – the buffered H2O2 solution will be incubated in an electric water bath at temperature 20°C (as this is the optimum temperature for this particular type of catalase as it is found in a plant). Also, the Buchner flask will be placed in an electric water bath of temperature 20°C so that, when the potato sample has been cut and placed in the Buchner flask it will remain at the optimum temperature.
Enzyme concentration – the enzyme catalase if found in the potato and therefore, by using the same sized potato chip with the same mass and surface area, and more importantly always from the same potato, the enzyme concentration will remain constant and therefore, not affect the results
Substrate concentration – the initial concentration of the H2O2 will always be the same, as I will always use the same volume of the same concentration of H2O2 (1%). During each experiment the substrate concentration will obviously decrease as it will be decomposed into its products however, this will happen the same amount in each and will therefore not affect the results. It can be thought of as systematic error.
Surface area of the potato chip – the potato samples will be cut with a cork borer, sized 4, producing cylinder shaped samples all with the same circumference. The chips will all be cut to a length of 1cm producing chips with the same surface area
Volume/mass of the potato sample – the 1cm long cylinders of potato will all be weighed so they are the same weight and therefore the same mass, thus creating the same volumes (assuming that the variations in density of the potato and water content due to osmosis are negligible).
Volume of the solution in which the potato sample is immersed in – this must remain constant a the solution is of buffer and H2O2 and therefore must be the same so that the enzyme has as the same amount of substrate to act upon.
Incubation period in the water bath – all of the buffered H2O2 solution in each repeat of the experiment must be kept in the water bath for at least 5 minutes in order to all the whole of the solution to reach an equilibrated temperature of 20°C for which the reaction will take place at.
Concentration of buffered solution in the buffered H2O2 solution added to the potato chip – the total volume of solution added to the potato chip amounts to 100cm3, in order to completely submerge the potato sample in the solution in a 200ml Buchner flask with approximate base diameter of 9cm. Also a larger volume will reduce percentage errors.

H2O2 solution naturally decays so the solutions used in this experiment must all be from the same sample.

DEPENDANT VARIABLE: if the volume of oxygen produced which is a method for measures the rate of the reaction. This depends on the pH level of the solution and is therefore, the dependent variable. It will be measured using a gas syringe in an airtight system in order to produce useful and reliable quantitative results. The method will be repeated in order to produce a full range of results.

Method

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H2O2 solution naturally decays so the solutions used in this experiment must all be from the same sample.

Method

NOTE: Goggles must be worn at all times and care taken when using the scalpel

Label a boiling tube pH3 (ignoring pH1 and pH2 due to preliminary experiments) and add 5cm3 of 1% hydrogen peroxide (H2O2) using a graduated pipette.
Add 5cm3 pH3 buffer solution to boiling tube pH3, seal with a rubber bung and shake vigorously.
Place the boiling tube in a rack in an electric water bath set at 20°C, start the stopwatch and incubate for period of at least 10minutes.
Cut a cylinder of potato using a cork borer (size 4), push out (at least 1cm) and then use a scalpel to cut an exact length of potato sample 1cm on a tile (leaving the rest of the potato cylinder in the cork borer to prevent water loss due to evaporation), ensuring no skin remains on the potato sample.
Weigh the boiling tube with side arm on a top-pan balance and record this weight to two decimal places (in table 1). Place the potato sample in the boiling tube and record the new weight. Calculate the weight of the potato sample by the difference in the two weights and record this value
Add this potato sample into the boiling tube with side arm and assemble equipment (as shown in diagram 1)
Remove all air in the gas syringe by pushing the plunger in all the way and then close the tap to the dropping funnel and check for air leaks by gently pulling the plunger out, which must spring back into place.
Pour 10cm3 of buffered H2O2 solution into the open top dropping funnel.

The following three stages must be carried out quickly:

When ready release the buffered H2O2 solution into the boiling tube with side arm and simultaneously start the stop watch
Record the initial volume measured in the gas syringe at eye level.
As soon as all of the buffered H2O2 is transferred into the boiling tube with side arm, immediately close the tap of the open top dropping funnel.
Undo the clamps holding the boiling tube with the side arm and continuously shake the boiling tube with side arm vigorously until no more oxygen is produced.
Record the new volume of oxygen in the gas syringe at eye level over a period of ten minutes at intervals of 30 seconds (on table 2), whilst repeatedly twisting the plunger of the gas syringe (at regular intervals of about 10 seconds) to stop friction having a restricting effect on the movement of the plunger.
When the reaction is complete or after ten minutes, dispose of the waste contents from the boiling tube with side arm, rinse and dry the boiling tube with side arm and dropping funnel and reset the plunger on the gas syringe to nought.
Repeat stage 1 – 14, twice, using fresh samples of 1% H2O2 and buffered pH3 solution. Cut a fresh potato sample from the remaining sample in the cork borer (N.B when this supply runs out cut another cylinder from the same area of the same potato).
Repeat stages 1 – 15 using increasing pH buffer solutions of intervals of 1 pH level, from pH4 to pH12 (chosen due to my preliminary investigation, where no reaction took place for the extreme pH buffer solutions i.e. pH1, 2, 13 and 14).
Record the data in table 2 for each buffered solution and calculate the averages for each individual buffered solution.
Construct a graph of the volume of oxygen produced against the pH level of the buffer solution for each individual buffer solution and work out the rate of the reactions by calculating the gradient of each graph.