The Effect of Alcohol Concentration on the Leakage of Pigment from Beetroot Cells - Report Example

The Effect of Alcohol Concentration on the Leakage of Pigment from Beetroot Cells The beetroot has high content of betalain. This betalain is the pigment responsible for the color of the beetroot. It is a red- purple pigment. Betalain is present inside the cell and it is protected by the tonoplast. Betalains are water-soluble and exists as internal salts in the sap vacuoles. The cell membrane is the most important part of a cell. It controls the movement of the molecules inside and outside of the cell. The cell membrane is linked to the inner organelles also. This cell membrane also acts like a wall to prevent the entry of the unwanted molecules. The cell membrane is made up of phospholipids molecules and proteins. Figure : The structure of the cell membrane showing the phospholipid bilayer with the protein molecules in between them. The phospholipids are present as a bilayer and that acts as the semi permeable membrane. The phospholipid bi-layer is formed because of the hydrophilic and the hydrophobic ends of the phospholipid molecule. The water loving hydrophilic head is present in the inner region of the membrane and the hydrophobic water hating tail region is present on the outer membrane region. This arrangement prevents the polar solutes from moving in and out of the membrane. This arrangement generally allows the passive diffusion of the hydrophobic molecules. The entire membrane is held intact because of the non-covalent interaction of the hydrophobic tails with a little fluidity.The betalains pigment of beet root is sequestered in the vacuole of the beet root cells. When the membrane condition is altered because of temperature variation, pH variations, the membrane becomes weak and loses its pigment content. The phospholipid bi-layer is soluble in an organic solvent. Ethanol is the most common organic solvent. When ethanol is used for the extraction of the pigment from the beetroot, it dissolves the phosphor lipid bi layer and thus makes the membrane permeable. The betalains thus diffuses out of the membrane due to the concentration gradient. As this is a water soluble pigment, it results in the color of the solution. The intensity of the red-purple color in the solution will indicate the diffusivity level of the pigment. Hence the effect of concentration of ethanol may increase the color of the solution. This is a predictive hypothesis. The predictive hypothesis will give a tentative answer to the question that is under the investigation. (faculty.uca.edu). So the experiment was designed such that the independent variable ethanol is applied to the experimental group and it is controlled in the control group. According to our hypothesis, the control group had 0% ethanol and the experimental group had the varying concentration of the ethanol from 20% to 100% varying at a concentration range of 20%. It was found from the experiments that the rate of diffusion of the betalain increases with the ethanol concentration. S.no Concentration of Alcohol in the solution (%) Absorbance value of the red pigment at 470 nm. 1 0% 0.079 2 20% 0.105 3 40% 0.351 4 60% 0.702 5 80% 1.175 6 100% 1.081 The pigment concentration in the solution was found to increase with the increase in the concentration of the ethanol starting from 0% to 100%. Here the cause and effect analysis is carried out. The dependent variables are the cause and the independent variables are the effects. The dependent variable here is the amount of leakage of the betalain pigment. The independent variable is the concentration of the ethanol. Even the independent variable can be the incubation time. If we obtain a linear or exponential curve then the cause has a good response and if non-linear then the response is very bad and our hypothesis is wrong. The graph used for the comparison is a line graph. Line graph helps us to indicate the maximum value more accurately with a greater comparison. The pigment concentration increased gradually from the 0% to 80%. When the concentration was 100%, the pigment concentration was found to decrease. So it is inferred that the maximum betalain pigment concentration in the ethanol is at 80%.There was a very less difference in the percentage of absorbance between the 80% and 100%.The percentage of variation between the two readings is 8%. This is a good percentage of variation between the results. But when the percentage of variation was found for the concentrations between 0-20%, 20-40%, 40-60%, 60-80%, it was found that they had a variation ranging from 25%, 71%, 50% and 41%. The reason for the greater difference in the variation for the treatment condition (i.e based on the effect) may be due to the variation in the time of treatment, small variation in the volume of the solvent used, the total number of the beet root discs used for a single treatment and the thickness of the beet root slices. Similarly the variation in the intensity of the color or the diffusion of the pigment may be due to many reasons. Some of them may be the time, volume of the solvent, degree of shaking of the tubes etc., (fed.cuhk.edu.hk) So