How enzyme activity is affected by pH
Investigating the effect of pH on enzymes.
Students are required to investigate how enzyme activity is affected by pH. This can be completed using the amylase and starch reaction as indicated by the colour change in iodine solution. Starch gives a blue-black colour when added to Iodine. Amylase eventually breaks down the starch into glucose, causing no colour change in the presence of Iodine. Students will test their solutions, testing a drop every 10 seconds after the reaction has begun. The use of a Bunsen burner to heat and maintain the temperature of a water bath satisfies the requirement for the safe use of heating equipment in the laboratory.
Equipment (per pair of participants)
• Safety goggles
• 11 x test tubes
• Test tube rack
• Water bath (electronic or a beaker of water)
• Spotting tile
• 5ml syringes
• Disposable pipettes
• 400ml beaker
• Glass rod
• Stopwatch
• Thermometer
• Chinagraph pencil or permanent pen
• Starch solution
• Amylase solution
• Iodine solution
• CLEAPSS Hazcards: 33, 40C, 54
Method
- Students should set up a water bath using a Bunsen burner and carefully heat some water in a 400ml beaker until it reaches 35°C.
- They then label 5 test tubes, each with the pH buffer values they are going to be using.
- 2mls of each buffer solution is added to the appropriately labelled test tubes and these are placed in the water bath.
- Five further test tubes are labelled as ‘Starch’, and 4mls of starch solution is added to each of these tubes and they are placed in the water bath with the other tubes. Placing a thermometer in one of these tubes allows the temperature to be monitored.
- Label a further test tube as ‘Amylase’ and add 12mls of Amylase to this tube, placing it in the water bath with the others.
- Allow the solutions to reach 35°C, monitoring this using the thermometer.
- In the meantime, one drop of Iodine is applied to each of the wells on the spotting tile. Adding a drop of starch solution to the first depression will give students a colour for comparison.
- Once the test tubes in the water bath are at temperature, take one of the test tubes of starch and add 2mls of the first buffer solution being investigated. Stir the mixture using a glass rod.
- Using a pipette, students then add 2mls of amylase solution to the mixture and start their stopwatch. Students must keep stirring the mixture with a glass rod throughout.
- Once ten seconds has elapsed, one drop of the mixture is removed using the glass rod and is added to the next cavity containing iodine on the spotting tile. The glass rod should be quickly rinsed with water.
- The spotting process is repeated, rinsing the glass rod in between, every ten seconds until the students no longer see a colour change when the drop is added to the Iodine spot.
- The number of drops should be recorded in a suitable table.
- The process is then repeated for the other buffer solutions.
- Multiply the number of drops by 10 to get the number of seconds it took for the amylase to fully digest the starch.
Video produced by @revisechemistrywithmrb practical GCSE Chemistry tutorials by a specialist Chemistry teacher with over 25 years experience teaching Chemistry and Biology.
Technician tips
Enzyme experiments can be tricky to get right. All reagents must be trialed before the practical session to ensure the practical will give the results needed in the given session time.
• All reagents should be at room temperature on entering the practical session.
Amylase and starch solutions must always be freshly prepared:
• 1% starch solution is made by making a paste using 1g of starch powder and a few drops of cold water (for every 100mls of solution). Boiling water from a kettle can then be used to dilute this to the final volume. Stir to dissolve the paste and boil further if the solution is cloudy. The starch solution must have been allowed to cool to room temperature before use in the classroom.
• 0.1% Amylase solution is made by dissolving 0.1g of Amylase powder in every 100mls of water. The concentration of Amylase solution can be reduced if the reaction occurs too quickly.
• CLEAPSS practical procedure document contains a printable spotting tile which offers a larger number of wells than a standard spotting tile. These could be printed and laminated for repeated student use: PP048 The Effect of pH on Amylase Digestion.
To do this practical, you will need
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