Catalase practical: a conclusion built on a confounded pH 7 trial
At a Year 12 practical, a student investigates how pH affects catalase activity in potato tissue. Five buffer solutions (pH 3, 5, 7, 9 and 11) are tested. For each pH, three potato discs are placed in 10 mL of buffer with 5 mL of 3% hydrogen peroxide, and the height of oxygen foam in the tube is measured after 2 minutes. Results: pH 3 = 5 mm; pH 5 = 31 mm; pH 7 = 48 mm; pH 9 = 22 mm; pH 11 = 4 mm. The student concludes that potato catalase has an optimum at pH 7. During moderation, the teacher notes: the pH 7 discs came from a different potato bought three days earlier; one pH 7 trial overflowed before 2 minutes; disc thickness varied by up to 3 mm; room temperature changed from 18°C to 24°C across the lesson; each pH was tested once.
1. Identify two methodological problems in this investigation and explain, for each, how it could affect the validity of the conclusion that the optimum is pH 7.
Read the worked answer
One problem is that the pH 7 discs came from a different, older potato. Catalase concentration and tissue condition can differ between potatoes, so the pH 7 result may be higher or lower because of the potato itself, not because pH 7 is the enzyme's optimum. This is a confounding variable: the independent variable (pH) is no longer the only difference between treatments. A second problem is that disc thickness varied by up to 3 mm. Catalase acts on hydrogen peroxide at the surface of the tissue, so a thicker or larger disc exposes more enzyme and can produce more foam. If the pH 7 discs happened to be thicker, the 48 mm result could overstate activity at that pH. The overflow in one pH 7 trial is also serious because the measured foam height is capped by the tube, so the true value may be higher and the replicate is not comparable. Temperature changes affect enzyme reaction rate, adding further variation. Together, these problems mean the conclusion is not strongly valid: the peak at pH 7 could be partly caused by the different potato, uneven disc size, measurement ceiling or temperature, rather than by pH alone.
2. Describe how the student should modify the investigation to improve reliability and validity, and explain how the modified data should be processed.
Read the worked answer
To improve validity, the student should use discs cut from the same potato, or at least the same batch, and standardise disc size using a cork borer and ruler. All trials should be run in a water bath at a fixed temperature, such as 25°C, with the same buffer volume, peroxide concentration and timing. A wider range of pH values around the suspected optimum, for example pH 5, 6, 7, 8 and 9, would better locate the peak. To improve reliability, each pH should be tested at least three times, with fresh peroxide each time, and the same person should read the foam height against a ruler at eye level. If foam overflows, a larger tube or a gas syringe should be used so the measurement is not capped. The data should be processed by calculating a mean and range for each pH, plotting mean foam height against pH with error bars, and identifying outliers. The student should compare the pattern across all replicates, not just the highest single value, before concluding where the optimum lies.