ib biology diffusion of beetroot pigment measured using visible spectrophotometer
DESCRIPTION
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.TRANSCRIPT
Candidate Name: Kelly Chaehyun Lee
Internal Assessment – Investigating the relationship between the
concentration of ethanol and the change in absorbance over time of beetroot
pigment.
Research Question:
How will changing the percentage of ethanol concentration affect the change
in 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 of
pigment is due to a red pigment in beetroot known as Betalin1. When ethanol
destroys the plasma membrane of ethanol, the red pigments flow out of the
cell down the concentration gradient2. Absorbance of beetroot pigment can be
measured using visible spectrophotometer, which is a device that measures
the absorbance of solutions. Beetroot pigment, which has red color, will
absorb wavelengths of other colors but will reflect wavelengths of red color.
The purpose of this experiment is to measure the different absorbance at
different concentration of ethanol, which will show the relationship between
percentage of ethanol concentration and the change in absorbance over time,
which is rate of diffusion.
1 " B e e t r o o t c o l o r . " B e e t r o o t - B e t a V u l g a r i s . S e l f s u f f i c i e n t , n . d . W e b . 5 O c t 2 0 1 1 .
< h t t p : / / w w w . s e l f s u f f i c i e n t i s h . c o m / b e e t r o o t . h t m > .
2 " D i f f u s i o n . " M e r r i a m - W e b s t e r . B r i t a n i c a , 2 0 1 1 . W e b . < h t t p : / / w w w . m e r r i a m - w e b s t e r . c o m / d i c t i o n a r y / d i f f u s i o n >
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. When
0% ethanol is used, plasma membrane will not break and the solution will
show no change in color. However, when 100% ethanol is used, the membrane
will break severely, allow more diffusion, and consequently turn the color of
solution into dark red. The visible spectrophotometer will detect highest
absorbance value for the ethanol concentration of 100%. Rate of diffusion is
the change in absorbance over time. Thus, as the concentration of ethanol
increases, the rate of diffusion will also increase.
Rate of diffusion, r = /
Rate of diffusion against concentration of ethanol
Conc. of Ethanol
Rate of diffusion
Figure 1: hypothesis of the effect of concentration of ethanol on the rate of diffusion
Variables:
Variable measured Method of measuring variable
Independent 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
time due to diffusion of red
pigment of beetroot
Rate of diffusion was measured by
change in absorbance over time.
Absorbance was measured by
spectrophotometer (absorbance
against concentration) using
Logger Pro 3.7.
Method of controlling variable
Controlled Variables
Amount of ethanol solution
3ml of ethanol solution was used
for each experiment. Micropipette
was used for accurate
measurement.
Size and type of beetroot
Same beetroot was used for the
entire experiment. For
consistency, only middle part
(bright red) of beetroot was used.
Beetroot was produced by using
cork borer in order to keep the
diameter consistent (diameter of
beetroot piece: 1cm)
Size and type of cuvette
Cuvettes of same type and equal
size were used throughout the
experiment.
Temperature
Experiment was performed at a
constant room temperature which
was approximately 25 .
Time
Each trial was given an hour for
diffusion to occur.
All beetroot segments were put
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 of measuring/controlling these variables
Apparatus:
- Visible spectrophotometer - 10cm3 pipette ( 0.03 cm3)
- Micropipette )
- Plate - Cuvette - Cork borer - Beaker - Test tube - scalpel
Materials:
- Beetroot - Ehanol (100%) - Distilled water
Procedure:
1. Prepare ethanol solution of different concentrations by doing 2-fold dilution as shown below (steps a ~ j).
Figure 2: shows 2-fold serial dilution of ethanol solution
Concentration, c/% Amount of
diluted ethanol,
ml
Amount of
distilled water, ml
Total
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 20.0
Table 2: shows the amount ethanol and distilled water needed to prepare different ethanol
concentration.
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.
100% 20 ml 100% ethanol
6.25% 10m 12.5% ethanol 10ml distilled water
12.5% 10ml 25% ethanol 10ml distilled water
25% 10ml 50% ethanol 10ml distilled water
50% 10ml 100% ethanol 10ml distilled water
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. 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.
