IB Biology diffusion of beetroot pigment measured using visible spectrophotometer


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IB Biology diffusion of beetroot pigment measured using visible spectrophotometer. Effect of ethanol on beetroot pigment quantify by absorbance change using visible spectrophotometer. Please cite and give proper reference to Kelly if you use her work.

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IB Biology diffusion of beetroot pigment measured using visible spectrophotometer

  1. 1. Candidate Name: Kelly Chaehyun LeeInternal Assessment – Investigating the relationship between theconcentration of ethanol and the change in absorbance over time of beetrootpigment.Research Question:How will changing the percentage of ethanol concentration affect the changein absorbance over time of beetroot pigment, represented as rate of diffusion,measured using visible spectrophotometer?Introduction:Beetroot contains red pigments that are soluble in water. The diffusion ofpigment is due to a red pigment in beetroot known as Betalin1. When ethanoldestroys the plasma membrane of ethanol, the red pigments flow out of thecell down the concentration gradient2. Absorbance of beetroot pigment can bemeasured using visible spectrophotometer, which is a device that measuresthe absorbance of solutions. Beetroot pigment, which has red color, willabsorb wavelengths of other colors but will reflect wavelengths of red color.The purpose of this experiment is to measure the different absorbance atdifferent concentration of ethanol, which will show the relationship betweenpercentage of ethanol concentration and the change in absorbance over time,which is rate of diffusion.1 "Beet ro ot co lo r." Beetroot-Bet a Vulga ri s. Selfsu ffic ient , n.d. Web. 5 Oct 2011. <http://www.selfsufficient ish.co m/beetroot.htm>.2 "D iffu sio n. " Merriam- Web ster. Br it an ica, 2011. Web. <ht t p://www. merr i a m-we bst er.co m/d ict io nar y/d iffu sio n >
  2. 2. Hypothesis:Ethanol is able to destroy the plasma membrane of the cells of beetroots. Thus,as concentration of ethanol is increased, the membranes will break even more,which will result in greater diffusion of beetroot pigments from the cells. When0% ethanol is used, plasma membrane will not break and the solution willshow no change in color. However, when 100% ethanol is used, the membranewill break severely, allow more diffusion, and consequently turn the color ofsolution into dark red. The visible spectrophotometer will detect highestabsorbance value for the ethanol concentration of 100%. Rate of diffusion isthe change in absorbance over time. Thus, as the concentration of ethanolincreases, the rate of diffusion will also increase.Rate of diffusion, r = /Rate of diffusion against concentration of ethanol Rate of diffusion Conc. of Ethanol Figure 1: hypothesis of the effect of concentration of ethanol on the rate of diffusion
  3. 3. Variables: Variable measured Method of measuring variableIndependent variable Concentration of Ethanol (%) 2-fold dilution was done. First, 100% ethanol was diluted to 50% by using distilled water. Then, 50% was diluted even further to 25%, 12.5%, and 6.25%.Dependent variable Change in absorbance over Rate of diffusion was measured by time due to diffusion of red change in absorbance over time. pigment of beetroot Absorbance was measured by spectrophotometer (absorbance against concentration) using Logger Pro 3.7. Method of controlling variable 3ml of ethanol solution was used for each experiment. Micropipette Amount of ethanol solution was used for accurate measurement. Same beetroot was used for the entire experiment. For consistency, only middle part Size and type of beetroot (bright red) of beetroot was used. Beetroot was produced by using cork borer in order to keep the diameter consistent (diameter ofControlled Variables beetroot piece: 1cm) Cuvettes of same type and equal Size and type of cuvette size were used throughout the experiment. Experiment was performed at a Temperature constant room temperature which was approximately 25 . Each trial was given an hour for Time diffusion to occur. All beetroot segments were put
  4. 4. into distilled water for about 10 minutes to make sure that pigments that had been produced due to damages of plasma membrane are washed away.Table 1: shows the independent, dependent, controlled variables, and the method ofmeasuring/controlling these variables
  5. 5. Apparatus: - Visible spectrophotometer - 10cm3 pipette ( 0.03 cm3) - Micropipette ) - Plate - Cuvette - Cork borer - Beaker - Test tube - scalpelMaterials: - Beetroot - Ehanol (100%) - Distilled water
  6. 6. Procedure:1. Prepare ethanol solution of different concentrations by doing 2-fold dilution as shown below (steps a ~ j). 100% 50% 25% 12.5% 6.25% 20 ml 100% ethanol 10ml 100% ethanol 10ml 50% ethanol 10ml 25% ethanol 10m 12.5% ethanol 10ml distilled water 10ml distilled water 10ml distilled water 10ml distilled waterFigure 2: shows 2-fold serial dilution of ethanol solution Concentration, c/% Amount of Amount of Total diluted ethanol, distilled water, ml ml 100 20.0 0.00 20.0 50 10.0 10.0 20.0 25 10.0 10.0 20.0 12.5 10.0 10.0 20.0 6.25 10.0 10.0 20.0Table 2: shows the amount ethanol and distilled water needed to prepare different ethanolconcentration. Steps for serial dilution a. Prepare 5 test tubes and label them: 100%, 50%, 25%, 12.5%, 6.25%. (100% = test tube 1/ 50%= test tube 2/ 25%= test tube 3/ 12.5%= test tube 4/ 6.25%= test tube 5) b. Put 10ml of distilled water into test tube 2 to test tube 5 using 10ml pipette. c. Put 20ml of 100% ethanol in test tube1. d. Take out 10 ml of 100% ethanol from test tube 1 and pour it into test tube 2. e. Seal the test tube and mix the solution (ethanol + water). Ethanol is soluble in water, so mixing the solution is unnecessary. But, to minimize any error, mix the solution. f. Take out 10ml solution from test tube 2 and pour it into test tube 3. Then mix the solution. g. Take out 10ml solution from test tube 3 and pour it into test tube 4. h. Take out 10 ml solution from test tube 4 and pour it into test tube 5.
