Core Practical 3 – From Topic 2 (Genes and Health)
Aim: To investigate membrane structure, including the effect of temperature on membrane permeability.
Independent Variable: The temperature of the water in the water bath (Degrees Celcius)
Dependent Variable: The percentage transmission of light through the resulting solution
- Volume of distilled water – 10cm³ of distilled water should be used to fill the boiling tubes each time
- Time left in water – leave each boiling tube containing beetroot for 30 minutes
- Size of beetroot piece – use a ruler and knife to cut cylindrical beetroot pieces of 1cm in length
- Colorimeter used – same colorimeter should be used on the same blue/green setting each time, measuring percentage absorbance. Calibration with distilled water should be carried out each time
- Volume of beetroot solution – 2cm³ of beetroot solution should be added to a cuvette each time
Why Use Beetroot?: Beetroot cells contain pigment called betalains in their vacuoles. We can observe the effect of temperature on cell membranes in beetroot by observing the leakage of this pigment, indicating the weakening of the cell membrane. Betalains display as a dark purple colour in this case.
- Raw beetroot
- Size 4 cork borer
- White tile
- Water baths
- Boiling tubes
- Colorimeter with cuvettes
- Distilled water
- Filter paper/tissue
Control: Keeping a beetroot piece in 10cm³ of distilled water at room temperature can provide control results.
- Use a cork borer and knife to cut 8 x 1cm lengthed cylinders of beetroot over a white tile.
- Place all the cut pieces in a beaker of distilled water and leave overnight to remove any dye (betalains) released when the beetroot was cut.
- Wash and blot dry (with filter paper or a tissue) the 8 pieces of beetroot.
- Fill 8 boiling tubes each with 10cm³ of distilled water and place them into 8 separate water baths of different temperatures (e.g. 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C).
- Once at the desired temperature, add a piece of beetroot to each boiling tube and leave for 30 minutes.
- Remove the beetroot pieces gently with a pair of forceps and then shake the tubes to disperse the dye.
- Set a colorimeter to percentage absorbance on the blue/green filter. Calibrate by filling a cuvette with distilled water first then add 2cm³ of beetroot solution from the first temperature to a new cuvette.
- Place this cuvette into the colorimeter to read the percentage absorbance. Repeat this for all other pieces.
Results & Calculations: In order to obtain the percentage transmission for each beetroot solution in the colorimeter, we can use the following equation:
Percentage transmission = 100 – percentage absorbance
Recordings can be noted down in an appropriate table as well as a graph.
Conclusion: As temperature increased, the percentage transmission slightly increased to a point at which it greatly increased.
The betalains pigment was contained in the vacuole of the beetroot cells. The cell membrane contains the contents of the cell like a barrier. The cell membrane is made up of the phospholipid bilayer and protein molecules. These proteins are made up of linked amino acids. The hydrogen bonds within the protein determines its 3D shape. However, as heat increased, the hydrogen bonds got weaker due to the increased kinetic energy of individual atoms. Greater kinetic energy created more gaps in the phospholipid bilayer for the betalains to leak through. At a certain point, broken hydrogen bonds caused proteins to change shape and denature – leaving larger holes in the cell membrane. This is when even greater amounts of betalains leaked through the membrane and so coloured a solution more strongly. In summary, an increase in temperature caused further destruction of the cell membrane, which allowed more pigment to leak out via diffusion.
- Some beetroot may have skin on affecting surface area (random error) – use a bigger beetroot and use cork borer to obtain pieces free from skin
- Difficulty in maintaining temperature (random error) – set water baths for higher temperatures and set refrigerators for lower temperatures
- Accurate reading of the colorimeter (systematic error) – use more precise colorimeter and close cap to ensure outside light does not interfere with reading. Make sure that distilled water is used for calibration
- Accurate size of beetroot (random error) – cut many different pieces from different beetroot and use the most similar sized pieces for the experiment
- From the different parts of the root (random error) – use a large beetroot and take all samples from the round part of the root
- Ensuring same amount of time at the different temperatures (random error) – have 7 other helpers to make sure all 8 boiling tubes are extracted at the same time after 30 minutes