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The Effect of Ethanol Concentration on the Permeability of Beetroot Cell Membranes to Betalain

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Introduction

Experiment to Investigate the Effect of Ethanol Concentration on the Permeability of Beetroot Cell Membranes to Betalain. Preliminary Experiment: I have undergone my preliminary experiment to help me decide on how I am going to do my actual experiment. My preliminary experiment has helped me investigate all the variables for my final experiment. I have found out a suitable length and diameter for my beetroot cylinders, the volume of ethanol I should use and the appropriate concentration range to use, I have also identified a suitable timing for: how long the cylinders should be washed for, how long the cylinders should be submersed in the ethanol for and how long each boiling tube should be shaken for. The method to my preliminary experiment was as follows: 1. Take a beetroot, with a cork borer (6mm diameter) and a cutting mat; bore 6 cylinders out of the beetroot. 2. With a scalpel cut the cylinders into lengths of 30mm, ensuring all are equal and cut at 90�. 3. Heat thermostatically controlled water bath to 40�C. 4. Take 6 boiling tubes; fill with 20 cm� of each concentration within the range 0% to 80% ethanol solution. 5. Put boiling tubes into water bath and leave to heat for 5 mins. 6. After 5 mins, take temperature of boiling tubes, if at correct temperature, put in cylinders in each boiling tube and start the time. 7. Every 1min of the 5mins submersion, remove each tube and shake. 8. After 5 mins remove all boiling tubes, place in boiling tube rack, remove all cylinders from boiling tubes and shake each tube thoroughly to distribute betalain (as betalain is denser than water). 9. Calibrate colorimeter using distilled water. 10. Take colorimetry tube (use same tube, and position exactly the same for each measurement due to change in thickness around glass and in differing tubes) and pour the solution of 0% to a cm off the top, wipe dry the boiling tube and insert in colorimeter, record reading. 11. ...read more.

Middle

Insert in the tube into the hole and mark the position in which it is put. As glass can vary in thickness around the tube and also all test tubes are different this one tube will be used for every measurement made and it has to be placed in the same place every time. Therefore, the tube must be thoroughly cleaned and dried (electricity and water causes a safety hazard, this will minimise the risk) after every solution is measured. Place the opaque cap on top to ensure no light enters and affects the 100% trasmition reading required. Now insert a blue filter, in the experiment I will be using a blue filter because I wish to measure the amount of betalain in the solution and betalain occurs as a pink/red colour when in water. Red and blue are on opposite ends of the visible light spectrum, if I were to use a red filter the light would transmit through easily, where as with a blue filter the blue filtered light will be absorbed by the red in the solution. The amount absorbed will depend on how much red pigment is present, and give a reading of how much blue light is successfully transmitting through. Now the tube is in place I can calibrate the colorimeter. On the left dial click to medium, and turn the right dial so that the hand of the gauge is on 100% exactly, go to eye level to make sure it is precise. If it does not reach 100% then click the left dial to high, and then adjust the right dial accordingly. The colorimeter is now calibrated, remove the test tube, empty, clean and dry it ready to be used again. Ensure that when taking readings insert test tube with solution in it, place hat on and when reading the gauge, go to eye level to ensure you get an exact reading. ...read more.

Conclusion

When beetroot is put into ethanol solutions the effects are as follows: The ethanol is able to form temporary bonds with the phospholipid heads in the bilayer as they are of opposite dipoles; this causes the phospholipids to pull slightly out of places causing big gaps to form allowing large molecules such as betalain to go out of the cell. The proteins, like enzymes, can become denatured by the ethanol due to the same affect as with the phospholipids. 'Denaturants disrupt the hydrogen bonds which maintain secondary structure; they do this by forming hydrogen bonds themselves with all the groups on the protein which can form hydrogen bonds' (http://www.science.siu.edu/microbiology/micr425/425Notes/11-Proteins.html) The slightly ? negative ethanol molecules cause the bonds between the amino acid chains' R-groups (those that can make hydrogen bonds) to break and form bonds with the ethanol instead, as the hydrogen is slightly ? positive; this therefore alters the structure of the protein, thus denaturing it. Also in higher concentrations, the proteins become soluble and begin to dissolve. This occurrence again causes the phospholipids surrounding the protein to move about as there is more room and produce large gaps in the layers to allow big molecules to pass. The cholesterol, as previously stated, was not soluble in water but to ethanol it is soluble, such that in high concentrations it actually begins to dissolve. As the cholesterol is no longer present and is used to hold the phospholipids together, the bilayer comes more fluid. Also as cholesterol generally helps to reduced fluidity, once removed there becomes large gaps in between the relatively freely moving phospholipids causing larger molecules to be able to diffuse out , similar to betalain, therefore the cell membrane is increasingly more permeable. Generally though, ethanol has little or no effect on the glycolipids and glycoproteins in the bilayer. Overall, as ethanol increases in concentration it does increase the permeability of the cell membrane, through denaturing, dissolving and bonding with the individual molecules forming the layers, thus causing large either momentary or permanent gaps, large enough to allow betalain to diffuse out. ...read more.

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Here's what a teacher thought of this essay

3 star(s)

This is a well written report that includes a lot of detail.
1. The introduction section should be at the start and the variables and safety section should be before the preliminary test
2. The sources of information need to be referenced using either the Oxford or Harvard systems
3. The running commentary should be removed as well as the demonstration table of results
***

Marked by teacher Luke Smithen 17/09/2013

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