In order to carry out this investigation safely, after using detergent, hands should be washed before coming in contact with the eyes. When looking up into the boiling tubes from underneath, to compare colour intensities, care must be taken so that no detergent solution is spilt or dripping from the boiling tubes, as it is likely this could come into contact with the eyes. Safety glasses should be worn.
Approximately half a beaker will be filled with detergent, another with distilled water, and another with the 20 beetroot discs. Then, ten of the boiling tubes will be set up in test tube racks. In the first, 10cm2 of 10% detergent solution (supplied) will be added, using the syringe, making a 10% detergent solution. This will be labelled 10%, with masking tape, for easy reference. In the second, using a syringe, 9cm2 of 10% detergent solution and 1cm2 distilled water will be added, making a 9% solution. This should be labelled 9%. In the third, 8cm2 of 10% detergent solution will be added, and 2cm2 of distilled water, using a syringe, making an 8% solution. This should be labelled 8%. 7% will be made by adding 7cm2 10% detergent solution and 3cm2 distilled water. 6% will be made by adding 6cm2 10% detergent solution, and 4cm2 distilled water. 5% will be made by adding 5cm2 10% detergent solution and 5cm2 distilled water. 4% will be made by adding 4cm2 10% detergent solution and 6cm2 distilled water. 3%will be made by adding 3cm2 10% detergent solution and 7cm2 distilled water. 2% will be made by adding 2cm2 10% detergent solution and 8cm2 distilled water. 1% will be made by adding 1cm2 10% detergent solution and 9cm2 distilled water. Distilled water will be used to eliminate impurities that may be found in tapwater. Each tube will be labelled with its respective concentration of detergent solution. A syringe will be used, as to make each concentration as accurate as possible. Each time a new concentration has been made, the syringe should be rinsed with water, so that small amounts of different concentrations do not mix together in the syringe. Although this may not be absolutely necessary, it would be desirable if time permits.
These concentrations have been used, as they cover quite a wide range of concentrations, therefore, a good range of results should be achieved. They are also relatively quick to make, in the available time. It is important that there is 10ml of liquid in each boiling tube in order to keep the test fair (see later). A volume of 10ml of liquid has been chosen, as it is enough to surround the beetroot, and can be made with the volume of detergent supplied.
Before I continue, it is important to mention the control.
Previously, another boiling tube had been set up, with 10ml of distilled water. Two of the fresh beetroot discs were placed in this. It was to be used as a control, so any leakage of pigment from the beetroot can be put down to the increased concentration of detergent, and no other factors. This was left for 30 minutes, and no pigment was in the water, so it can be concluded that any loss of pigment from the beetroot is due to the effect of the detergent.
Small discs of fresh beetroot, of approximate diameter 5mm, are supplied. These have been left to soak for several hours, to remove any excess pigment that would have leaked when the beetroot was cut. The discs must be of identical size and shape, so that, for each disc, there is the same surface area is in contact with the detergent solution. The discs must also be identical as a larger disc would contain more cells containing betacyanins, which would make the colour intensity higher than expected.
In each boiling tubes just set up, using the forceps two discs of beetroot will be placed, and left engulfed for 30 minutes (see problem below). The reason for using two pieces is because not all the beetroot pieces may be of exactly the same thickness so using two pieces would produce a more accurate ‘mean’ amount of beetroot per tube, which will be more comparable to the other tubes. Another reason is that it will also make comparing colour intensities easier, as there will be more pigment let out in each tube. Therefore using two pieces per boiling tube will improve the accuracy of the results, when read by the human eye.
There is a slight problem with the timing procedure. If the stop watch is started straight after the two discs of beetroot have been placed in the 10% solution, by the time the beetroot has been placed in all the other tubes, a few minutes will have passed, meaning the 10% would get the full 30 minutes, whilst the 1% (done last) may only get about 27 minutes – i.e. less time to let out the betacyanins. This is a considerable difference in timing. To overcome this problem, the stop clock will still be started directly after the beetroot has been added to the 10% solution, but the time on the stop clock will be recorded after the beetroot has been added to the 9% detergent solution, and so on. 30 minutes will be added on to each time. This will ensure each concentration will be able to get a full 30 minutes engulfed in the detergent.
During the 30 minutes, two of the unused boiling tubes will be taken. Two identical scales (in mm) will be drawn on two individual pieces of masking tape. The scales should go up to 80mm (8cm). This is so the height of the columns can be measured later on. These scales will then be stuck onto the two boiling tubes. The scales will be stuck on after they have been drawn, so that they are being drawn on a flat surface – not whilst on the side of the boiling tube. The scales must be stuck on straight up (i.e. at 90 degrees to the work surface), and must be drawn accurately. The scales will start from just above the curvature of the boiling tubes.
