To investigate one of the factors that affects the permeability of cell membranes.

        Effect of chloroform and paraffin oil:

Paraffin oil and chloroform are also organic solvents, and so they dissolve the lipids in the cell membrane in a similar way to that of alcohol. This means that they also increase the permeability of the cell membranes and cause more of the pigment to leak out in the same way that alcohol does.

Effect of pH levels:

The pH levels of the solution which the beetroot cells are left in will also have an affect on the amount of pigment which comes out. Cell membranes have proteins embedded in them, and different pH levels can affect the integrity of proteins and change their shape, which could allow gaps to be formed in the membrane, allowing more of the pigment to come out and therefore increase the permeability.

Effect of detergents:

Detergents affect the permeability because they cause the cell membrane to break down by dissolving the lipids and proteins of the cell, and disrupting the bonds that hold the membrane together. They disrupt the non-covalent interactions that hold the cell membrane together. The extrinsic proteins on the cell membrane can be washed away by salt, but salt cannot remove the intrinsic proteins which go right through the membrane. Only using a harsher method, such as a detergent, can these intrinsic proteins be removed. When the detergent has broken down the lipids and proteins it forms complexes with the lipids and proteins which causes them to precipitate out of solution. Because the detergents break down the membrane like an increase in alcohol and an increase in temperature does, it will allow more of the pigment from the beetroot out of the cells, because there will be gaps in the membrane. This therefore means that an increase in concentration of detergents will mean an increase in the permeability of cell membranes.

(Sources of information:

chem.dept.uwsp.edu/tzamis/lab/onion_dna_lab.pdf)

Effect of salt (sodium chloride):

Both chloride and sulphate salts can affect the membrane stability and conformation. When there is a high concentration of salt, the sodium can displace part of the cell membrane. In cotton root hairs for example, the sodium displaces calcium from the plasma membrane, which increases the permeability, allowing larger molecules to leak through. Therefore when the concentration of salt around the beetroot cell increases, the permeability will increase also, and the pigment molecules will leak through the membrane into the surrounding solution.

The factor which I am going to investigate is the effect of alcohol on the permeability of cell membranes.

My hypothesis is that as the concentration of alcohol increases, the permeability of the membranes will increase, and more of the pigment will leak out.

Equipment:

• 6 Test Tubes
• Distilled water
• Alcohol
• Beetroot
• Measuring cylinder (10ml)
• Stopwatch
• Colorimeter
• Knife
• Cork borer
• Ruler

Method:

Here is a diagram to show how the experiment will be set up:

I will begin the experiment by cutting up the beetroot into 6 equal pieces. This will be done using a cork borer to ensure they are of an equal size, and the beetroot cut from the cork borer will be cut into six 2mm strips. These will then be rinsed under the tap to wash off all the excess pigment released from cutting the beetroot, so that this pigment does not affect the results. When I have all the beetroot pieces I will measure out the different solutions which they will be placed in, and set up the test tubes. The first one will be a control experiment, where the beetroot is placed in 10ml of water, with no alcohol, so that the results can be compared to this control result. I will use the measuring cylinder to measure out 10ml of water and put this in test tube 1. The next tube, 2, will have 2ml of alcohol and 8ml of water, giving it a 20% alcohol concentration. I will then measure out the solution for test tube 3, which will have 40% alcohol concentration, with 4ml alcohol and 6ml water. Test tube 4 will have 60% alcohol concentration with 6ml of alcohol and 4ml of water. Test tube 5 will have 80% concentration of alcohol with 8ml of alcohol and 2ml of water, and the final tube will have 10ml of alcohol and no water. When each of these 6 tubes have been set up, I will place a piece of beetroot in each of the tubes and begin timing using the stopwatch. I will then leave the experiment for 30 minutes.

When the 30 minutes is up, I will take the beetroot pieces out of each of the tubes, and I will then begin to measure how much pigment has come out. There are two ways in which this can be done. The first is a very subjective method, in which you give each tube an arbitrary value for the redness of the solution, with the darkest red being 10, and the lightest red being 1. The second method is more scientific and will give more accurate results to work from. Using a colorimeter I will measure the amount of light which will be transmitted through each solution. This is done by putting a sample of the solution into the colorimeter, and sending a beam of light through the solution, then reading off the value showing the amount transmitted through. The more light which transmits through, the less pigment present in the solution. After I have done the whole experiment once, I will repeat the experiment a further two times, so that I will have three sets of results. This will help to improve the validity of the results.

