Practical - pH and the rate of enzyme controlled reactions


To determine the optimum pH at which an enzyme's activity is greatest.


  1. Set a waterbath to 35 °C.
  2. Now put two drops of iodine solution into each spot of a spotting tile.
  3. Add 5 cm3 of amylase enzyme solution to a test tube, and place in the waterbath.
  4. Place 10 cm3 of starch solution and 5 cm3 of pH buffer solution to a second test tube. The buffer keeps the pH at the same level. Place this test tube in the waterbath.
  5. Mix the solutions in the two test tubes once they have reached the correct temperature and replace in the waterbath. Start a stop clock.
  6. Use a pipette to remove a few drops of solution every 30 seconds from the test tube and put them into a different well of the spotting tile.
  7. Repeat until the iodine solution stops turning black which shows when all the starch has been broken down.
  8. Record the time this takes.
  9. Repeat with buffer solutions of different pH.


  • Independent variable - pH level
  • Dependent variable - Time taken for starch to be broken down.
  • Control variables - Temperature, volume and concentrations of enzyme solution and starch solution.


  • Iodine solution is an irritant. If it touches skin it should be washed off.
  • Goggles should be worn.


pH of buffer solutionTime taken for starch to disappear (s)

The enzyme amylase breaks down starch into maltose. If the enzyme is working effectively, this will happen quickly. The shortest time for the starch to disappear is at pH 7. This is therefore the optimum pH for amylase.