The aim of the experiment that was carried out was to see, at what temperature and in which standard solution, does the amount of dye left in the boiling tube had the highest percentage transmission. Beetroot was used for the experiment because it was easier to see the dye after the practical was completed. Putting beetroot cylinders into different boiling tubes, which contained three different standard solutions of Ethanol, they were left for half an hour at a certain temperature to get the dye out.
Once the time was up, the solutions was added to cuvettes and inserted into the colorimeter to test how much light was passed through the sample. Preliminary Work: Before the experiment was started, a preliminary experiment was needed to see if the actual experiment would give a good enough range of results. The preliminary was the same as the experiment but only did the extremes; at 0°C and at 70°C.
From the results, it was easy to see that there is a clear range as at 0°C the highest was at 99% which indicated that hardly any betalain left the cell membrane and the sample in the cuvette was translucent. But then at the other end of the scale at 70°C, the lowest was only 2% transmission, meaning that most of the betalain in the membrane was released and left the cell and mixed with the Ethanol. So from the preliminary work, it was certain to say that there was a range from the highest and lowest temperatures and that for the experiment, there would be a suitable results table to create well presented graphs. Hypothesis:
For the experiment it was believed that ethanol would have the effect on the cell membrane than water and that the more ethanol present, the lower the percentage transmission. So, pure ethanol should have the lower results, than the others and water should have the highest. The phospholipid bilayer which makes up the cell membrane can be described as a fluid structure in which proteins can move about dependent upon the fluidity of the lipids. In a fluid membrane one protein can change place with another or move to attach to another, thus facilitating function. Clearly the amount of fluidity of the membrane influences the function.
Diminishing the fluidity decreases the ability of the proteins to move and interact, while enhancing the fluidity may lead to a disordered mess. Ethanol is shown to fluidize the bilayer and therefore decreasing the proteins ability to move. [1] The ethanol might also impair particular proteins or denature the structure as protein has a tertiary structure. Higher concentrations of ethanol would also destroy hydrogen bonds that clasp the protein structure. As the lipids and proteins in the cell membrane would be destroyed, the pigment is able to leak from the cell at a higher rate due to there being no cell membrane holding it in the cell. 2] The red dye that is in the beetroot called betalain is found within the vacuoles of the eukaryotic cell which leaves the cell once the ethanol has broken the cell membrane down and passes through into the cell and reaches the vacuoles cell and thus damaging its membrane. This process happens much quicker with the more ethanol and therefore meaning that pure ethanol will release more betalain than 50% ethanol. This is because there is twice as much ethanol and can then destroy more of the cell membrane and can then also allow more ethanol to reach the vacuoles.
For 0% ethanol, which is just pure distilled water, osmosis occurs; the movement of water molecules from a region of high concentration to a region of low concentration through a partially permeable membrane. [3] This happens because the water molecules are small enough to pass through the membrane without having to destroy it. When the water goes into the membrane it gets into the vacuoles and therefore pushing all the betalain out of the cell into the beaker and the water replaces it in the beetroot. Risk Assessment: * Using Ethanol – harmful and highly flammable, wear gloves and goggle when handling the chemical.
Try to avoid any contact with the skin and if contact is made, wash off with water immediately.
- Knife – Watch the fingers when cutting the beetroot and also be careful when carrying the knife around the work area.
- Temperature Of Water – Due to the high temperature, the glass beakers and boiling tubes will get hot to handle, so hold them using the rim of the glass. Also, try not to spill any water from the water baths onto the skin as it will be hot and may burn.
- Corer – Keep fingers out of the way when coring the beetroot as the corer is sharp enough to pierce the skin.
Method
- Sections were cut from raw beetroot using a size 4 corer and 15, 1cm length slices were taken from these sections.
- They were then placed in a beaker of distilled water. Left overnight to wash away and excess dye. And after so many hours, the water was changed to help stop dye leaking out.
- The next day, 5 boiling tubes were put into 3 different test tube racks. Then filled one of the racks with 10cm? of pure Ethanol, the second with 3 with 5cm? pure Ethanol and 5cm? of distilled water and then fill the final rack with 10cm? of distilled water. This made up the 100%, 50% and 0% Ethanol).
- Either an ice or water bath was needed so that the correct temperature was to be achieved. The water baths were left for 10 minutes to allow them to get to the right temperature.
- Three beakers were filled with water and placed in either the ice bath or the water bath dependant on what temperature was done. The 5 boiling tubes with pure ethanol were transferred into one beaker, the 5 with half Ethanol and water were put into the second beaker and the 5 with just pure water were placed into the third beaker.
- After some time to allow the water in the beaker and the solutions to get to the same temperature as the ice/water bath, each beetroot slice was added to the boiling tubes. They were left for 30 minutes.
- After the 30 minutes, the boiling tubes were put back into the correctly labelled racks. Each boiling tube was shaken a little to mix the solution and the betalain together.
- Using pipettes, 2cm? of each boiling tube were added to 15 individual cuvettes. An extra cuvette was used as a reference and was filled with pure water. Making sure the colorimeter was at the right setting, each cuvette was placed into the colorimeter with the clear side facing forward.
- Each reading was record, put into a table and then an average table was made. Graphs were created from the average table and then error bars were added to show the range.
Equipment List
- Electric Colorimeter
- Pure Ethanol
- Distilled Water
- Ice
- 15 Boiling Tubes
- 3 Test Tube Racks
- 16 Cuvettes
- 17 Pipettes
- Size 4 Corer
- Knife
- White Tile
- Tweezers
- 4 Glass Beakers
- Water Bath 2 Measuring Cylinders
- A Bunch of Raw Beetroot
- Stopwatch
- Thermometers
Variables
There were a few variables throughout the procedure which were controllable and uncontrollable. The controllable variables were ones that as such were human errors, meaning that if followed correctly; either wouldn’t have happened or were simple errors that with a minor adjustment would have given more reliable and accurate results. Examples of controllable variables were;
- Reading of the meniscus
- Size of the beetroot slices Making sure the colorimeter is at the same setting each time
- Ensuring that I put the cuvettes into the colorimeter correctly.
The uncontrollable variables were those that weren’t because of human error and possibly could’ve been unable to stop the error from occurring. These errors individually, may not change the results significantly, but as an overall view, they could all, together, reduce the accuracy and reliability of the results. Examples of uncontrollable variables were;
- Accuracy of the colorimeter
- Keeping the beetroot the same
- Effect light had on the colorimeter reading. Accuracy of the thermometer
- Accuracy of the water bath
