Cells and organisation · GCSE Biology
Amylase required practical
Run the GCSE Biology amylase–starch practical: iodine end-point, water baths, buffers, 1/time as rate, and how to investigate temperature or pH without mixing up the variables.
Iodine is blue-black with starch and orange-brown without it. The time until the orange-brown end-point is shorter when amylase works faster. Rate can be compared as 1/time.
The important bits
What you need to know
- 1
Aim: investigate the effect of pH (or temperature) on the rate of starch breakdown by amylase.
- 2
Independent variable (AQA RP): pH, using buffer solutions (for example pH 4, 5, 6, 7, 8, 9). Temperature must then be controlled with a water bath. Some centres investigate temperature instead; then pH is buffered and temperature is the IV.
- 3
Dependent variable: time taken for starch to be broken down, judged by iodine no longer turning blue-black (end-point). Rate = 1/time.
- 4
Control variables: volumes and concentrations of starch and amylase, temperature (if pH is IV), pH (if temperature is IV), mixing, and sampling interval.
- 5
Method idea: equilibrate amylase and starch in a water bath, mix, then spot samples into iodine in a dimple tray at regular times (for example every 10 s) until the iodine stays orange-brown.
- 6
Iodine is orange-brown with no starch and blue-black with starch. Do not use Benedict’s test here; Benedict’s detects reducing sugars, not the disappearance of starch.
- 7
A shorter end-point time means a faster rate: more starch–amylase collisions with an intact active site. Very long or infinite time means the enzyme is denatured or pH is far from optimum.
- 8
Plot rate (1/time) against pH or temperature. Expect a peak at the optimum (often near pH 7 for salivary amylase, near 37 °C for temperature).
Quotations worth analysing
Short evidence. Real method.
“Iodine solution stays blue-black while starch is present and turns orange-brown when starch has been broken down.”
The end-point is the first time the iodine does not go blue-black. That time is your raw result; 1/time is the rate.
“rate = 1 / time”
A time of 40 s gives rate 0.025 s⁻¹. A time of 20 s gives 0.050 s⁻¹, twice as fast. Do not plot raw time if the question asks for rate.
“Use a buffer to control pH and a water bath to control temperature.”
One independent variable at a time. If both drift, you cannot say which caused the change in rate.
Go deeper
How do I describe the method so a stranger could repeat it?
Measure equal volumes of starch solution and amylase (for example 2 cm³ each) with a syringe. Place each in a water bath at 35 °C for five minutes so they reach the same temperature. Add a stated volume of buffer of known pH to the amylase. Mix starch into the amylase, start a stopwatch, and every 10 seconds use a pipette to add one drop of the mixture to a drop of iodine in a spotting tile. Record the time when the iodine remains orange-brown. Repeat at each pH, then calculate mean time and rate as 1/time. Safety: iodine stains; water baths can scald. Validity: same starch concentration, same amylase concentration, same temperature, same sampling interval. If you investigate temperature, keep the buffer at pH 7 and change only the bath temperature, including a run above 60 °C to show denaturation.
Go deeper
How should I process and plot the results?
Raw times are inversely related to rate: a good enzyme finishes quickly. Convert each mean time to 1/t so the graph peaks at the optimum instead of dipping. Units of rate are s⁻¹ (or min⁻¹). Draw a smooth curve. For pH, expect a bell shape; for temperature, a climb then a crash. If one pH never reaches the end-point in 5 minutes, you can treat time as >300 s and rate as approximately zero — the enzyme is denatured or far from optimum. Anomalous fast times often mean iodine was too dilute or the tile well was contaminated with an already-digested drop. Repeats and a mean matter. Do not use Benedict’s quantitative colour as a substitute unless the paper specifically sets that alternative method.
Go deeper
What evaluation points do examiners actually want?
The iodine test is discrete (every 10 s), so the true end-point may sit between spots: use a shorter interval to improve resolution. Spotting delays the next sample; a colorimeter measuring iodine absorbance would be more precise. Volumes from dropping pipettes vary; use syringes. Starch suspensions can settle; swirl. Amylase batches differ in activity; use one bottle. Temperature in a beaker water bath drifts; a thermostatically controlled bath is better. Buffers can be labelled wrong; check with a pH probe. The investigation measures disappearance of starch, which is a fair proxy for amylase activity if iodine and starch concentrations stay constant. It does not measure maltose appearing unless you add a sugar test. State that limitation honestly.
See the idea in action
At pH 5, 7 and 9, mean times to the iodine end-point are 120 s, 40 s and 180 s at 35 °C. Rates (1/t) are 0.0083, 0.025 and 0.0056 s⁻¹. Optimum in this range is pH 7: complementary active site and most successful collisions. At pH 9 the active site is losing shape (denaturation), so starch remains longer and iodine stays blue-black for longer. Independent variable: pH. Dependent: time to end-point (then rate). Controls: 35 °C water bath, 2 cm³ starch, 2 cm³ amylase, 1 cm³ buffer, 10 s sampling.
Exam technique
Turn knowledge into marks
State IV (pH or temperature), DV (time to iodine end-point / rate = 1/time), and controls (the other of pH or temperature, volumes, concentrations). Describe blue-black to orange-brown. Never write that iodine tests for sugar.
Common mistakes
Do not give these marks away
- 01
Using Benedict’s reagent instead of iodine to follow starch disappearance.
- 02
Plotting time on the y-axis when the question asks for rate, or forgetting that a long time means a slow rate.
- 03
Changing temperature and pH together, or forgetting a water bath and buffer as controls.
In the amylase practical, iodine in a spotting tile stays blue-black. What does that show?
AAll the starch has been broken down
BStarch is still present
CThe enzyme has been used up
DMaltose is absent
Show the answer
Starch is still present. Iodine is blue-black with starch. The end-point is when it stays orange-brown, meaning amylase has broken the starch down. Enzymes are not used up.
Quick questions
If this is the bit you searched
How does the iodine test work in the amylase practical?
Iodine is blue-black if starch is present and orange-brown if it is not. The time until orange-brown is the end-point for starch digestion.
How do you calculate rate from the amylase experiment?
Rate = 1 / time to the end-point. A shorter time means a higher rate.
What is the independent variable in the AQA amylase required practical?
Usually pH, changed with buffers. Temperature is controlled with a water bath. Some classes investigate temperature instead and hold pH constant.
Why use a buffer in the amylase practical?
Buffers hold pH still so you can compare rates fairly, or so you can set pH as the independent variable without it drifting as the reaction proceeds.