Data Collection: Qualitative Data Higher the concentration of ethanol concentration, the redder the color was. After beetroot was placed in different ethanol concentration for an hour, 0% ethanol concentration, which was distilled water, was transparent. 6.25% ethanol was light pink but hard to distinguish from water. 12.5% was pink. 25% was pinkish red. 50% was bright red. Finally, 100% was dark crimson red. Data Collection: Quantitative Data
Diluted Ethanol
Concentration, c/%
Absorbance (at 477.0 nm) Mean absorbance
1 2 3 Mean
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 0.011
Table 3: shows mean absorbance of triplicate trials
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-1
Ethanol 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 0.011
Table 4: shows the relationship between the ethanol concentration and rate of diffusion
Data Presentation
Absorbance against Concentration
Figure 2: Data that shows the relationship between absorbance and concentration of ethanol measured using spectrophotometer and Logger Pro 3.7
program
Data Presentation
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
(a)
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
Table 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”).
Ethanol Concentration,
c/%
Percentage uncertainty for Volume use Total percentage
uncertainty, %
Concentration
with
percentage
uncertainty, %
Concentration
with absolute
uncertainty, % Volume of ethanol added using pipette, ( /
cm3
Percentage uncertainty in volume, %
Volume of distilled water added using pipette, ( /
cm3
Percentage Uncertainty in Volume, %
100 20 x100= 0.15
0 (a)1.8 + 0.15 + 0 = 1.95
100 100
50 10 x100= 0.3
10 x100= 0.3
1.8 + 0.3 + 0.3= 2.4
50 50
25 10 x100= 0.3
10 x100= 0.3
1.8 + 0.3 + 0.3= 2.4
25 25
12.5 10 x100= 0.3
10 x100= 0.3
1.8 + 0.3 + 0.3= 2.4
12.5 12.5
6.25 10 x100= 0.3
10 x100= 0.3
1.8 + 0.3 + 0.3= 2.4
6.25 6.25
0 0 20 x100= 0.15
1.8 + 0 + 0.15 = 1.95 0 0
Conclusion According to graph 1, as the concentration of ethanol solution increases, the rate of diffusion also increases, which validates my hypothesis. This graph generally has a positive relationship but the line of best fit is not a positive linear graph. From concentration 0% to 25%, the rate is relatively constant because the change in rate is not very significant. Absorbance for 0%, 6.25%, and 12.5% were all similar. This suggests that ethanol is not going to have significant impact on plasma membrane until a larger quantity is used. Thus, when larger quantity of ethanol was used, which was 50%, rate of diffusion 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 huge impact on breaking down plasma membrane of beetroot cells which allowed the diffusion of red pigments from the cell. However, although there was a significant increase of absorbance when the concentration increased from 25% to 50%, the change wasn’t very big when the concentration increased from 50% to 100%. The graph started to level off from 50%. This shows that concentration has limit to increasing the absorbance: once the concentration reaches a high point, absorbance doesn’t increase significantly but rather increases gradually and then level off.
Evaluation Since it is impossible to be free from systematic or random errors while performing a lab, triplicate trials were done to get a more reliable average of data. There was a relatively high standard deviation for 100% ethanol solution because the absorbance for each cuvette varied a lot.
Table 6: shows the limitations and improvement
Limitations Improvements
Beetroot segments were not consistent. Although best efforts had been put to extract the middle, red part of beetroot segments, some of them had darker pigments while others didn’t. For example, even though all the segments were from the middle part of the beetroot, some segments had blackish red spots. This might have affected the absorbance because each segment had different amount of pigment. Some were darker than others.
It is very difficult to get a perfect sample that has completely homogeneous red color. However, in order to improve this problem, beetroot can be more carefully selected. For example, the color of beetroot can be more cautiously examined by comparing the color with a sample that was already chosen.
The beetroot segments were not put into the cuvettes (filled with ethanol solution) at the same time. 15 cuvettes had been used to get a more accurate result by finding average of many trials. However, the usage of so many cuvettes was a problem because it was difficult to place beetroots into all 15 cuvettes at the same time. As a result, some beetroots were placed in ethanol solution for longer time than others. This might have affected the absorbance because each beetroot was given slightly different amount of time for diffusion.
In order to reduce this human error, a more systematic method should be used. For example, if there are more people and each one of them is responsible for 2~3 cuvettes, then beetroots can be released at the same time. Another method is to reduce the number of cuvettes used, so that it will be easier to handle them.
Distilled water in ethanol solution might have evaporated. Although evaporation was reduced by sealing the test tubes with parafilm, water might have evaporated during the process of serial dilution. Evaporation can affect the concentration of ethanol solution had have impact on the change in absorbance.
Evaporation can be controlled by sealing the opening of the test tubes more tightly with parafilm. It is better to stretch the parafilm completely so that there are no wrinkles on the surface.