Investigating Temperature & Enzyme Activity

This is a required practical. You are finding out how temperature changes the rate at which an enzyme works.

• Enzyme used: amylase (found in saliva and the pancreas).
• Substrate used: starch (a large carbohydrate).
• Reaction: amylase digests (breaks down) starch into maltose (a sugar).

How we see the reaction happening — the iodine test:

• Iodine solution turns blue-black when starch is present.
• When all the starch has been broken down, iodine stays orange-brown (its normal colour).

So we time how long it takes for the blue-black colour to stop appearing — that is the point when all the starch has been digested.

Key idea — what "rate" means here:

• A short time to digest the starch = a fast reaction (high rate).
• A long time = a slow reaction (low rate).
• We work out rate of reaction = 1 ÷ time so we can plot rate against temperature.

Exam tip. Examiners want you to be precise: iodine is orange-brown → blue-black with starch. Saying iodine "turns black" or "tests for sugar" loses marks.

For a valid (fair) test you must control everything except the one factor you are investigating.

Type of variable	In this experiment	Why
Independent (you change)	Temperature of the water bath	This is what we are testing
Dependent (you measure)	Time taken for starch to be fully digested	Tells us the rate
Control (keep the same)	pH, concentration of amylase, volume of amylase, volume of starch, starch concentration	A fair test — only temperature affects the result

Setting the temperatures — use water baths:

• Use a range, e.g. 10, 20, 30, 40, 50 °C (more values = a better graph).
• A water bath keeps each tube at a steady temperature — better than just leaving tubes in the room.
• Let the amylase and starch reach the bath temperature before mixing them, so the reaction starts at the right temperature.

Why control variables matter (a top-grade point):

• If the pH or enzyme concentration also changed, you would not know whether temperature or that other factor caused the change in rate.
• Controlling them lets you say the change in rate was caused only by temperature.

Exam tip. "Same amount of amylase" is too vague — write same volume and same concentration of amylase. Examiner reports flag this constantly.

A clear, repeatable method scores method marks. Here is a reliable version:

1. Add iodine drops to each dimple of a spotting tile (this is your test surface).
2. Measure a fixed volume of starch solution into one tube and a fixed volume of amylase into another.
3. Place both tubes in a water bath at the first temperature (e.g. 10 °C) and leave for 5 minutes to reach that temperature.
4. Mix the amylase and starch together and start a stopwatch.
5. Every 30 seconds, remove one drop of the mixture and add it to a fresh dimple of iodine.
6. Repeat sampling until the iodine stays orange-brown (no blue-black) — this is the end point (all starch digested).
7. Record the time taken.
8. Repeat at each temperature (20, 30, 40, 50 °C).
9. Repeat the whole experiment and calculate a mean time for each temperature.

Why the spotting tile? It lets you sample the reaction repeatedly without affecting the tube, so you catch the exact moment the starch disappears.

Exam tip. Two marks are routinely lost here: (1) forgetting to let the tubes reach temperature before mixing, and (2) not stating you repeat and take a mean.

Recording results — a results table with temperature, time taken, repeats and a mean, then a calculated rate (1 ÷ time):

Temperature (°C)	Mean time (s)	Rate = 1 ÷ time (s⁻¹)
10	300	0.0033
20	150	0.0067
30	90	0.0111
40	60	0.0167
50	240	0.0042

The expected pattern (plot RATE against temperature):

• As temperature rises to the optimum (~37 °C), the rate increases — molecules have more kinetic energy, move faster and collide more often with the substrate.
• At the optimum, the rate is highest (shortest digestion time).
• Above the optimum, the rate falls steeply — the high temperature denatures the enzyme: bonds holding its shape break, the active site changes shape, and the substrate no longer fits.

Exam tip. The curve is not symmetrical: it rises gradually then drops sharply after the optimum because denaturing is sudden. Use the words kinetic energy / collisions for the rise and denatured / active site for the fall.

Higher-mark questions ask you to evaluate the method. Use these three ideas:

1. Reliability (can you trust the results?)

• Repeat each temperature and take a mean — this reduces the effect of random errors and lets you spot anomalies.
• More repeats = more reliable.

2. Validity (is it a fair test?)

• Control pH, enzyme concentration and volumes — otherwise you can't be sure temperature alone caused the change.
• Use a water bath (steady temperature) rather than relying on room temperature.

3. Sources of error and improvements

Problem	Improvement
Judging the colour end point is subjective	Use a colorimeter, or always compare against the same reference
Tube temperature drifts during the test	Keep tubes in the water bath throughout
Sampling every 30 s may miss the exact end point	Sample at shorter intervals (e.g. every 10 s) near the end
Reaction starts before tubes reach temperature	Pre-warm both solutions before mixing

Going deeper for A*: explain why an improvement works. "Sampling every 10 s gives a more precise end-point time, reducing the uncertainty in the measured time and so in the calculated rate."

Exam tip. Don't just say "repeat to make it accurate" — say repeat and take a mean to improve reliability and identify anomalies. Examiners reward precise evaluation language.