Facilitated diffusion occurs when large molecules and charged ions cannot pass freely through the cell’s lipid-based plasma membrane and result in carrier proteins binding to a diffusing molecule. This binding, produces a change in the shape of the carrier that allows the molecules to pass through the protein and across the plasma membrane, being released at the other side.
The effect of temperature on a membrane is similar to that of an enzyme, in that they both denature at high temperatures. The protein pumps allow any molecule through as they lose their specific shape and denature as the bonds that hold their tertiary structures together are broken, and they increase in kinetic energy. Loss in specificity increases the number of protein channels that allow passage of the pigment molecule, as any molecule may enter. If the proteins in the plasma membranes of the beetroot cells become denatured, this is likely to affect the rate at which the beetroot pigment escapes from the cells. As stated in the book ‘Revise AS Biology for OCR, by Fosbery, Gregorgy and Stevens,’ particles are in a state of random motion. Temperature will have an effect on the movement of these particles; namely an increase in temperature will increase the speed of their random motion and therefore increase the rate of diffusion. Phospholipids increase in fluidity as the temperature increases. This results in the molecules moving faster and increasing in kinetic energy.
Prediction:
With all the background information taken into account I feel that as the temperature increases, the colour intensity of the solution will increase also. I predict that at a lower temperature, the beetroot pigment particles will be moving in random motion more slowly, and as a result, diffusion will take longer. As the temperature increases, the particles will move more quickly and diffusion will be faster. This will be evident by an increased pigment leakage from the beetroot, and the solution turning darker. As the proteins in the membrane denature there will be an increased diffusion rate. This is due to the fact that the membrane will no longer be effective in regulating the amount of substances that pass through it and therefore pigment will diffuse out of the beetroot cells at a much quicker rate. Once you reach a temperature where proteins become denatured, the rate of diffusion should be much faster as you have the effect of particles moving quickly, as well as a damaged cell barrier. However, at lower temperatures, you have the effect of fast moving particles, but they still have to cross the intact cell plasma membrane.
My hypothesis is that as the temperature increases, so will the rate of diffusion, and I predict that the colour intensity of the solution will increase in abitrary units.
Preliminary Work:
The experiment involved placing pieces of beetroot in water at various temperatures for a set time then measuring the amount of pigment released, using a colorimeter.
There are many factors to think about in this experiment such as how many pieces of beetroot should I use and what size? Also how am I going to cut the beetroot into uniform pieces? Moreover, how much water do I need to immerse the beetroot in? and how will I measure out the water? The temperature is a major factor and should consist of at least 5 sensible variables. However, I needed to decide how I was going to keep the temperature constant.
I began my preliminary investigation by measuring out 10cm3 of distilled water into 5 sterile test tubes. I did this by using a 10cm3 pipette which is highly accurate, ensuring the best possible results. However, I found that 5cm3 of distilled water emphasised a trend in results more clearly. I labelled each test tube with my name and the corresponding temperature, before placing them in their water baths to equilibrate. The test tube measuring room temperature was left in the test tube rack.
I found it quite difficult to cut identical pieces of beetroot, so I used a ruler to make the width easier to see and measure. After trying an experiment with 3 pieces of beetroot in each test tube, I decided to carry out one with only one piece in each tube and found out that the trend in results was easier to see and the colorimeter did not flash as the absorbance did not exceed 2 arbitrary units. I decided to time the experiment for 3 minutes and shook the test tube every 30 seconds. This increased the rate of diffusion enabling me to deduce a trend from the results. The test tube and its contents did not cool down, as I only took them out of the water bath every 30 seconds to shake them, and I replaced them promptly. As soon as the experiment was finished, and the 3 minutes had expired, I poured the solution into a curvette straight away, to eliminate the chance of further diffusion from the beetroot piece. This would decrease the accuracy of my results.
Here is a table of my results collected during my preliminary investigation:
Apparatus:
Variables:
The independent variable in this experiment will be the temperature of the distilled water. The dependent variable, and therefore the one which I am choosing to investigate, is the rate of diffusion of the beetroot pigment in each temperature condition.
Risk Assessment:
- Water with higher temperatures may cause scolding to exposed skin and should be handled sensibly and carefully.
- The cork borer should be handled with care as it is sharp and may cause injuries if misused.
- The knife used to cut the beetroot is a potential risk if misused as it is very sharp and could cause a serious injury.
Method:
Use a cork borer to cut out a cylinder of beetroot, then use a ruler and scalpel to cut the cylinder into a series of discs with uniform thickness (5mm is probably best) you will need at least 24 discs.
-
Measure out 5cm3 of distilled water into 6 sterile test tubes using a 5 cm3 graduated pipette. Label the test tubes clearly with a waterproof marker stating your name and the corresponding temperature.
-
Place each test tube in a water bath set at the right temperature and leave for a minimum of 5 minutes to equilibrate. The test tube measuring 20oC can be left in a test tube rack to equilibrate at room temperature, as can the control test tube.
- While the test tubes of water are left to equilibrate, the beetroot should be cut into identical discs. To achieve this, simply push a cork borer, facing downwards on a tile, into a whole fresh beetroot. Slide the cylinder of beetroot out carefully onto the tile.
- Using the blade, cut off 10mm from each end of the cylindrical beetroot sample in order to ensure that the outer edges of the root are not used in the experiment, as it may be possible that they could contain a smaller amount of pigment.
- Using the ruler, measure out a minimum of 15 discs of beetroot, all 5mm in width. Try to be as accurate as possible and produce discs of uniform thickness. You may have to bore another cylinder from the beetroot. Try and only use one beetroot as different beetroots naturally contain different levels of pigment.
- Rinse the beetroot discs by placing them in a beaker and adding tap water. This eliminates any pigment which has spilled out of the cut cells. Place the beetroot discs on the tile so that they are ready to use.
- Select a test tube containing distilled water, of a chosen temperature, and using forceps, place one piece of beetroot inside. Start the stop watch immediately and shake the test tube once every 30 seconds. Ensure that any test tube in a water bath is put back while the experiment is taking place, this prevents the test tube or its contents decreasing in temperature and affecting the accuracy of the results.
- After 3 minutes has passed, immediately pour the solution into a cuvette, to eliminate the chance of further diffusion.
- In order to produce a control colorimeter reading, draw some water from the control test tube using a pipette, and transfer to a cuvette. Insert the cuvette into the colorimeter and obtain a reading, taking care to avoid touching the glass as much as possible, to reduce grease deposits on the glass as this may affect the colorimeter reading. The reading should be 0.00 arbitrary units.
-
Pour the solution from the experiment into another cuvette and measure the absorbance.
- Repeat steps 7-10 until each of the temperature have been repeated at least three times.
- Record your results in a table similar to the one below.
Analysis of Results:
- Find the mean absorbance at each temperature
- Plot a graph, showing mean absorbance against temperature.
Bibliography:
Revise AS Biology for OCR, Fosbery, Gregorgy and Stevens, Heinemann
Tools, Techniques and Assessment in Biology, Nelson Advanced Science: Biology, Adds, Larcom et al, Nelson Thornes
Website: