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Better Equipped Practical Teaching Guides
A complete A Level Biology practical guide for investigating the effect of pH, temperature and substrate concentration on enzyme activity using catalase and hydrogen peroxide, with apparatus, method, results tables, graph skills, troubleshooting, risk assessment, technician notes and exam support.
Teacher note: This resource supports practical teaching and equipment planning. Teachers should adapt the procedure and risk assessment to match their exam board specification, local laboratory rules and departmental policies.
This practical investigates how enzyme activity is affected by pH, temperature and substrate concentration. Students use catalase and hydrogen peroxide to produce oxygen gas, which can be measured using a gas syringe. The volume of oxygen produced over a fixed time provides a quantitative measure of enzyme activity.
Enzymes are biological catalysts. They speed up reactions by lowering activation energy and are not used up during the reaction. Each enzyme has a specific active site where a complementary substrate binds to form an enzyme-substrate complex. The products are then released and the enzyme can be reused.
The substrate fits precisely into the active site because the active site has a complementary shape.
The active site changes shape slightly when the substrate binds, helping to form the enzyme-substrate complex.
Catalase reaction: 2H2O2 → 2H2O + O2. The oxygen produced can be collected and used as an indicator of enzyme activity.
Check that gas syringes move freely, tubing connections are airtight, bungs fit securely and water baths have reached the correct temperature before students begin.
The same basic workflow can be adapted for the pH, temperature and substrate concentration investigations. Students should change one independent variable at a time, keep control variables constant, record oxygen production over the same time period and repeat trials for reliability.
Independent variable: pH
Dependent variable: volume of oxygen produced in 60 seconds
Control variables: temperature, catalase concentration, hydrogen peroxide concentration, reaction volume and reaction time.
| pH | Trial 1 / cm³ | Trial 2 / cm³ | Trial 3 / cm³ | Mean / cm³ |
|---|---|---|---|---|
| 4 | ||||
| 5 | ||||
| 6 | ||||
| 7 | ||||
| 8 | ||||
| 9 |
Independent variable: temperature
Suggested temperatures: 10°C, 20°C, 30°C, 40°C, 50°C and 60°C.
Expected trend: at low temperatures, reaction rate is low because particles have less kinetic energy and fewer successful collisions occur. As temperature increases, rate rises until the optimum temperature. Above the optimum, enzyme denaturation causes activity to decrease rapidly.
| Temperature | Trial 1 / cm³ | Trial 2 / cm³ | Trial 3 / cm³ | Mean / cm³ |
|---|---|---|---|---|
| 10°C | ||||
| 20°C | ||||
| 30°C | ||||
| 40°C | ||||
| 50°C | ||||
| 60°C |
Independent variable: hydrogen peroxide concentration
Dependent variable: volume of oxygen produced in 60 seconds
Suggested concentrations: 0.5%, 1%, 2%, 3%, 4% and 5%.
Expected trend: rate initially increases rapidly as more substrate molecules are available. Eventually, all active sites become occupied and the rate plateaus because enzyme concentration becomes the limiting factor.
| Concentration | Trial 1 / cm³ | Trial 2 / cm³ | Trial 3 / cm³ | Mean / cm³ |
|---|---|---|---|---|
| 0.5% | ||||
| 1% | ||||
| 2% | ||||
| 3% | ||||
| 4% | ||||
| 5% |
Mean = total results ÷ number of results. Students should repeat trials, calculate a mean and identify any anomalies before drawing conclusions.
Rate = oxygen produced ÷ time. Example: 40 cm3 oxygen in 60 seconds gives a rate of 40 ÷ 60 = 0.67 cm3 s-1.
Practical conclusion: Catalase activity depends on environmental conditions. Each enzyme has an optimum pH and optimum temperature, and enzyme activity becomes limited at high substrate concentration when active sites are saturated.
| Problem | Possible cause | Solution |
|---|---|---|
| No oxygen produced | Enzyme inactive or hydrogen peroxide degraded | Use fresh catalase and fresh hydrogen peroxide. |
| Variable results | Timing inconsistent | Use the same timing method and start timing immediately after mixing. |
| Gas leaks | Loose tubing or poorly fitted bung | Check seals, tubing and bung fit before starting. |
| Unexpectedly low rates | Incorrect temperature or solutions not equilibrated | Re-equilibrate solutions in the water bath before mixing. |
| Little effect of pH | Buffers incorrect or mislabelled | Check buffer labels and use fresh buffer solutions. |
Higher temperature always increases rate.
