5.1.5Enzymes

Enzymes and pH — Cambridge IGCSE Co-ordinated Sciences 0654 - Biology Revision Notes & Topical Questions

5 min read 8 parts Lesson 6 of 6

📚 Cambridge IGCSE Co-ordinated Sciences 0654 - Biology Hub / Topical Revision

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Part 1 of 6The aim and the principle

This practical investigates how pH changes the rate at which the enzyme amylase digests starch.

Starch is broken down to maltose.

Blue-black

Iodine solution is used to detect starch: it turns blue-black when starch is present and stays orange-brown when starch has gone.

The time taken for the starch to disappear gives a measure of the rate: a shorter time means a faster rate.

Part 2 of 6Method

Place a drop of iodine solution in each well of a spotting tile.

Measure 2 cm³ of amylase solution into a test tube and add 2 cm³ of buffer solution at a chosen pH (for example pH 4, 5, 6, 7, 8 and 9).

Buffers keep the pH constant.

Place the tube in a water bath at 35 degrees Celsius for a few minutes so that all solutions reach the same temperature.

Add 2 cm³ of starch solution, mix and start a stopwatch.

Every 30 seconds use a clean dropping pipette to transfer one drop of the mixture to a fresh well of iodine solution.

Continue until the iodine remains orange-brown, showing that all the starch has been digested. Record this time.

Repeat for each pH, and repeat each pH at least twice to calculate a mean.

Part 3 of 6Variables and control

The independent variable is the pH, which is changed using buffers.

Dependent variable

The dependent variable is the time taken for starch to disappear. Control variables that must be kept the same include temperature (using a water bath), volume and concentration of the amylase and starch,

and the stirring.

Control

A control can be set up using boiled and cooled amylase or water instead of enzyme,

which should show that starch is not broken down without a working enzyme.

The rate can be calculated as 1000 / time (s) or 1 / time, so that larger numbers mean faster reactions and graphs are easier to interpret. For example, if starch disappears in 120 s, rate = 1 / 120 = 0.0083 per second.

Part 4 of 6Results and conclusion

pH	Time for starch to disappear (s)	Rate (1000 / time)
4	no change at 600 s	0
6	240	4.2
7	90	11.1
8	210	4.8
10	no change at 600 s	0

With data like these, the rate is highest at about pH 7, so the optimum pH of amylase is close to 7. At pH values far from this the enzyme is denatured and the starch remains. The conclusion should state the optimum and refer to the results, and the explanation should refer to the effect of pH on the shape of the active site.

When describing the trend, quote data from the table: for example, "the starch took 90 s to disappear at pH 7 but did not disappear at pH 4 and pH 10".

Results and conclusion

Then explain that extreme pH altered the bonds in the enzyme, so that the shape of the active site changed and starch could no longer fit.

If asked about validity, point out that the experiment is only valid if all other variables stay the same,

and that a more precise optimum could be identified by testing pH 6.5, 7 and 7.5.

Part 5 of 6Safety, accuracy and evaluation

Wear eye protection because iodine stains and can irritate.

Take care with hot water baths and wipe up spills.

Possible sources of error include judging the end point, as the colour change is gradual,

which is a subjective decision, and the time taken between sampling.

To improve reliability, repeat the experiment, use narrower pH intervals near the optimum to find it more accurately, and keep temperature constant.

Another way to measure the rate is to use a colorimeter to track the loss of colour.

Anomalies should be identified and omitted when calculating the mean, and the range of repeat results shows the precision.

The same method can be used to investigate temperature by keeping pH constant and changing the water bath temperature. Using a buffer ensures that the pH stays constant as the reaction proceeds.

Part 6 of 6Exam-style questions

Why is a buffer solution used in this investigation? [1]1 mark
Suggest why the amylase and starch solutions are placed in a water bath before mixing. [2]2 marks
Describe how you would improve the reliability of the results. [2]2 marks

Key terms

Term	Meaning
Buffer	A solution that keeps pH constant.
Independent variable	The variable that is deliberately changed.
Dependent variable	The variable that is measured.
Control variable	A variable kept the same to make the test fair.
Iodine solution	A reagent that turns blue-black with starch.
Reliability	How repeatable the results are.
Anomaly	A result that does not fit the pattern.

Avoid these mistakes

Changing temperature as well as pH. Only the independent variable should change.

Using the same pipette for all samples, which transfers starch or enzyme between wells.

Saying the enzyme "dies" at the wrong pH. It is denatured.

Stating the end point colour wrongly. Starch gives blue-black with iodine; no starch stays orange-brown.

Last updated: 20 October 2026