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IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Candidate Name Candidate Number Date of Practical

:Yoojin Lee :002213-067 :March 14, 2010

Internal Assessment – Determining the relationship between ethanol concentration and rate of diffusion of Betanin pigment of beetroot, using the visible spectrophotometer Research Question: How will changing ethanol concentration affect the rate of diffusion of beetroot pigment, Betanin 1 , from beetroot cubes placed in water, measured using visible spectrophotometer?

Introduction: Visible spectrophotometer 2 is a device that measures the absorbance of solutions. Some wavelengths of light pass through, but some wavelengths of light reflect back. For example, beetroot pigment, which is red in color, reflects wavelengths that code for red and absorbs other wavelengths that code for different colors. The detector records the reflection of light.

Betanin is responsible for the red pigment in a beetroot. It is a glycoside composed of sugar and colored portion. It is water soluble, which lets diffusion possible in aqueous environment. Betanin is found in vacuoles in plant cells. When the plasma membrane of the plant cell is denatured by ethanol, the Betanin pigments will flow out of the cell, down the concentration gradient.

The purpose of this experiment is to test the different absorbance at different concentration of

1

George Pucher, Lawrence Curt is and Herbert Vickery, “The Red Pig ment of the Root of the Beet (beta Vulgaris),” The Journal of Biological Chemistry, http://www.jbc.org/content/123/ 1/61.full.pdf (accessed March 14, 2010). 2

“Ultravio let–visible spectroscopy,” Wikipedia, the free encyclopedia,http://en.wikipedia.org/wiki/ Ult raviolet%E2%80%93visible_spectroscopy (accessed March 14, 2010). 1


IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

ethanol, hence find out the relationship between the rate of diffusion and the ethanol concentration. The beetroot piece after washed with distilled water has its plasma membranes around the cells to protect and to resist leaking of red pigments. However, when ethanol is added to the solution surrounding the beetroot piece, ethanol molecules will destroy the plasma membranes and make red pigments to come out to the solution. In this experiment, the relationship between ethanol concentration and the rate of reaction, which is represented by absorbance of beetroot pigment, will be tested.

The Beer-Lambert Law 3 states A=ebc, in which A represents the absorbance, e represents molar absorbtivity, b represents the path length of the cuvette, and c represents the concentration of solution. In this experiment, the molar absorbtivity and cuvette are constant, because beetroot solution is the only solution to be tested and the same cuvette is used for each trial. Thus, with two constant variables, the Beer-Lambert Law states that the absorbance is directly proportional to the concentration. However, the linear relationship between the absorbance and the concentration is deviated at high concentrations, so in this experiment, only solutions of low concentration are valid.

3

“Beer's Law,� Sheffield Hallam University,http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm (accessed March 14, 2010). 2


IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Hypothesis: Rate of diffusion is represented by the change in absorbance in an hour. Since ethanol has an ability to destroy plasma membrane of beetroot cells, the increase in ethanol concentration will destroy the membranes more severely, which will result in excess diffusion of beetroot pigments from the cell. Thus, when the identically cut beetroot pieces are put into solutions with different concentration, 0% ethanol solution will remain transparent, while 100% ethanol solution will have the darkest red color. When put into the visible spectrophotometer, ethanol solution will have absorbance value that is extremely close to 0, while 100% ethanol solution will have the highest absorbance value. The relationship between ethanol concentration and absorbance is directly proportional. Hence, as the ethanol concentration goes up, the rate of diffusion will increase accordingly. Rate of diffusion, r =

∆ Absorbance −1 /h Time

Figure 1 shows the predicted relationship between rate of diffusion and ethanol concentration

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IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Variables: Variables Independent

Description Ethanol Concentration (%)

Dependant

Rate of diffusion of Betanin pigments from beetroot cells

Controlled

Method of Measuring 100% ethanol was diluted to 50% using distilled water. Then the diluted ethanol was further diluted to prepare 20%, 40%, 60%, 80%, and 100%. Distilled water was used for control (0% ethanol). Triplicate trials were performed on each concentration to obtain the mean. Rate of diffusion is represented by the change of absorbance in an hour. Absorbance was measured using the

Rate of diffusion, r ∆ Absorbance −1 = /h Time

visible spectrophotometer at λ max 480.5nm. Only one cuvette was used for each trial to reduce systematic errors.

Size and type of beetroot

Beetroot pieces of identical shape and size (0.5cm) was prepared using cork borer. Only the middle part of the beetroot was used. Same beetroot was used for all 3 trials.

