The Enzyme Run That Peaked at a Tube Nobody Trusts
Ananya is investigating the effect of pH on salivary amylase. She labels five tubes, puts 5 cm³ of 1% starch solution and 1 cm³ of buffer at pH 4, 5, 6, 7 and 8 into each, adds 1 cm³ of freshly diluted saliva, and starts a timer. Every minute she removes one drop and tests it with iodine on a spotting tile, recording the time until no blue-black colour appears. Results: pH 4 - 18 min; pH 5 - 9 min; pH 6 - 4 min; pH 7 - 12 min; pH 8 - no blue-black colour at zero minutes. She concludes that the optimum pH is 6. Her partner points out that the pH 8 tube was never blue at all, that every tube was read by eye against a white tile, and that the saliva was diluted 'until it looked about right'. The teacher asks for one graph and a conclusion that can be defended.
1. State exactly what Ananya's results support and what they do not, paying particular attention to the pH 8 tube.
Read the worked answer
Only the middle three readings can carry weight. pH 6 gave the shortest time (4 min) among valid results, with pH 5 at 9 min and pH 7 at 12 min. That supports the claim 'activity is highest at pH 6 among the pH values tested'. It does not support 'the optimum pH is 6', for two reasons. First, the pH 8 tube showed no blue-black colour at zero minutes, which means either starch was missing from the start, the iodine had failed, or something other than the intended reaction removed the starch. That reading is invalid, not fast - yet if it were plotted as a valid 'instant' result, the graph would peak at pH 8 and contradict her own conclusion. Second, readings exist only at whole pH units, so the true peak could lie at 5.5 or 6.5. A defensible conclusion names both the invalidity and the gap.
2. List the controls and protocol changes needed before the experiment can be repeated, and explain what each one rules out.
Read the worked answer
Add: (i) a zero-time tube at each pH, read before the enzyme goes in, proving that starch and iodine are working - this would have caught the pH 8 anomaly immediately; (ii) a boiled-enzyme tube at pH 6, showing that colour loss is enzymatic rather than acid hydrolysis of starch; (iii) a buffer-only tube at each pH with no enzyme, separating pH effects on the starch–iodine reaction from effects on the enzyme; (iv) one enzyme batch, one dilution, one temperature (a 37 °C water bath), one starch volume and concentration across all tubes - 'diluted until it looked about right' is not a measurable variable; (v) at least three replicates per pH. Reading should be quantitative: a colorimeter tracking absorbance of the iodine–starch complex at fixed intervals yields a rate, not a stopwatch judgement against a white tile. Then rerun pH 8 with fresh reagents to see whether the anomaly repeats.
3. Why does the one-minute sampling interval matter, and how should she locate the peak more precisely?
Read the worked answer
The measurement is coarse and uneven. A one-minute interval means 4, 9 and 12 minutes differ by only a handful of sampling steps, so a true peak between pH 5 and pH 7 could be sharper or flatter than the five-point graph suggests, and small differences between adjacent tubes may be within reading error. Exam answers earn credit for stating the resolution of the instrument and what it can and cannot localise. She should test pH 5.5, 6.0 and 6.5, plus 7.5 to confirm the fall, using the same protocol, and plot rate (1/time, or absorbance change per minute) against pH with error bars from replicates. If the extra points still put the maximum near 6, she may write 'optimum approximately 6.0'; if 5.5 and 6.5 turn out equal, she should say the optimum lies between them. That is the difference between describing four numbers and defending a conclusion.