A letter ‘S’, using another piece of masking tape, will also added to one of the tubes, that tube being used for the standard.
After 30 minutes on the stop clock, the liquid from the 10% detergent concentration will be decanted, so that no further leakage can take place. This will be done by taking the final new boiling tube, and gently pouring the liquid into it, discarding the beetroot from the original tube, and then pouring the liquid back into the original tube. This procedure will be repeated for the 9%, when the stopwatch reads (start time for 9% + 30 minutes). This method of decanting will be repeated for all the other tubes, right down to 1%.
Once all the liquid in each of the ten tubes has been decanted, enough of the 10% will be transferred into the empty boiling tube labelled S, so that the height of liquid in this boiling tube is 1cm. (This is why the scale was needed). This will be the standard, as indicated by a letter S on the tube. The liquid will be transferred by pouring, so that the flow of liquid from to the standard can easily be controlled. Using a syringe would create extra bubbles which would affect the accuracy of the results, as more light would be absorbed by more bubbles.
Now the other tube that has the scale on it will be taken. Enough of the 9% will be added (using a syringe, so that it is easy to control the flow of liquid), so the colour intensity is equal to that of the standard. To make the colour intensities the same, there must be approximately the same amount of pigment particles in the standard and in the comparison.
To do this, enough liquid will be added so that the comparison tube is approximately 1cm high. Then, both boiling tubes will be held above eye level, and will be looked up from underneath. The comparison tube should have a lower intensity than the standard. Therefore, more of the 9% will be added (still using a syringe). If the colour intensity of the 9% becomes greater than that of the standard, some of the 9% will be removed. It is important to note that this cannot be done with the syringe, because as well as the bubbles problem described earlier, the syringe will not be able to get right to the bottom of the boiling tube. Through this method of trial and error, enough of the 9% will be added to make it the same colour intensity of the standard, when viewed from below. The column height of the 9%, in mm will be recorded. This procedure will be repeated for 8% down to 1%, with the column height being recorded in each case, when the colour intensity of each concentration is equal to that of the standard.
In order to keep the test fair, the same volume of liquid must be added to each boiling tube, as a higher volume in one tube would make the colour intensity less than with the correct volume. The liquid must be, as far as possible, the same temperature, otherwise, a higher temperature could mean the membrane is broken due to the heat, and not solely the detergent. The same size discs of beetroot must be used throughout, otherwise larger pieces would leak more betacyanins and make a given detergent concentration have a higher colour intensity, in comparison to the other detergent concentrations. This would affect the accuracy of the results, as a smaller volume of a higher colour intensity would be needed to make it the same as that of the standard.
The following formula will be used to find the percentage intensity:
percentage intensity = (10/height of column) x 100
This will show the percentage intensity of each of the ten different detergent solutions. Height of column will be measured in mm.
This procedure will be repeated with the 8% detergent, successively down to the 1% detergent solution.
Prediction
I predict that an increase in detergent concentration will lead to more damage to the cell membranes, increasing their permeability, and therefore allowing more of the pigment to be released. I predict that if the concentration of detergent is doubled, double the cells will be (at least partly) damaged, which will lead to double the amount of betacyanins being let out of the cells. As explained earlier, the cell membrane is made up of a phospholipid bi-layer. The hydrophobic tails of the detergent molecules are taken up by this bi-layer. The detergent’s hydrophilic heads will then repel the individual phospholipids and this will mean they are ‘disrupted’. The detergent will then enter the cell, and the tonoplast surrounding the membrane.Therefore, the higher concentration of detergent, the more detergent molecules, and this means there is more chance of the extra detergent molecules being taken up by the cell membranes. If more detergent is taken up by the cell membranes, more phospholipids will be repelled, and more cells will have damaged membranes, so more betacyanins will be able to leak out. This means the colour intensity will be greater, and therefore less liquid will be required to make the colour intensity the same as that of the standard.
Bibliography
Biology: A functional approach (second edition) – M.B.V. Roberts
Biology 1 – Cambridge Advanced Sciences/OCR
'Dictionary of Biology' - M. Thain and M. Hickman (Penguin, 1995)
Results
Analysis
From the graph, it can clearly be seen that as the detergent concentration increases, the colour intensity becomes greater – i.e. of a higher percentage. This was because higher detergent concentrations meant more molecules of detergent were taken up by the phospholipids of the cell membrane, therefore more cells had their bi-layer disrupted. This meant than more betacyanins were allowed to leak out of the cell, resulting in a higher colour intensity.