Fair Test:

To keep the experiment a fair test the only factor which I will change each time will be the concentration of the alcohol. I will have to ensure that all other factors are kept the same. For example, from the research I have done, I have found out that temperature can increase permeability, so I will have to ensure that each experiment is done at the same temperature to stop the permeability increasing or decreasing more than it should. Another factor which I will have to keep the same is the amount of time that each experiment is left for. If one experiment is left for longer than the others then it will leak out more of the pigment simply because it has more time for it to leak out, not because the permeability has increased. To make sure the results are as accurate as possible I will change only the concentration of alcohol each time, and make sure that every other variable is kept constant for each experiment.

Reliability:

To make the results as reliable as possible so that valid conclusions can be made I will try to repeat the experiment as many times as possible. I will try to get three sets of results, so that an average can be taken. This will help to make the results more reliable and make the possibility that the results happened by chance less likely.

Safety:

To ensure that I carry out the experiment safely I will take a number of safety precautions. I will wear goggles, and I will be careful when using the alcohol as it is flammable. As I will be using glassware, I will be careful not to break it, and will be careful when using sharp instruments such as the cork borer and the knife.

Implementing:

While carrying out the practical I ensured that I did the following for safety reasons:

• Wear goggles because I was using glass equipment and alcohol.
• Work away from people using naked flames, because alcohol is flammable.
• Put bungs in all test tubes because alcohol is flammable.

When making measurements, the accuracy of the measurements depended on the equipment that I used, so I tried to use equipment that would give me as accurate results as possible. For measuring out the distilled water and the alcohol I used a 10cm³ pipette, and made measurements to ±0.05cm³. When making measurements using the colorimeter, I made them to ±1%, because that was all the equipment would allow.

Results:

Conclusion:

My results show that, generally, as you increase the concentration of alcohol in the solution the beetroot is placed in, the light transmitted through the solution decreases. This decrease in light transmitted through the solution is because more pigment molecules from the beetroot are getting in the way, so when you increase the concentration of alcohol, more pigment leaks out. Therefore I can conclude that increasing the concentration of alcohol increases the permeability of cell membranes.

My graph shows that at 0% alcohol, around 56.7% (3dps) of the light is transmitted through. As you then increase the percentage of alcohol the amount of light transmitted decreases, and the line of best fit starts with a curve. However when you reach 40% alcohol concentration, as you increase the concentration of alcohol further the amount of light transmitted through continues to decrease, but begins to be directly proportional to the alcohol concentration, creating a straight line on the graph. The lowest amount of light transmitted through was 21%, which was with a concentration of alcohol of 100%.

Using scientific knowledge, I can say that this pattern is generally correct, and the permeability should increase as you increase the alcohol concentration. Alcohol is an organic solvent, which means it is carbon based. I have found out that cell membranes are actually a phospholipid bilayer with proteins embedded in them. 45% of the membrane is made of phospholipids, and, as an organic solvent, alcohol can dissolve this phospholipid part of the membrane. Beetroot cells have a red pigment in them, which gives the beetroot its red colour. The pigment molecules cannot normally leave the cell because they are too big. However, when alcohol is present, part of the membrane can be dissolved, which will destroy it, leaving gaps big enough for the molecules to escape out of and enter the solution. Here is a diagram to explain this:

When you increase the concentration of alcohol, there will be more alcohol molecules surrounding the membrane, and so there is more chance that the alcohol molecules will reach the membrane and dissolve the phospholipid layer. If more of the membrane is dissolved when you increase the alcohol concentration, then there will be more gaps for the pigment to leak out of, and so more pigment molecules will escape into the surrounding solution. Therefore, the permeability is increased. When more molecules have escaped into the surrounding solution there are more pigment molecules in the way, to block the light, which is why the amount of light transmitted decreases as you increase the concentration.

If the alcohol dissolves the membrane then when there is no alcohol present, as in my control experiment, no pigment molecules should be able to escape because there will be no damaged membrane, and so 100% of the light should transmit through. I did not however find this was the case, so there must be some other explanation as to why pigment molecules still escaped when there was no alcohol. I cannot find any scientific reason, but it might be due simply to the fact that, although I tried to wash most of the pigment off, when the beetroot was cut, the outer membranes of the slice were all damaged and so a lot of pigment was released. If not all this pigment was removed, it may have entered the distilled water, and so perhaps, the line should not curve in the way it does, and perhaps my results were anomalous due to inaccuracy. Only further investigation and repeated experiments could determine whether this is in fact the case.

