Amylase practical

Question: how does pH affect amylase?

Amylase digests starch to sugars. Investigate how pH affects the time taken for starch to disappear. A buffer controls pH; a water bath controls temperature.

Use continuous sampling

Put iodine drops in the wells of a spotting tile. Equilibrate the starch, amylase and buffer at the same controlled temperature. Mix measured volumes of starch, amylase and buffer; start the timer. Every 30 seconds, place a fresh sample of the reaction mixture into a fresh iodine drop.

Recognise the endpoint

At first iodine turns blue-black because starch is present. The endpoint is the first sample where iodine stays orange-brown: no starch is detected. Record the time. Use fresh iodine wells and a clean sampling technique to avoid contamination. Do not add iodine to the reaction tube.

Fair test and rate

Change only the pH. Keep temperature, enzyme/starch concentrations and volumes the same. Repeat each pH, identify anomalies and calculate a mean. Sampling every 30 s limits time precision.

These are illustrative results. Shorter time = faster rate. For a fixed endpoint, relative rate = 1 ÷ time. An optimum is the pH giving the fastest rate in the tested range.