Correction: rate only increases up to the optimum temperature.
Denatured enzymes have simply stopped working temporarily.
Correction: denaturation changes the active site shape, often irreversibly.
More substrate always increases rate.
Correction: rate eventually reaches a maximum when active sites are saturated.
pH only affects the substrate.
Correction: pH affects the enzyme active site structure.
Extension activity: Introduce Michaelis-Menten theory. At low substrate concentration, many active sites are available. At high substrate concentration, active sites become saturated and maximum rate, Vmax, is reached.
Theory introduction: 10 mins | Teacher demonstration: 10 mins | Experiment setup: 15 mins | Data collection: 25 mins | Analysis: 15 mins | Evaluation: 10 mins
Show oxygen production at room temperature, then compare this with boiled catalase so students can clearly observe the effect of denaturation.
Overall risk: low. This practical presents a low level of risk when standard laboratory procedures are followed and appropriate PPE is worn. Schools should complete their own risk assessment according to local policies.
| Hazard | Risk | Control measure |
|---|---|---|
| Hydrogen peroxide solution, typically 1-3% | Eye irritation or skin irritation if splashed | Wear safety goggles and gloves. Avoid contact with skin and eyes. Wash spills immediately with plenty of water. |
| Glassware | Cuts from broken glass | Handle carefully, check for cracks before use and clear breakages using a brush and dustpan. |
| Gas syringe apparatus and glass delivery tubes | Breakage causing cuts or unstable apparatus | Secure apparatus with clamps and stands. Keep the work area uncluttered. |
| Rubber bungs under pressure | Bung may be forced out if pressure builds rapidly | Use appropriate concentrations and volumes. Do not block apparatus or point it towards others. |
| Water baths | Burns from hot water or hot equipment | Use water baths below 70°C where possible. Handle hot equipment carefully and allow cooling before moving. |
| Biological enzyme source | Minor contamination risk or allergic reaction in sensitive individuals | Avoid ingestion, wash hands after the practical and dispose of biological material appropriately. |
| Spillages of liquids | Slips, falls or contamination of work area | Wipe up spills immediately and keep benches tidy and dry. |
| Electrical equipment | Electric shock if liquids contact electrical components | Keep liquids away from electrical equipment and dry hands before using plugs or switches. |
Emergency actions: For eye contact with hydrogen peroxide, irrigate with water for at least 10 minutes and inform the teacher immediately. For skin contact, wash thoroughly with water. Report cuts from glass immediately and follow school first aid procedures.
| Skill assessed | Example evidence |
|---|---|
| AO2 application | Setting up gas syringe apparatus correctly. |
| AO3 analysis | Calculating reaction rates and means. |
| AO3 evaluation | Identifying limitations, anomalies and improvements. |
| Maths skills | Using rates, units and graph interpretation. |
| Practical technique | Controlling variables and collecting quantitative data. |
Catalase breaks down hydrogen peroxide into water and oxygen. The volume of oxygen produced over time gives a measurable indication of reaction rate.
Changing one independent variable allows students to link any change in enzyme activity to that factor only.
At high substrate concentrations, all enzyme active sites become occupied. Adding more substrate no longer increases the rate because enzyme concentration is limiting.
Boiling denatures catalase, changing the shape of the active site so the enzyme can no longer catalyse the reaction effectively.
Explore more A Level Biology practical guides, equipment support and laboratory planning resources from Better Equipped.
This guide was written and reviewed by Better Equipped's technical team, drawing on experience supplying practical science equipment to schools, colleges, laboratories and science departments throughout the UK. Our technical team include ex-school laboratory technicians and are here to support schools, colleges and laboratories across the UK. If you have feedback on this guide, please contact us.
Last reviewed and updated: July 2026
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