Practical - The effect of pH on the rate of reaction of amylase

Aim

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

Method

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 its absence. 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 you might set up the experiment:

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.

Hazards

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

Example results

pHTime for starch to disappear (s)
5120
640
730
850
9150

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

It will give us an indication of the rate, but it is the inverse (new) of the rate – the shorter the time taken, the greater the rate of the reaction.

We can calculate the rate of the reaction by dividing the number one by the time taken in seconds. The calculation is therefore 1/t.

For example, for the pH 5investigation, the calculation would be:

1/120 = 0.0083

So, from the results:

pHTime for starch to disappear (s)Rate of starch breakdown (1/t)
51200.0083
6400.0250
7300.0333
8500.0200
9500.0067

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, for instance, you could monitor pH change – lipids are broken down into fatty acids and glycerol. As the reaction proceeded, the release of fatty acids would mean that the pH would decrease.