AQA GCSE Biology · Organisation
Enzymes required practical
Investigate the effect of pH on the rate of reaction of amylase.
Amylase breaks down starch. You time how long the starch takes to disappear at each pH, using iodine on a spotting tile. The fastest rate is at the enzyme’s optimum pH.
Equipment
- Amylase solution and starch solution
- Buffer solutions at pH 5, 6, 7, 8 and 9
- Spotting tile with a drop of iodine in each well
- Water bath at 35 °C and a thermometer
- Test tubes, syringes and pipettes
- Stopwatch
Variables
- Independent you change
- pH of the buffer solution
- Dependent you measure
- Time taken for the starch to be broken down (iodine stays orange)
- Control you keep the same
- Temperature (35 °C water bath)
- Volume and concentration of amylase
- Volume and concentration of starch
- Time between samples
Method
- 1
Put one drop of iodine solution into each well of a spotting tile.
- 2
Put 2 cm³ of amylase, 2 cm³ of starch and 1 cm³ of pH buffer in separate tubes in the water bath for 5 minutes to reach 35 °C.
- 3
Mix the amylase and buffer with the starch and start the stopwatch.
- 4
Every 30 seconds, use a pipette to put a drop of the mixture into the next well of iodine.
- 5
Stop when the iodine stays orange-brown: all the starch has been broken down. Record the time.
- 6
Repeat at each pH, then work out the rate as 1000 ÷ time.
Worked example
Time for the iodine to stay orange-brown at each pH, at 35 °C.
| pH | Time (s) | Rate (1000 ÷ time, s⁻¹) |
|---|---|---|
| 5 | 180 | 5.6 |
| 6 | 90 | 11.1 |
| 7 | 60 | 16.7 |
| 8 | 120 | 8.3 |
| 9 | 270 | 3.7 |
Working
Rate = 1000 ÷ time
pH 7: 1000 ÷ 60 = 16.7 s⁻¹
pH 9: 1000 ÷ 270 = 3.7 s⁻¹
Conclusion
The rate is fastest at about pH 7, the optimum for this amylase. Further from pH 7, the shape of the active site changes, so starch fits less well and the reaction slows; at extremes the enzyme is denatured.
Risks
- Iodine stains and irritates eyesWear eye protection.
- Amylase can cause an allergic reactionAvoid skin contact and wipe up spills.
Common mistakes
- Timing in big gaps. Testing every 30 seconds means the real time could be up to 30 s earlier, so the result is only accurate to 30 s.
- Not warming the solutions first. If they start cold, the temperature isn’t controlled at the start.
- Saying the enzyme is “killed”. Enzymes aren’t alive; they are denatured when the active site changes shape.
Better results
- Sample every 10 or 20 seconds to time the end point more precisely.
- Repeat each pH three times and calculate a mean.
- Test more pH values close to the optimum (e.g. 6.5 and 7.5) to find it more precisely.
Launching November 2026
7 exam questions on this practical.
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