from the above results it was concluded that the reasons for the leakage of the pigment may be due to the concentration of the solvent, pH of the solution, temperature of the solution, presence of the amphipathic molecules, the thickness of the slices used in the experiment, the slices were from the different parts of the beetroot, age of the beetroot, light intensity, colorimeter etc., (merspi.com.au, Hamilton 2007). The reason for the smaller variation between the results for the 80% and 100% concentration of the ethanol may be due to the fact that the cell membrane of the beetroot might have got completely broken down at the 80% concentration itself and increasing the concentration of the ethanol beyond that point have no effect on the cell membrane. The second reason for the small variation may be due to the fact that the beetroot slices might have not been uniform in thickness and length. If the thickness of the beetroot slice is varied then the region of exposure to the solvent also varies. This results in the errors. The third reason for the small variation of the result may be due to the surface area of the slices. The larger is the surface area, the greater is the exposure. Hence the surface area must be uniform for all the slices. To increase the surface area, the beetroot must be cutted down to the smallest piece possible. But while doing so, the eruption of the cell membrane due to the level of damage increases. This also results in the variation of the result. (saps.plantsci.cam.ac.uk 2010) Another reason for the variation in the result may be due to the error in the handling. When the beetroot was cutted into small pieces, the cell membrane damage may result in the diffusion of the pigment, before the solvent is added. This pigment will stick to the test tubes and may not get completely transferred from one test tube to another. (Symes) The most important reason is the permeability of the membrane. When the temperature of the solution is increased, the molecules will vibrate faster. The lipid molecules present in the cell membrane liquefies at the higher temperature. This results in the increase in the permeability of the membrane. The cell membrane is the best example for the mosaic fluid model. The fluidity of the membrane determines the movement of the protein molecules present in them. The decrease in the fluidity of the membrane results in the decrease in the ability of the protein to move and interact. The increase in the fluidity of the molecule leads to the damage of the cell membrane. The ethanol dissolves the lipid molecules that are present in the cell membrane. As a result the fluidity increases and the permeability of the membrane decreases and the pigment diffuses out. Similarly the protein molecule’s tertiary structure is affected by the ethanol. The altering and denaturing effect on the protein by the ethanol results in the altered function of the protein. This is because the ethanol affects the hydrogen bonding which links the lipid molecule and the protein molecule. (White 2004). The pH of the solution also affects the diffusivity level of the pigment. The pH affects the protein molecules present in the cell membrane. Protein molecules constitute about 70% of the total cell membrane. The proteins are made up of amino acids. The amonoacids are much sensitive to the change in the pH of the medium. (Cevc 1993). They are amphoteric and tend to stabilize the pH of the membrane always. They loose their H+ ions at the COOH part of the molecule to lower the pH and will gain an H+ ion at the NH2 region to increase the pH. If there is a change in the pH of the solution, then the functions of the protein molecules are altered. This alteration favors the cell membrane to increase the diffusivity of the molecules. This is the main reason for the solubility of the membrane when ethanol is added. References: Cevc,G 1993, Phospholipids handbook, 1st Edition, CRC Press. faculty.uca.edu, The Scientific Method: Hypothesis Formulation, Experimental Design and Data Analyses, viewed on April 12, 2010 from http://faculty.uca.edu/johnc/Hypothesis1440.htm fed.cuhk.edu.hk, Dinning out Teacher’s Notes, viewed on April 12, 2010 from http://www.fed.cuhk.edu.hk/~johnson/tas/investigation/membrane_teacher.htm , Hamilton, M 2007, Effects on the Membrane by Temperature, pH, alcohol and other chemicals, viewed on April 12, 2010 from www.eqzone.ezyrewardsplus.com/fatal/report.doc, merspi.com.au, What is the effect of varying pH/temperature/solutions on beetroot membranes? viewed on April 12, 2010 from http://merspi.com.au/questions/1313/what-is-the-effect-of-varying-ph- temperature-solutions-on-beetroot-membranes saps.plantsci.cam.ac.uk 2010, Question and Answer Archive, viewed on April 12, 2010 from http://www-saps.plantsci.cam.ac.uk/records/rec82.htm Symes, A, Biology Coursework, viewed on April 12, 2010 from http://www.studyzones.com/uploadfiles/11597x1597.doc, last retrieved on April 12, 2010. White, I 2004, Why an how do certain pH ranges affect the beetroot cell membrane? viewed on April 12, 2010 from http://www.madsci.org/posts/archives/2004-03/1080223472.Cb.r.html, Last Read More