Biology 9700/36 — October/November 2014

Prepare the space below to show:

• the concentration of each enzyme solution
• the volumes of E
• the volumes of W.

Read step 1 to step 13 before proceeding.

Proceed as follows:

1. Prepare all the concentrations of enzyme solutions you have listed in (a)(i) in the containers provided.

You are required to investigate the effect of different concentrations of enzyme solutions on the hydrolysis of triglycerides in milk. The appearance of fatty acids can be detected using the indicator solution B.

Every 30 seconds, for a total of 180 seconds, a sample of milk will be removed from the test-tube and placed on a tile. You will need to have prepared this tile before you add the enzyme solutions.

1. Wipe the tile clean with a damp paper towel and then dry the tile. Label the tile as shown in Fig. 1.1. The numbers indicate the sampling times in seconds. Put two drops of indicator solution, B, on the tile above each sampling time, as shown in Fig. 1.1.

1. Prepare a water-bath between $35\ ^{\circ}\text{C}$ and $40\ ^{\circ}\text{C}$. You will need to add hot water/cold water to maintain the temperature of the water-bath between $35\ ^{\circ}\text{C}$ and $40\ ^{\circ}\text{C}$ for steps 7 to 12.
2. Put $2.5\ \text{cm}^3$ of M into a test-tube.
3. Put $2.5\ \text{cm}^3$ of A into the same test-tube.
4. Repeat steps 4 and 5 for each of the concentrations of enzyme solution you are going to investigate, including the 5% concentration, E.
5. Put all the test-tubes into the water-bath. Allow 2 minutes for the contents of the test-tubes to reach the same temperature as the water-bath.

The reaction will start as soon as you add the enzyme solutions so read steps 8 to 13 before proceeding.

1. Put $2.5\ \text{cm}^3$ of the 5% concentration, E, into one of the test-tubes, leaving the test-tube in the water-bath. Stir the contents of the test-tube and start timing.
2. After 30 seconds use the pipette to transfer two drops of the contents of the test-tube to the drop of B, labelled 30.
3. Repeat step 9, at 30 second intervals, until a drop changes to yellow (end-point).
4. Record the time taken to reach the end-point.
5. If the drop at 180 seconds does not reach the end-point, record 'more than 180' as your result.

Once you have recorded a time for the end-point remove this test-tube from the water-bath and proceed to step 12.

1. Wipe the tile clean with a damp paper towel and then dry the tile. Label the tile again as shown in Fig. 1.1.
2. Repeat steps 8 to 12 with the remaining concentrations of enzyme solutions.

Prepare the space below to record your results.

Identify one significant source of error in measuring the dependent variable in this investigation.

Describe one improvement to this investigation which would increase the confidence in your results.

State two other variables that need to be standardised in this investigation.
Describe how you would standardise each of these variables.

The results are shown in Table 1.1.

Table 1.1

Using the results in Table 1.1, calculate the rate of production of glucose between:

• 4 and 12 minutes

rate = ______ $\text{mg min}^{-1}$

• 12 and 20 minutes.

rate = ______ $\text{mg min}^{-1}$

Plot a graph of the data in Table 1.1.

Explain the reasons for the change in mass of glucose between:

• 0 and 12 minutes
• 12 and 20 minutes.