PAG 5.1.

Question 1

Equipment

Answer 1

• Thermostatically controlled water baths at 30°C, 40°C, 50°C and 60°C containing test tube racks

Equipment (per 4-6 students/groups)

• Colorimeter and green filter

Equipment (per student/group)

• Thermometer
• 10 beetroot cylinders, 30 mm long
• Knife / Scalpel
• Ruler
• White tile
• Paper towel
• 5 test tubes
• 5 cuvettes
• Distilled water
• 10 cm3 syringe
• Marker pen / Chinagraph pencil / OHP pen / Stickers
• Forceps
• Timer
• Test tube rack

Question 2

Procedure

Answer 2

Health and Safety

Take care with the knife when trimming the cylinders of beetroot to the correct length. The water in the water baths over 40°C will be hotter than hand-hot so be aware of this.

1. Take the 5 test tubes and label each with one of the temperatures from 20°C to 60°C.
2. Add 10 cm3 distilled water to each tube using the syringe.
3. Place the correctly labelled test tube in its corresponding water bath for 5 minutes to allow the water to equilibrate to the correct temperature. Record the actual temperature with the thermometer. Leave the tubes in the water baths.
4. Collect 10 beetroot cylinders and trim them all to 30 mm using the knife and ruler on the white tile.
5. Rinse the cylinders under a running tap and pat dry using paper towel.
6. Add 2 cylinders to a tube in each temperature and leave for 15 minutes.
7. Label each of the cuvettes with one of the temperatures from 20°C to 60°C.
8. Remove the tubes from the water bath, carefully swirl once and use the forceps to remove the cylinders. Throw the cylinders into a waste receptacle.
9. One at a time, carefully pour the remaining liquid into the cuvette with the corresponding temperature labelled on it. You should now have 5 cuvettes of showing differing intensities of pigment in the water.
10. Use the colorimeter to measure the absorption for each temperature and record this information in a suitably designed table. The absorption is measured in arbitrary units (AU).
11. Plot a graph of temperature against absorption and draw an appropriate line.

Question 3

Describe the relationship between temperature and the rate of leakage of the pigment from the beetroot cells.

Answer 3

Increasing the temperature leads to more pigment being released. Is non-linear. Students could quote data and units.

Question 4

2. Explain your results and the shape of your graph using theory about membrane structure.

Answer 4

2. Cell surface membrane and tonoplast both become more fluid at higher temperatures. More pigment is released from the beetroot cells as the temperature is increased and the phospholipid bilayer becomes progressively more disaggregated because an increase in kinetic energy increases the movement of the phospholipid molecules that make up the membrane. Higher temperatures (for example, from 55 °C) increased the permeability of the tonoplast and plasma membrane, which have the same structure. Membrane (cell surface membrane and tonoplast) proteins become denatured. Both these create spaces for pigment leakage by diffusion. This occurs faster i.e. more pigment leaks out at higher temperatures.

Question 5

3. What effect might a solvent such as ethanol have on membrane permeability? Explain your answer using ideas about membrane structure.

Answer 5

3. Ethanol will cause membrane disintegration as it forms temporary bonds with the phospholipid heads in the membrane. This causes the phospholipids to move out of place so large gaps form in the membrane allowing pigment to leak out. Ethanol also affects bonding in the membrane proteins leading to denaturation causing further gaps in the membrane. Cholesterol, important in membrane structure, is

Question 6

4. State four limitations of this procedure.

Answer 6

4. Limitations include not washing all the pigment from the beetroot surface, pigment being present in different amounts in different parts of the beetroot, the beetroot cylinders being left in the water for slightly different lengths of time, different amounts of drying/patting, an insufficient temperature range, age of beetroot may vary if more than one is used per student/group, might not have equilibrated to the temperature in the time allowed.