Size and type of cuvette

The same cuvette for each trial was used, which was calibrated at the beginning of the trial.

Volume of ethanol solution

Time

Ethanol and water to obtain was mixed carefully. Equal volume of the total solution, 2.5cm3 , was prepared for all trials. Micropipette was used for accurate measurement. An hour was given for all trials for diffusion. Trials were simultaneously stopped by taking out beetroot pieces at the same time. Experiment was conducted in the lab at a constant room temperature, which is

Temperature

approximately 25℃. λ max λ max was fixed at 480.5nm, because the absorbance is measured relatively to the λ max. The maximum absorbance is determined by the λ max value. Table 1 shows the independent, dependent, and controlled variables and the methods of measuring

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IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Apparatus:

Materials:

 Visible spectrophotometer  Micropipette (± 0.006cm3 )  25cm3 Pipette (± 0.03cm3)  Microplate  Cuvette  Cork Borer (0.7cm diameter)  Beaker

 Beetroot  Ethanol (100%)  Distilled water

Procedure: 1. Using the cork borer, extract several strands of beetroot and cut the middle part, 0.5cm wide. 2. Put the beetroot pieces into distilled water to wash away pigments that are produced due to damages of plasma membrane. 3. Dilute 100% ethanol, using 25cm3 pipette. 4. Prepare different solutions of diluted ethanol in the microplate. Concentration, c/% 100

Volume of diluted ethanol, V1 /cm3 2.50

Volume of distilled water, V2 /cm3 0.00

Total Volume, V/cm3 2.50

80.0

2.00

0.50

2.50

60.0

1.50

1.00

2.50

40.0

1.00

1.50

2.50

20.0

0.50

2.00

2.50

0.00

0.00

2.50

2.50

Table 2 shows the volume used to prepare solutions of different ethanol concentration 5. Place beetroot pieces into each solution using a tweezers and wait for an hour to let diffusion occur. 6. Calibrate cuvette and warm up the spectrophotometer at λ max 480.5nm 7. Using one cuvette for each trial, place the entire solution in the cuvette and measure the absorbance. 8. Repeat steps 6 and 7 to obtain the mean for the triplicate trials. 5


IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Data Collection and Processing: Quantitative Data Absorbance at λ Max 480.5nm

Diluted Ethanol Concentration, c/%

1

2

3

Mean(a)

100

0.937

0.933

0.978

0.949

0.949 ± 0.020

80.0

0.902

0.854

0.905

0.887

0.887 ± 0.023

60.0

0.403

0.443

0.436

0.427

0.427 ± 0.017

40.0

0.070

0.068

0.082

0.073

0.073 ± 0.006

20.0

0.035

0.029

0.038

0.034

0.034 ± 0.004

0.00

0.043

0.029

0.045

0.039

0.039 ± 0.007

Mean ± SD(b)

Table 3 shows mean absorbance of triplicate trials at λ max 480.5nm. Mean: average of triplicate trials for each set. (b) SD: standard deviation for triplicate trials. (a)

Qualitative Data Higher ethanol solutions had redder and darker color than lower ethanol solutions. In fact, 0% ethanol concentration, which is distilled water, seemed transparent while 100% ethanol became darker as time went on. Since the Betanin pigment concentration was higher around the beetroot, the solution had to be homogenized well before measuring the absorbance.

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IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Data Processing  Calculation of 100% ethanol concentration for the mean of triplicate trials. Mean (x) = =

Trial 1+Trial 2+Trial 3 3 0.937 1+0.933+0.978 3

= 0.949

 Calculation of 100% ethanol concentration for the standard deviation of triplicate trials Standard deviation =

=

(X trial − x ) 2 3 (0.937−0.949) 2 + (0.933 −0.949) 2 + (0.978−0.949) 2 3

= 0.020

 Calculation of 100% ethanol concentration for the rate of diffusion Rate of diffusion = =

∆Absorbance 1hr 0.949−0.000 1hr

= 0.949 hr−1

Subsequent calculations were performed on 80%, 60%, 40%, 20%, and 0%.