To conclude, I have found that the presence of alcohol does affect the permeability of cell membranes. As you increase the concentration of alcohol, the permeability of a cell membrane increases, because the alcohol dissolves the phospholipid bilayer, creating gaps in the membrane which can allow large molecules which cannot usually pass through, to escape from the cell.

Evaluation:

I generally obtained the results that I expected for my practical and therefore the procedure was generally suitable for the task which I used it for. I have drawn a line of best fit on my graph, which all my points are very near to, but as I said in my conclusion, I am unsure about my result at 0% alcohol concentration, and there is a possibility that the result is incorrect because it does not relate to scientific knowledge, and the curve part of my line of best fit is wrong. Therefore my results for 0% alcohol concentration and 20% alcohol concentration may be anomalous.

There are many factors which could have affected my results and caused error. Here are the sources of error (greatest first) that I think may have caused my possible anomalous results or affected my other results:

• Size and shape of beetroot pieces:

Although I used a cork borer to cut through the beetroot to ensure the strips were all of an equal diameter, when cutting this down into the 6 beetroot strips the only equipment I had to use was a knife and ruler. This resulted in the chips not being of a totally even size due to the difficulty of cutting and measuring with the equipment available. This could have a big impact on the results because if a strip has a bigger surface area, then there is a bigger area for the alcohol to work on and for the pigment to leak out of so it would seem as though the permeability was greater. To minimise this error you could use better equipment, such as machinery programmed to cut the tube cut from the cork borer into identical sized slices. This would then rule out this factor as a major source of error.

• Rinsing of freshly cut beetroot:

After cutting the beetroot, there was a lot of excess pigment, because of the membranes which were damaged in the process. I tried to wash all the beetroot pieces after cutting to remove this excess pigment because it would be added to any pigment released from membranes destroyed by alcohol. However I perhaps did not wash them for long enough, and this could be the reason why I got the anomalous results at 0% and 20% alcohol concentration. If there was no alcohol present then no pigment should have been released, but quite a lot of pigment was present in the water. This could be explained by the beetroot piece not being washed for long enough, and so the pigment present in the water was from when the beetroot was first cut. To minimise this error you would have to try and wash the beetroot pieces off for longer, for example 5 minutes, to ensure all the pigment was washed away, so that it could no longer affect the results.

• Accuracy of measurements taken from colorimeter:

The colorimeter had an accuracy of ±1%. This did give me satisfactory results, but using a colorimeter that was perhaps digital, and gave readings to a number of decimal places might give more accurate results. The dial on the colorimeter which I used was quite small and could be confusing, so there was a chance that errors were made when taking readings off the colorimeter. Therefore using one which was digital would make this procedure more accurate.

 

• Time which the experiments were left for:

All the tubes were left for just about the same amount of time, (30 minutes) however it was impossible to put the beetroot pieces in at exactly the same time, and take them out at exactly the same time, so there was up to around 30 seconds difference between the times that the first and last tubes were left for. Although this would not have a great effect on the results, it may have given the one left for longer the chance to lose a bit more pigment. To minimise this error you could do the experiments at different times, so that you could concentrate on just one tube at a time. However this would be very time consuming, and was not possible for me to do.

• Accuracy of measurements of alcohol and water:

I used a fairly accurate pipette to measure out the quantities of alcohol and water which I needed to use. The accuracy was ±0.05cm³. Therefore the measurements should have been fairly accurate. The only way that this could really become a source of error in the procedure and possibly have created the anomalous results is if I made a mistake in measuring out the quantities. However repeating the experiment would show if this was the case. The only way to minimise human error such as this is to repeat the experiment a number of times.

For reliability of my results I repeated the experiment 3 times. If there were any anomalous results during one experiment due to human error this could be discarded if I could see that during the other two experiments a completely different result was obtained. Although it would not help if the problems encountered were due to the limitations of the procedure itself, I think that by repeating my experiment this number of times and taking an average, I have been able to gain results that I can presume are fairly accurate.

I think that because of the number of sources of error I have found within my practical procedure, and the fact that results which could be anomalous were obtained, I cannot really draw valid conclusions from my practical. With the greater sources of error such as the size and shape of the beetroot pieces and time that they were washed for potentially affecting my results to a significant extent, I do not think I can say that my final conclusions drawn are entirely valid.