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Chloe Kazantzis

How does temperature affect the diffusion of pigment across beetroot cell walls?
Introduction The dark red and purple pigments in beetroot are located in the cell vacuole and are chemical compounds called betalains. The colour dye is held together by the membrane structure which maintains the red rich colour in beetroot. The pigments cannot pass through membranes, but can pass through the cellulose cell walls if the membranes are disrupted; this occurs if for example they are heated, which will denature an enzymes active site. I predict that the increase of the temperature on the beetroot will increase the rate of diffusion of the colour dye in the beetroot into the solution. Variables The independent variable in this experiment is the temperature of the water bath, which we will need to keep as accurate to the desired temperature as possible. This will be done by making sure no windows or doors are left open to ensure the room temperature remains as constant as possible. We will make sure that the temperature is correct for each water bath using a thermometer. The dependent variable in this experiment is the absorbency of pigment. This measurement will be recorded using a colorimeter. Controlled variables in this experiment will be the size of the beetroot pieces (their surface area and volume), the volume of water in the test tubes, and the length of time the beetroot pieces spend in the water baths. We will need to make sure that we handle the beetroots with the same treatment so that it is fair. Safety Lab coats should be worn as beetroot stains are difficult to remove from clothing. Care should be taken when handling knives and borer as they can cause damage if not handled correctly. The water baths at high temperatures should also be used mindfully, as this could burn skin. Spillages of water on the floor should be cleaned as soon as possible to avoid people tripping over.

Chloe Kazantzis

Equipment list

Raw beetroot Cork borer White tile Knife Ruler Beaker Water baths at 070C (10C difference) 8 boiling tubes 8 boiling tube racks Crushed ice Thermometer (1 per bath) Colorimeter

Specimen needed to use as a model of cell membranes in order to investigate the effect temperature has on diffusion of betalains To precisely cut the beetroot into the same shaped cylinder To cut beetroot on so that it does not stain work surface or clothing To precisely cut the pieces of beetroot To measure precisely each piece of beetroot so they are the same length To keep the beetroot soaked in water overnight Water baths are needed to maintain the temperature of water, which are needed to investigate the effect of temperature on beetroot Used to keep each sample of beetroot in 5cm of distilled water Used to keep each boiling tube upright when placed in water bath In order to maintain a 0C temperature In order to check each water bath is the correct temperature, also to measure when bath is at desired temperature In order to measure pigment absorbency, which would not be accurate enough to do with naked eye alone Tubes which are put in to colorimeter, in which we will pour the coloured water from the beetroot in the test tubes In order to measure 30 minutes exactly, for the time of which we needed to keep the beetroot samples in the water baths Needed in order to add to the beetroot pieces, so that pigment can diffuse through water and can then be measured using the colorimeter In order to measure the distilled water precisely To prevent staining on hands. Also they ensure cleanliness, some oils on hands may affect the results Prevents staining on clothes

Cuvettes Stop clock

Distilled water Pipettes Gloves Lab coats

Chloe Kazantzis

Method 1. Use a size 4 cork borer to cut sections from the beetroot. Place these on the white tile, and then carefully cut them into 1cm long slices. Using a ruler, ensure that all pieces are equal in length. 2. Place the slices into a beaker of distilled water, and leave overnight to get rid of excess dye. 3. On the following day, use the pipette to measure 5cm of distilled water into each of the boiling tubes. 4. Label the tubes with temperatures of water baths and place them accordingly. Leave each inside the water for 5 minutes so that the water reaches the required temperature. Use the thermometers to ensure that the distilled water is at desired temperature. 5. Place one of the beetroot sections into each of the boiling tubes. Set the stop clock and leave for 30 minutes in the water baths. 6. Carefully remove beetroot. It can be done with tweezers or any other technique that ensures gentle handling of the slices, so that more pigment won't leak. 7. Shake the solutions to disperse the dye evenly. 8. Turn on the colorimeter and set it to % absorbance. 9. Set the filter to blue/green. 10. Fill a cuvette with distilled water. . Place this in the colorimeter and adjust to read 0 absorbance for clear water. 11. Fill 2cm of dye solution into a cuvette and take a reading. Record the results. 12. Repeat the readings for all of the temperatures.


Temperature C

Absorbency (%)
2nd 3rd Repeated tests Mean

0 10 20 30 40 50 60 70

0.06 0.10 0.16 0.33 0.32 0.47 0.70 1.12

0.09 0.15 0.15 0.21 0.28 0.64 0.92 1.22

0.04 0.13 0.37 0.41 0.65 0.82 0.97 1.11

0.12 0.32 0.22

0.06 0.13 0.14 0.29 0.27 0.64 0.86 1.15

Chloe Kazantzis

Interpretation The results have shown that as temperature is increased the rate of diffusion also increases. This shows that the higher the permeability of the membrane, the more pigment leaks out of the cell. From the graph one can see that from 0-20 C the absorbency percentage has only raised slightly, which is because the membrane is rigid and so not much pigment can leak out. From 20-40 C the absorbency percentage has increased due to some proteins which are starting to denature and so more pigments can leak out. Finally, from 40-70 C the absorbency gradient is at its steepest. The water expands due to the rise in temperature, and this puts pressure on the membrane. Most of the proteins have denatured, the structure of the membrane is damaged, and so much more of the pigment is able to pass through. Evaluation There were some errors in our experiment which could compromise the reliability of our results. Such as, we had no control over what beetroot we were given, so we may have been using a different variety of beetroot which may have had a more permeable membrane to begin with, which would give us less accurate results. Also, because our data was a collective of the best results from the class, each group may have treated their beetroot differently; we may not have each followed the exact same procedure for the experiment. For example we may have used different amounts of distilled water, which would affect the absorbency readings. Therefore, this has limited the quality of our results. To make it more reliable in the future we should carry out our own repeated tests, and then we will know that we have carried out is the same each time. Our results could have been more accurate had we of known how to use the colorimeter machine. Because this piece of apparatus was unfamiliar to us, we may have used it incorrectly, for example we may not have reset the machine correctly, and therefore this could mean inaccurate readings. If we were to use the machine more accurately this could reduce our error margins. However, taking everything into account, I believe our experiment to be fairly valid. We have repeated this experiment three times, which meant that we can take into account the outliers, and we have made repeated tests for these results. If we were to carry out more tests, it would make it easier to identify outliers.