How enzyme activity is affected by pH
Investigating the effect of pH on enzymes.
Students are required to investigate how enzyme activity is affected by pH. This can be completed using the amylase and starch reaction as indicated by the colour change in iodine solution. Starch gives a blue-black colour when added to Iodine. Amylase eventually breaks down the starch into glucose, causing no colour change in the presence of Iodine. Students will test their solutions, testing a drop every 10 seconds after the reaction has begun. The use of a Bunsen burner to heat and maintain the temperature of a water bath satisfies the requirement for the safe use of heating equipment in the laboratory.
Equipment (per pair of participants)
• Safety goggles
• 11 x test tubes
• Test tube rack
• Water bath (electronic or a beaker of water)
• Spotting tile
• 5ml syringes
• Disposable pipettes
• 400ml beaker
• Glass rod
• Stopwatch
• Thermometer
• Chinagraph pencil or permanent pen
• Starch solution
• Amylase solution
• Iodine solution
• CLEAPSS Hazcards: 33, 40C, 54
Investigating the Effect of pH on Amylase Activity
Method
- Preparation
- Set Up Water Bath
- Heat water in a 400ml beaker using a Bunsen burner until it reaches 35°C.
- Monitor the temperature using a thermometer.
- Label Test Tubes
- Label 5 test tubes with the pH buffer values being used.
- Label 5 additional test tubes as ‘Starch’.
- Label a final test tube as ‘Amylase’.
- Preparing Solutions
- Buffer Solutions
- Add 2ml of each buffer solution to the appropriately labelled test tubes.
- Starch Solution
- Add 4ml of starch solution to each of the ‘Starch’ tubes.
- Amylase Solution
- Add 12ml of Amylase solution to the ‘Amylase’ test tube.
- Water Bath
- Place all test tubes (buffers, starch, and amylase) into the water bath to allow the solutions to reach 35°C, checking with the thermometer.
- Setting Up Iodine Comparison
- Spotting Tile Preparation
- Place one drop of Iodine solution into each well of the spotting tile.
- Add one drop of starch solution to the first well as a colour reference.
- Conducting the Reaction
- Mix Buffer and Starch
- Once the test tubes have reached 35°C, take one starch test tube and add 2ml of the first buffer solution.
- Stir the mixture with a glass rod.
- Add Amylase
- Using a pipette, add 2ml of amylase solution to the buffer-starch mixture.
- Start the stopwatch immediately and continue stirring the mixture with the glass rod.
- Spotting for Starch Breakdown
- After 10 seconds, use the glass rod to take one drop of the mixture and add it to the next well of the spotting tile.
- Rinse the glass rod with water after each use.
- Repeat the Process
- Continue adding drops every 10 seconds until there is no colour change in the Iodine well. This indicates the starch has been fully digested.
- Recording Results
- Count Drops
- Record the total number of drops required for the reaction to complete in a suitable table.
- Convert to Time
- Multiply the number of drops by 10 seconds to calculate the total time it took for the amylase to digest the starch at each pH level.
- Repeat for Other Buffers
- Repeat the entire process using the remaining buffer solutions, ensuring all measurements are accurate and consistent.
Video produced by @revisechemistrywithmrb practical GCSE Chemistry tutorials by a specialist Chemistry teacher with over 25 years experience teaching Chemistry and Biology.
Technician tips
Enzyme experiments can be tricky to get right. All reagents must be trialed before the practical session to ensure the practical will give the results needed in the given session time.
• All reagents should be at room temperature on entering the practical session.
Amylase and starch solutions must always be freshly prepared:
• 1% starch solution is made by making a paste using 1g of starch powder and a few drops of cold water (for every 100mls of solution). Boiling water from a kettle can then be used to dilute this to the final volume. Stir to dissolve the paste and boil further if the solution is cloudy. The starch solution must have been allowed to cool to room temperature before use in the classroom.
• 0.1% Amylase solution is made by dissolving 0.1g of Amylase powder in every 100mls of water. The concentration of Amylase solution can be reduced if the reaction occurs too quickly.
• CLEAPSS practical procedure document contains a printable spotting tile which offers a larger number of wells than a standard spotting tile. These could be printed and laminated for repeated student use: PP048 The Effect of pH on Amylase Digestion.
To do this practical, you will need
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