Investigating the effect of pH on an enzyme-catalysed reaction

Phenolphthalein is an indicator that is pink in alkaline solutions of about pH10, but turns colourless in pH conditions less than 8.3. In this investigation, an alkaline solution of milk, lipase and phenolphthalein will change from pink to colourless as the fat in milk is broken down to form fatty acids (and glycerol) thus reducing the pH to below 8.3. The time taken for this reaction to occur is affected by temperature.

Apparatus

Milk, full-fat or semi-skimmed

Phenolphthalein in a dropper bottle

lipase solution (5g/ 100cmP 3 P)

Sodium carbonate solution (0.05 mol dm –3)

5x Test tubes and rack

2 x 10cm3 syringes/measuring cylinders

2cm 3 syringe

Stirring rod

Thermometer

Water baths set to 15oC, 25oC, 35oC, 45oC and 55oC. Ice

Method

1. Place a beaker of lipase solution in the 25oC water bath.

2. Place 5 cm3 milk, in a test tube.

3. Add 5 drops of phenolphthalein to the test tube.

4. Add 7 cm3 of sodium carbonate solution.

5. Place the test tube in the 25oC water bath for 10 minutes to equilibrate.

6. Add 1 cm3 of lipase from the beaker in the water bath and start the stop clock.

7. Stir the contents of the test tube until the solution loses its pink colour, record the time taken.

8. Repeat steps 1 – 7 for 15oC, 35oC, 45oC and 55oC.