The human digestive system: Students should use a continuous sampling technique to determine the time taken to completely digest a starch solution at a range of pH values. Iodine reagent is to be used to test for starch every 30 seconds. Temperature must be controlled by use of a water bath or electric heater.

The idea

This required practical investigates how pH affects the rate at which amylase digests starch. You set up a series of tubes, each containing starch solution, amylase and buffer solution of a chosen pH, and keep them all at the same temperature using a water bath or electric heater. At 30-second intervals you remove a small sample from each tube, add it to iodine reagent on a spotting tile, and record the colour. Starch gives a blue-black colour with iodine; as digestion proceeds the colour becomes progressively less intense, and the time at which no blue-black colour remains is the time taken for starch to be completely digested. Repeating across a range of pH values lets you compare digestion times and identify an optimum pH.

What earns the marks

• A continuous sampling technique is used: small samples are removed from the reaction mixture at regular intervals and tested, rather than testing the whole mixture once at the end.
• Iodine reagent is added to each sample and the colour observed; blue-black indicates starch is still present, while a yellow-brown colour indicates starch has been completely digested.
• Samples are taken and tested every 30 seconds, and the time is recorded when the iodine no longer turns blue-black.
• A range of pH values is investigated, for example using buffer solutions of pH 3, 5, 7, 9 and 11, so that the effect of pH on digestion time can be compared.
• Temperature is controlled by placing tubes in a water bath or using an electric heater, and the same temperature is used for every pH so that temperature is not a confounding variable.
• The volume and concentration of starch solution and amylase, and the volume of buffer, are kept the same in each tube to make the comparison valid.
• Results are presented so that the time taken for complete digestion at each pH can be compared, and a conclusion about the optimum pH is drawn.

Common mistakes and how to put them right

• Testing the whole reaction mixture each time instead of removing a small sample.

  Put it right: Remove a small, equal volume sample with a clean pipette or stirring rod at each 30-second interval so the reaction mixture is not disturbed or used up.

• Recording the first colour change as the end point.

  Put it right: Continue sampling until the iodine stays yellow-brown, because partial digestion still gives a blue-black or purple colour with iodine.

• Allowing tubes to sit at room temperature and assuming this is controlled.

  Put it right: Place all tubes in the same water bath or electric heater at a fixed temperature and allow them to reach that temperature before adding amylase.

Exam tips

• State clearly that the iodine test detects starch, not the enzyme or the product, and describe the colour change from blue-black to yellow-brown.
• Explain why each control matters: same temperature, same volumes and same concentrations make the pH the only variable being changed.
• When describing the method, use the phrase every 30 seconds and make clear that sampling is continuous until the end point is reached for each pH.