  7. 7. i. Seal test tubes 1~5 with parafilm to make sure evaporation doesn’t happen. Evaporation might change the concentration of ethanol solution. j. Place the test tubes in the test tube rack while preparing beetroot.2. Cut the beetroot in half using scalpel.3. Extract several strands of beetroot by using cork borer. Use cork borer that has diameter of 1cm. First push in the cork borer vertically into the beet. Then, use thin stick such as spatula to take out cylindrical beetroot from the cork borer.4. Cut the beetroot into small segments (1cm in length). Use ruler for accuracy. Make sure to only use the middle part of the beetroot (middle, red part) for consistency of the experiment. Prepare 15 segments.5. Fill a beaker with distilled water.6. Put beetroot segments into the beaker to wash away any pigments that had been released due to damages of plasma membrane.7. Take out beetroot segments after 10 minutes.
  8. 8. 8. Prepare a cuvette and label it “100%”.9. Take out 3ml of 100% ethanol solutions from test tube (prepared in step 1) and pour them into the cuvette (prepared in step 8).10. Put 1 beetroot segment into the cuvette.11. Seal the cuvette using parafilm (to prevent evaporation) and wait 1 hour for diffusion to occur.12. While waiting for diffusion to happen, prepare computer for data collection.13. Connect visible spectrophotometer to computer.14. Calibrate by using distilled water.15. When 1hour had passed, remove parafilm from the cuvette.16. Place one cuvette into spectrophotometer and collect data for absorbance.17. Step 8 through 16 are repeated 3 times (triplicate trials) for the same concentration to compute the average.18. Step 17 is repeated for all different concentrations.
  9. 9. Data Collection: Qualitative DataHigher the concentration of ethanol concentration, the redder the color was. After beetroot wasplaced in different ethanol concentration for an hour, 0% ethanol concentration, which was distilledwater, was transparent. 6.25% ethanol was light pink but hard to distinguish from water. 12.5% waspink. 25% was pinkish red. 50% was bright red. Finally, 100% was dark crimson red.Data Collection: Quantitative Data Diluted Absorbance (at 477.0 nm) Mean Ethanol absorbanceConcentration, 1 2 3 Mean c/% 100 1.269 1.376 0.907 1.184 1.184 0.201 50 0.945 1.100 0.731 0.925 0.925 0.151 25 0.067 0.064 0.116 0.082 0.082 0.024 12.5 0.010 0.041 0.018 0.023 0.023 0.013 6.25 0.009 0.015 0.023 0.016 0.016 0.006 0 0.014 0.038 0.038 0.030 0.030 0.011Table 3: shows mean absorbance of triplicate trials
  10. 10. Data Processing Calculation of the mean of the triplicate trials for 100% ethanol. Mean ( ) = = = 1.184 Calculation of the standard deviation of triplicate trials for 100% ethanol. Standard deviation = = = 0.201 Calculation of rate of diffusion for 100% ethanol Rate of diffusion, r = = = 1.184 hr-1Ethanol Concentration, c/% Rate of Diffusion, r/hr-1 100 1.184 0.201 50 0.925 0.151 25 0.082 0.024 12.5 0.023 0.013 6.25 0.016 0.006 0 0.030 0.011Table 4: shows the relationship between the ethanol concentration and rate of diffusion
  11. 11. Data Presentation Absorbance against ConcentrationFigure 2: Data that shows the relationship between absorbance and concentration of ethanol measured using spectrophotometer and Logger Pro 3.7program
  12. 12. Data Presentation (a)Graph 1: shows the average rate of diffusion against ethanol concentration(a) Vertical error bar represents the standard deviation of the triplicate trials for the rate of diffusion
  13. 13. Uncertainties Uncertainty due to use of micropipette: Uncertainty due to the use of pipette: 0.03cm3 Each cuvette contains these uncertainties: - Uncertainty due to use of pipette to measure ethanol solution (serial dilution). - Uncertainty due to use of pipette to measure distilled water (serial dilution). - Uncertainty due to use of micropipette to transfer 1ml of ethanol solution from test tube to cuvette 3 times (because micropipette can carry maximum of 1ml each time. Need 3ml total). Sample calculations  Total percentage uncertainty for concentration of ethanol solution of 50%:= (percentage uncertainty due to pipet to measure ethanol solution) + (Percentage uncertainty due to pipet to measure water) =( )+( ) = 0.6  Absolute uncertainty for concentration of ethanol solution of 50%: =( ) = 0.06  Percentage uncertainty due to use of micropipette to transfer 1ml of ethanol solution from test tube to cuvette 3 times (because micropipette can carry maximum of 1ml each time): = (Percentage uncertainty of 1ml) + (Percentage uncertainty of 1ml) + (Percentage uncertainty of 1ml) =( x 100) + ( x 100) + ( x 100) = 1.8
