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.