Required practical 4 · Biology · Biology Paper 1, both tiers
Amylase and pH
Investigate the effect of pH on the rate of reaction of amylase.
The exam trap. Keep temperature fixed so only pH changes. The true end point can be up to one sampling interval earlier.
Variables
- Independent (you change)
- pH of the buffer solution
- Dependent (you measure)
- Time taken for the starch to be broken down (iodine stays orange-brown)
- Control (you keep the same)
- Temperature (water bath at, for example, 35 °C)
- Volume and concentration of amylase
- Volume and concentration of starch
- Sampling interval
Apparatus
- Spotting tile with a drop of iodine in each well
- Amylase solution and starch solution
- Buffer solutions of different pH
- Water bath or electric heater
- Test tubes, syringes or pipettes
- Stopwatch
Method, and why each step matters
Put a drop of iodine solution in each well of a spotting tile.
Why Iodine shows when starch is still present (blue-black).
Add amylase, starch and a buffer of known pH to separate tubes and leave them in the water bath for a few minutes.
Why The reactants must reach the same temperature before mixing, because temperature is controlled.
Mix the amylase, buffer and starch and start the stopwatch at once.
Why The reaction starts on mixing, so timing must too.
Every 30 seconds, put a drop of the mixture onto a fresh drop of iodine.
Why Regular sampling shows when the starch has gone.
Record the time when the iodine stays orange-brown.
Why That is when all the starch has been broken down.
Repeat with each buffer pH, and calculate rate = 1000 ÷ time.
Why Rate is inversely related to time, so it can be compared across pH values.
Risks
| Hazard | How to reduce the risk |
|---|---|
| Iodine is an irritant. | Wear eye protection. |
| Hot water from the bath can scald. | Keep the bath below 50 °C and handle tubes carefully. |
A worked set of results
| pH | Time for starch to go in s | Rate (1000 ÷ time) |
|---|---|---|
| 5 | 180 | 5.6 |
| 6 | 60 | 16.7 |
| 7 | 90 | 11.1 |
| 8 | 240 | 4.2 |
- The optimum pH gives the shortest time (here pH 6).
- Away from the optimum the active site changes shape, so fewer enzyme-substrate complexes form.
Mistakes that cost marks
Changing temperature and pH together, or not controlling temperature at all.
Instead Use a water bath so only the pH changes.
Recording a time that is really the sampling interval: the true end point could be up to 30 s earlier.
Instead Sample more often (every 10 s) or use a colorimeter for a more precise end point.
Saying the enzyme is killed at the wrong pH.
Instead Enzymes are not alive: the active site changes shape (denatures).