The Final Experiment
Diagrams of Apparatus and Equipment Required:
Apparatus List:
Method for Final Experiment:
- Take cork borer (6mm diameter) and bore 90 cylinders out of the beetroots using a cutting board. With a scalpel and cutting matt cut the ends off the tubes and cut each one to 3cm in length to the nearest mm, cut at a 90° angle to ensure the same surface area on cylinders.
- Rinse cylinders in tap water using a sieve for 2 mins.
- Roll for 30 seconds in tissue paper.
- Repeat stages 2 and 3, six times. I will make sure I have removed all the pink as this can affect my 0% ethanol solution reading.
- Heat a water bath to 40°C.
- Label 9 pairs of chop sticks with each concentration.
- Make 200cm³ of each 0% to 80% ethanol solutions by mixing according to the table below. Using a syringe measuring to the nearest 1 cm³ and a 200cm³ beaker.
Remember: After using ethanol, cover up, as it can evaporate into the air and affect people. Also when leaving solutions, cover up, as the evaporation can also change the concentration of the solution, I will use a bung to stop evaporation. When using syringe, ensure that there is no air bubble as it can alter the actually amount in the syringe.
- Take 10 boiling tubes, label for each concentration and put 20 cm³ of each ethanol solution in to them.
- Place the test tubes in the water bath to warm up to the correct temperature for 5mins, and check the temperature to ensure that the correct temperature is reached.
- Begin stop clock when adding the first cylinder to 0% solution, wait 15 secs before adding the next one continue until all 9 cylinders are placed, thus giving an equal time in the solution as I must remove them at the end after the same amount of time – staggered start.
- After 1 min remove 0% and shake for 10 secs in a side to side oscillation and put then return it to the water bath, at 1 min 15 secs remove the next and shake for 10 secs return and carry this on every 15 secs till all have been shaken.
- Repeat step 10 for every minute within the 5 mins.
- After 5mins remove 0% solution and with chop sticks remove beetroot cylinder, again continue every 15 secs to remove test tube and remove cylinder, use the labelled chop sticks according to solution to not mix solutions.
- Give each solution a thorough shake as betalain is denser than water, so to distribute the pigment.
- Calibrate colorimeter using blue filter and distilled water.
- To calibrate and use a colorimeter read section below.
- Take the calorimeter tube and fill to about 1cm from the top with 0% ethanol solution, place in colorimeter in exact position, put the cover on top and read the dial, record result to the nearest 1%, pour the liquid back in the boiling tube and clean and dry the tube thoroughly as other solutions may affect the following result.
- Repeat step 16. for all solutions.
- Repeat stages 9. to 17. ten times.
- Produce an average of each percentage of transmition for every concentration of ethanol by adding all the results up for 1 concentration and dividing by 10.
- Record results in the table below.
Calibrating and Using the Colorimeter
Take the colorimeter tube, and fill with distilled water up to 1cm from the top. Wipe dry the test tube with tissue paper and ensure that there are no marks or moisture on the tube as these could effect the calibration. 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. The more transmition percentage, the less blue light is being absorbed and so the less betalain in the solution.
Constant Variables
There will be some variables which I must ensure remain the same for every part of the experiment as they will have an effect on the results; these are on the following pages.
Safety
Prediction
‘Electron microscopic examinations of cell membranes have led to the development of the lipid bilayer model (also referred to as the fluid-mosaic model). The most common molecule in the model is the phospholipid, which has a polar (hydrophilic) head and two non-polar (hydrophobic) tails.’
(http://www.emc.maricopa.edu/faculty/farabee/biobk/BioBookCELL2.html)
Functions of the molecules:
The phospholipids molecules are made of 1 polar head and 2 fatty acid chains, they form a double layer called a bilayer and are able to move around by diffusion in their layer. The hydrophobic tails are position on the inside of the bilayer as they are repelled from water and form a non-polar hydrophobic interior,
‘Some of the phospholipid tails are saturated and some are unsaturated. The more unsaturated they are, the more fluid the membrane. This is because the unsaturated fatty acid tails are bent and therefore fit together more loosely’ Biology 1
The hydrophilic heads are positioned on the outside of the bilayer facing the aqueous medium surrounding the membrane as they are attracted to it. This accumulation of phospholipids forms the beetroot cell membrane and is 7nm thick and acts as a barrier to most water-soluble substances allowing lipid-soluble substances to pass through freely.
The proteins in a beetroot cell membrane are irregularly arranged and most float around in the bilayer, they can be embedded in the cell membrane in two ways forming either: Extrinsic proteins, were the protein remains on the outside of a bilayer never going completely through the membrane, and are used for structural support or along side glycoproteins as receptors. Or intrinsic proteins were the protein spans through the cell membrane, form one side to another. Most act as transport or channel proteins allowing polar molecules to pass through them, were as others act as enzymes. They also: ‘form ion channels for sodium potassium etc. Act as energy transducers, form recognition sites by identifying cells help cell s adhere to each other’ Essential AS Biology, Glenn and Susan Toole, pg 57.
The cholesterol in the beetroot cell membrane, like phospholipids, has a polar head and non-polar tail and so fits in-between the phospholipids and so also is not soluble to water. These help to regulate the fluidity of the cell membrane, preventing an extreme state occurring by holding the phospholipids tails together but not allowing them to become too rigid, restricting their lateral movement, also they help keep it mechanically stable as at high temperatures they make the membrane less fluid. Cholesterol also prevent leakages of water and soluble ions.
Finally glycolipids and glycoproteins are where a phospholipid or a protein has a short carbohydrate chain attached to them. These only occur on the outside of the beetroot cell membrane, they form hydrogen bonds with the water and so help to stabilise the cell and can help cells attach to one another. Also they form receptor molecules which bind with things such as hormones or neurotransmitters, but these are specific to the cell as to what they bind to. Glycoproteins form antigens which allow cells to recognise each other too.
In a beetroot cell the betalain is such a large molecule that it is too big to fit through any normal membrane therefore making the cell impermeable to the pigment. As a result the substance resides in the vacuole of the beetroot cell. The path in which the betalain must take to diffuse out of the cell into the ethanol solutions is through the tonoplast, the cell membrane and finally the permeable cell wall.
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 δ 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 δ 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.
References: Biology 1, Mary Jones, Richard Fosbery, Dennis Taylor. Pgs: 52 – 54.
Essential AS Biology, Glenn and Susan Toole. Pgs: 56 & 57
http://www.emc.maricopa.edu/faculty/farabee/biobk/BioBookCELL2.html
Final Prediction:
The results from my preliminary experiment are graphed and shown below:
These results show the steady decline in the transmition of light, and then a levelling off.
I predict that as the concentration of ethanol rises the more betalain will be present absorbing the blue light and lowering the transmition percentage. Though more precisely, I predict that the ethanol only gradually increase the permeability at the lower concentrations of 0% to 40% as it is denaturing more and more molecules and bonding with an increasing amount of phospholipids and so the first 4 points on my final graph will show a steady decline from close to 100% transmission down to 30 or 40% transmition. Then a relatively straight line as of 50% ethanol, as the ethanol can have no longer an effect on the cell membrane as it is completely broken down and so the betalain levels remain at a constant as shown in my graph above and below is a smoother estimate of my predictions