Evaluation
Although this experiment did contain some errors and areas that have room for improvement, I believe that on the whole the results were reasonably accurate and reliable. They give a clear idea of effect of detergent on the permeability of the cell membrane – it increases its permeability.
Although experimental procedure used was good enough to allow a set of reasonable results to be obtained, two of the results obtained were anomalous.
The first anomalous results was for the 6% detergent solution. Here, the result was 22mm, and 45.4% colour intensity. This was too low to fit the line of best fit. An intensity of near to 52% would have been required in order to fit the graph. This result does not support the prediction, as it is of a too low colour intensity for the detergent concentration. The reason for this was that the pieces of beetroot were smaller than the average size. This means there were less cells, therefore there was less betacyanins being stored within the beetroot. Therefore there were less to diffuse out into the detergent solution, which would make the colour intensity lower than it should have been.
The other anomalous result was for the 9% detergent solution. Here, the result, of 71% was much too high. Although this result should have been higher than 8% (Which had a colour intensity of 58.8%), a figure of 62% colour intensity would have been expected, in order to fit the line of best fit. The reason that this was too high was due to tears in the beetroot discs in this tube. This meant that there was a higher surface area of the beetroot in contact with the detergent solution, and therefore more of the betacyanins were able to leak out. This lead to a higher colour intensity, which meant less liquid had to be added in order to get it to the same colour intensity of the standard. As there were just enough beetroot discs to go around the group, it was extremely difficult to find twenty identical pieces. To get over this problem, a microtome should be used. This is a electronic, high precision knife which would cut the beetroot to identical size, meaning each piece of beetroot would have the same (or very similar) amounts of betacyanins. As all pieces would be the same size and shape, each piece would have the same amount of surface area in contact with the detergent. Again, these were not available in school not least due to the high cost of purchase.
Other than the problems just stated there were other problems with the method, which could be improved in order to obtain more accurate results.
The main problem was comparing the two solutions (comparison and standard). This was done by the human eye, which was a rather inaccurate way of measuring the results, not least because there are variations in ordinary human eyesight. To overcome this problem, a colorimeter should have been used. This is an electronic, computer controlled device, which is capable of measuring colour intensity much more accurately than the human eye. A liquid with a higher colour intensity absorbs more light given out by the colorimeter. The colorimeter monitors how much of the light given out is received by the photocell. A higher percentage of light hitting the photocell means the colour intensity is lower, as the liquid has absorbed less light, and therefore more light is hitting the photocell. It is important to note that the same colorimeter should be used throughout the experiment, and must be calibrated correctly. These were not available in school due to the cost and specialist knowledge required.
There were other, more minor problems with the method. One of these problems is the temperature. A higher temperature in one tube would lead to more betacyanins being released, as heat also breaks up the cell membrane. However, this is unlikely to have had an effect on the investigation, as all the water was obtained from the same tank of distilled water. During the 30 minutes, however, changes in room temperature could have changed the water temperature. Again, this is very insignificant, as even if there were small changes it would affect all the tubes equally.
Another problem was that the liquid each tube, after decanting had bubbles on the surface. This may have distorted the light when looking up into the tube from underneath. This may have distorted the light meaning less was able to reach and pass through the solution, making it appear to have a higher colour intensity.
A more serious problem is concerned with the curvature in the boiling tubes. The scale was stuck on above the curvature on each boiling tube. It was difficult to ensure the scale was in the same place on each of the two boiling tubes. This meant that the scale would be slightly inaccurate, meaning the heights in mm were not correct. Using test tubes would overcome this problem, as these are flat bottomed, and therefore the scale could be more accurately applied. Measuring cylinders with pre-printed scales could also be used. However, neither of these pieces of apparatus was available during the investigation.
Finally, a potential minor problem is that if any of the pieces of beetroot were not washed thoroughly, some pigment would have leaked into the detergent solution immediately. Again, this did not happen, as the beetroot was left to soak overnight.
Overall, the main problem was that the investigation was reliant on human eyesight in order to compare the colour intensities. This meant that the results may have been slightly inaccurate. The other problems, such as temperature and whether the beetroot had been washed were much less significant. The other significant problem was the scale on the boiling tube. Overall, despite the problems with the human eyesight, and with the scale, a reasonable set of fairly accurate results were obtained.
Repeating the entire experiment would have been desirable in order to obtain more accurate results, but there was simply not time.
