Specified practical 1.3A: Investigate factors affecting enzyme action

Investigate the effect of pH on the rate of reaction of amylase enzyme


To determine the rate of the amylase activity at different pHs.


You will investigate the breakdown of starch by amylase at different pHs.

The different pHs under investigation will be produced using buffer solutions. Buffer solutions produce a particular pH, and will maintain it if other substances are added.

The amylase will break down the starch.

A series of test tubes containing a mixture of starch and amylase is set up at different pHs.

A sample is removed from the test tubes every 10 seconds to test for the presence of starch. Iodine solution will turn a blue/black colour when starch is present, so when all the starch is broken down, a blue-black colour is no longer produced. The iodine solution will remain orange-brown.

Diagram showing three test tubes containing amylase, buffer and starch solutions.

A control experiment must be set up - without the amylase - to make sure that the starch would not break down anyway, in the absence of an enzyme. The result of the control experiment must be negative - the colour must remain blue-black - for results with the enzyme to be valid.

When the starch solution is added:

  • Start timing immediately.
  • Remove a sample immediately and test it with iodine solution.
  • Sample the starch-amylase mixture continuously, for example every 10 seconds.

This is how the experiment could be set up:

Diagram showing how to set up the experiment and to monitor the results

For each pH investigated, record the time taken for the disappearance of starch, ie when the iodine solution in the spotting tile remains orange-brown.


  • Wear safety goggles.
  • Amylase solution may cause allergic reactions.
  • Iodine solution is an irritant.
  • Avoid contact with skin and eyes.

Example results

pHTime taken for the disappearance of starch in s

The time taken for the disappearance of starch is not the rate of reaction.

It will give us an indication of the rate, but is the inverse of the rate - the shorter the time taken, the greater the rate of the reaction.

We can calculate the rate of the reaction by calculating  \frac{1}{t}, obtaining a measure of the rate of reaction by dividing one by the time taken for the reaction to occur.

So, from the results:

pHTime taken for the disappearance of starch in sRate of breakdown of starch  \frac{1}{t}

Plot a graph of rate of reaction against pH.

Graph showing the rate of reaction against pH

A similar experiment can be carried out to investigate the effect of temperature on amylase activity.

Set up a series of test tubes in the same way and maintain these at different temperatures using a water bath - either electrical or a heated beaker of water.

Depending on the chemical reaction under investigation, you might monitor the reaction in a different way. If investigating the effect of temperature on the breakdown of lipid by lipase, you could monitor pH change - lipids are broken down into fatty acids and glycerol. As the reaction begins, the release of fatty acids will mean that the pH wil decrease.