Effect of pH on amylase activity
GCSE Biology (8461) · Required practical 5 — method, variables, the marks examiners report students losing.
Investigate the effect of pH on the rate at which amylase breaks down starch, using iodine to detect when the starch has gone.
Apparatus
- Amylase solution and starch solution
- pH buffer solutions across a range (e.g. pH 4 to pH 10)
- Iodine solution in the wells of a spotting tile
- Water bath at about 35 degrees C, test tubes and a stopwatch
- Pipettes and a thermometer
Method
- 1Place drops of iodine solution in the wells of a spotting tile.
- 2Mix amylase, starch and one pH buffer in a test tube and start the stopwatch.
- 3Keep the tube in a water bath at a constant temperature (about 35 degrees C).
- 4Every 30 seconds, remove a drop of the mixture and add it to a well of iodine; note the colour.
- 5When the iodine stays orange-brown (no longer blue-black), all the starch has been digested — record the time.
- 6Repeat with each pH buffer, keeping everything else the same.
Variables
Independent
pH of the buffer solution
Dependent
Time for the starch to be digested (used to find the rate)
Control
- Temperature (kept constant in a water bath)
- Concentration and volume of amylase and starch
- Volume/interval of sampling onto the iodine
Results & processing
- Rate of reaction = 1 / time taken for the starch to be digested (in s−1).
- The pH that gives the shortest time (highest rate) is the optimum pH for the enzyme.
- A graph of rate against pH peaks at the optimum and falls away on either side as the enzyme works less well.
Where students lose marks
Not controlling temperature.
Fix: Use a water bath so temperature does not change the rate — only pH should vary.
Sampling at uneven time intervals.
Fix: Test a drop on the iodine at fixed intervals (e.g. every 30 s) so the end point is consistent.
Saying a high pH denatures the enzyme without explaining it.
Fix: Away from the optimum, the active site changes shape (denatures), so starch no longer fits and the rate falls.
Improve the method
- Repeat each pH and take a mean time to reduce the effect of random error.
- Sample at shorter intervals to pinpoint the end point more precisely.
- Use a colorimeter to judge the iodine end point objectively.
Try it — exam-style
At pH 6 the starch is fully digested in 120 seconds. Calculate the rate of reaction in s−1.
Explain why amylase stops working at a pH far from its optimum.
Questions are written in the style of past AQA papers — never copied from them.
Drill it properly
Stuck on effect of ph on amylase activity?
The rate calculation and the denaturing explanation are the marks students miss here — I drill both, and your first lesson is free.