  14. 14. Ethanol Percentage uncertainty for Volume use Total percentage Concentration ConcentrationConcentration, uncertainty, % with with absolute c/% Volume of Percentage Volume of Percentage percentage uncertainty, % ethanol added uncertainty in distilled water Uncertainty in uncertainty, % using pipette, volume, % added using Volume, % ( / pipette, cm3 ( / cm3 (a)100 20 0 1.8 + 0.15 + 0 = 100 100 x100= 0.15 1.9550 10 10 1.8 + 0.3 + 0.3= 50 50 x100= 0.3 x100= 0.3 2.425 10 10 1.8 + 0.3 + 0.3= 25 25 x100= 0.3 x100= 0.3 2.412.5 10 10 1.8 + 0.3 + 0.3= 12.5 12.5 x100= 0.3 x100= 0.3 2.46.25 10 10 1.8 + 0.3 + 0.3= 6.25 6.25 x100= 0.3 x100= 0.3 2.40 0 20 1.8 + 0 + 0.15 = 1.95 0 0 x100= 0.15Table 5: shows percentage uncertainty and absolute uncertainty for different ethanol concentrations(a) 1.8 = Percentage uncertainty due to use of micropipette to transfer 3ml of ethanol solution from test tube to cuvette (Refer to “sample calculation”).
  15. 15. ConclusionAccording to graph 1, as the concentration of ethanol solution increases, the rate of diffusion alsoincreases, which validates my hypothesis. This graph generally has a positive relationship but the lineof best fit is not a positive linear graph. From concentration 0% to 25%, the rate is relatively constantbecause the change in rate is not very significant. Absorbance for 0%, 6.25%, and 12.5% were allsimilar. This suggests that ethanol is not going to have significant impact on plasma membrane untila larger quantity is used. Thus, when larger quantity of ethanol was used, which was 50%, rate ofdiffusion showed a sudden increase. From 50%, the rate continued to increase. And, as expected,100% showed the highest absorbance rate. This is due to the fact that 100% ethanol had hugeimpact on breaking down plasma membrane of beetroot cells which allowed the diffusion of redpigments from the cell. However, although there was a significant increase of absorbance when theconcentration increased from 25% to 50%, the change wasn’t very big when the concentrationincreased from 50% to 100%. The graph started to level off from 50%. This shows that concentrationhas limit to increasing the absorbance: once the concentration reaches a high point, absorbancedoesn’t increase significantly but rather increases gradually and then level off.
  16. 16. EvaluationSince it is impossible to be free from systematic or random errors while performing a lab, triplicatetrials were done to get a more reliable average of data. There was a relatively high standarddeviation for 100% ethanol solution because the absorbance for each cuvette varied a lot.Limitations ImprovementsBeetroot segments were not consistent. It is very difficult to get a perfect sample that hasAlthough best efforts had been put to extract completely homogeneous red color. However, inthe middle, red part of beetroot segments, some order to improve this problem, beetroot can beof them had darker pigments while others more carefully selected. For example, the colordidn’t. For example, even though all the of beetroot can be more cautiously examined bysegments were from the middle part of the comparing the color with a sample that wasbeetroot, some segments had blackish red spots. already chosen.This might have affected the absorbancebecause each segment had different amount ofpigment. Some were darker than others.The beetroot segments were not put into the In order to reduce this human error, a morecuvettes (filled with ethanol solution) at the systematic method should be used. For example,same time. 15 cuvettes had been used to get a if there are more people and each one of them ismore accurate result by finding average of many responsible for 2~3 cuvettes, then beetroots cantrials. However, the usage of so many cuvettes be released at the same time.was a problem because it was difficult to place Another method is to reduce the number ofbeetroots into all 15 cuvettes at the same time. cuvettes used, so that it will be easier to handleAs a result, some beetroots were placed in them.ethanol solution for longer time than others.This might have affected the absorbancebecause each beetroot was given slightlydifferent amount of time for diffusion.Distilled water in ethanol solution might have Evaporation can be controlled by sealing theevaporated. Although evaporation was reduced opening of the test tubes more tightly withby sealing the test tubes with parafilm, water parafilm. It is better to stretch the parafilmmight have evaporated during the process of completely so that there are no wrinkles on theserial dilution. Evaporation can affect the surface.concentration of ethanol solution had haveimpact on the change in absorbance.Table 6: shows the limitations and improvement