RP5: Required practical activity 5: Amylase and pH
Not started yet — this one needs some love.
Aim: investigate the effect of pH on the rate of reaction of amylase enzyme using continuous sampling.
Independent variable: pH of the buffer solution (e.g. pH 4, 5, 6, 7, 8).
Dependent variable: time taken for amylase to completely digest the starch (seconds); rate of reaction = 1 ÷ time (s⁻¹).
Control variables:
temperature (kept constant using a water bath, e.g. 25 °C)
amylase concentration
starch concentration
volume of each solution
sampling interval (every 30 seconds)
Method
Place one drop of iodine solution into each depression on a spotting tile.
Place labelled test tubes of buffer solution, amylase solution and starch solution in the water bath. Allow them to reach the target temperature.
Add 2 cm³ of buffer solution to a clean test tube.
Use a syringe to add 2 cm³ of amylase solution to the buffer. Mix with a glass rod.
Use a separate syringe to add 2 cm³ of starch solution to the amylase/buffer mixture. Start the stop clock immediately.
After 30 seconds, use the glass rod to remove one drop of the mixture and place it in the first iodine depression on the spotting tile. Rinse the rod with water.
While starch is still present, the iodine turns blue-black. Continue taking one drop every 30 seconds, each time into the next iodine depression.
The endpoint is when the iodine no longer turns blue-black but stays orange-brown — starch has been completely digested. Record the time.
Repeat with buffer solutions of different pH values. Calculate the rate for each: rate = 1 ÷ time (s⁻¹).
Plot a graph of rate (y-axis) against pH (x-axis). The highest rate occurs at the optimum pH.
Safety
Iodine solution is an irritant — wear safety goggles throughout; wash any spills off skin immediately.
Hot water bath — risk of burns and scalds; handle test tubes with a test tube holder; take care removing them from the water bath.
Enzyme allergy — some people are allergic to amylase; inform the teacher if you have a known enzyme allergy before starting.
Naked flames — if using a Bunsen burner to heat a water bath, keep hair tied back and avoid loose clothing near the flame.
Common exam mistakes
Iodine is used to test samples from the reaction mixture on a spotting tile; it is not added to the whole amylase/starch mixture.
Change pH deliberately because it is the independent variable; keep temperature constant because it is a control variable. Temperature also affects enzyme rate, so uncontrolled heating changes the conclusion.
The endpoint is when iodine stays orange-brown because starch has gone; do not say the iodine digests starch or that amylase is the substrate.
Rate is commonly calculated as 1 / time taken. Use consistent units and do not round too early in calculations.