The human digestive system: Required practical activity 4: investigate the effect of pH on the rate of reaction of amylase enzyme.

The idea

Amylase breaks down starch to maltose. In this practical, starch solution, amylase and buffer solutions of different pH are mixed at a controlled temperature. At intervals, a drop of the mixture is placed on a spotting tile with iodine solution. While starch remains, iodine turns blue-black; when starch is fully digested, iodine stays yellow-brown. The time to reach this end point is measured for each pH, and rate can be compared as 1 ÷ time. A pH close to the enzyme’s optimum gives the shortest time and fastest rate; extremes of pH slow the reaction because the enzyme is denatured. Temperature, starch and amylase volumes and concentrations must be controlled.

What earns the marks

• Amylase catalyses the breakdown of starch to maltose, so starch concentration falls as the reaction proceeds.
• Iodine solution is used to monitor the reaction: blue-black shows starch is still present, while yellow-brown shows starch has been digested.
• The time from mixing to the first yellow-brown result is recorded for each pH, and rate can be compared using rate = 1 ÷ time.
• Buffer solutions keep pH constant, allowing the effect of pH alone to be investigated.
• Temperature, volumes and concentrations of starch and amylase are controlled variables because they also affect enzyme activity.
• The optimum pH is identified as the pH giving the shortest time or fastest rate; away from the optimum, enzyme activity decreases and the enzyme may denature at extremes.

Common mistakes and how to put them right

• Assuming the iodine end point means all substrate has become maltose.

  Put it right: Iodine only shows starch has disappeared, not the identity of the products.

• Using a different temperature for each pH.

  Put it right: Keep temperature constant, for example using a water bath, so pH is the only independent variable.

• Recording the time when the mixture first turns blue-black.

  Put it right: Record the time when iodine no longer turns blue-black, which is the end point.

Exam tips

• Write a clear method that names the independent variable (pH), dependent variable (time or rate) and at least two control variables.
• Present results in a table and calculate rate as 1 ÷ time, using consistent units such as s⁻¹ or min⁻¹.
• Explain the shape of a rate–pH graph by linking the optimum pH to fastest enzyme action and denaturation at extreme pH values.