Ethanol concentration, c/%

Rate of diffusion, r/hr-1

100

0.949 ± 0.020

80.0

0.887 ± 0.023

60.0

0.427 ± 0.017

40.0

0.073 ± 0.006

20.0

0.034 ± 0.004

0.00

0.039 ± 0.007

Table 4 shows the relationship between the ethanol concentration and the rate of diffusion

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IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Data Presentation:

Rate of Diffusion,r/hr-1 against Different Concentration, c/% of Diluted Ethanol at 位 Max 480.5nm

y = 0.010x - 0.131 R虏 = 0.873

1.2

1

Rate of Diffusion, r/hr-1

(b)

0.8

0.6 (a) 0.4

0.2

0 0

-0.2

10

20

30

40

50

60

Ethanol Concentration, c/%

Graph 1 shows the rate of diffusion at 位 max 480.5nm Vertical error bar shows the standard deviation of the triplicate trials for the rate of diffusion (b) Horizontal error bar shows the uncertainty in ethanol concentration (a)

8

70

80

90

100


IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Uncertainties: Diluting 100% ethanol % uncertainty for volume use

Volume of 100% ethanol using 25cm3 pipette, (ΔV = ± 0.03)/cm3

25

% uncertainty in volume, %

Volume of distilled water using 25cm3 pipette, (ΔV = ± 0.03)/cm3

% uncertainty in volume, %

(0.03/25) x100 = 0.12

25

(0.03/25) x100 = 0.12

Table 5 shows the percent uncertainty for diluting ethanol.

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Total % uncertainty, %

0.12 + 0.12 = 0.24


IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Uncertainty for volume use % uncertainty for volume use Ethanol Concentration, (c = Âą 0.24)/%

100

Volume of ethanol using micropipette, (ΔV = ¹ 0.006)/cm3 2.50

% uncertainty in volume, % (0.006/0.5) x5x100 =6 (0.006/0.5) x4x100 = 4.8 (0.006/0.5) x3x100 = 3.6 (0.006/0.5) x2x100 = 2.4 (0.006/0.5) x100 = 1.2

Volume of distilled water using micropipette, (ΔV = ¹ 0.006)/cm3

% uncertainty in volume, %

0.00

0.0

Total % uncertainty, %

Concentration with % uncertainty, %

Concentration with absolute uncertainty, %(a)

0.24 + 6 + 0 = 6.24

100Âą6.24

100Âą6.24

(0.006/0.5) 0.24 + 4.8 + 1.2 x100 80.0Âą6.24 = 6.24 = 1.2 (0.006/0.5) 0.24 + 3.6 + 2.4 60.0 1.50 1.00 x2x100 60.0Âą6.24 =6.24 = 2.4 (0.006/0.5) 0.24 + 2.4 + 3.6 40.0 1.00 1.50 x3x100 40.0Âą6.24 = 6.24 = 3.6 (0.006/0.5) 0.24 + 1.2 + 4.8 20.0 0.50 2.00 x4x100 20.0Âą6.24 = 6.24 = 4.8 (0.006/0.5) 0.24 + 0 + 6 0.00 0.00 0.0 2.50 x5x100 0.00Âą6.24 = 6.24 =6 Table 6 shows percent uncertainty and absolute uncertainty for volume use for different ethanol concentrations 6.24 (a) Absolute Concentration Calculation: 100 Ă— đ??¸đ?‘Ąâ„Žđ?‘Žđ?‘›đ?‘œđ?‘™ đ??śđ?‘œđ?‘›đ?‘?đ?‘’đ?‘›đ?‘Ąđ?‘&#x;đ?‘Žđ?‘Ąđ?‘–đ?‘œđ?‘› 80.0

2.00

0.500

10

80.0Âą4.99

60.0Âą3.74

40.0Âą2.50

20.0Âą1.25

0.00Âą0


IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Conclusion: The data suggests that as the ethanol concentration increases, the rate of diffusion increases and that my hypothesis is valid. The linear regression and the R2 value show that there is a positive correlation between the rate of diffusion and the ethanol concentration. However it cannot be proved that the correlation is always directly proportional. When observing the first three data on 0%, 20%, and 40% there was no obvious increases. Perhaps, this might be due the fact that low ethanol concentration was not enough to destroy the plasma membrane effectively. The graph slowly increased in the beginning and in the end, while the gradient in the middle is extremely steep, which represents a huge change in absorbance in 60% ethanol range. The results tell that when the ethanol concentration is high, the plasma membrane gets more damaged, which leads to more out flux of Betanin pigment, which depends on the concentration gradient. Thus, the results lead to a conclusion that the rate of diffusion is directly proportional when the ethanol concentration is higher than 40%.

Evaluation: The experiment is justifiable because reliable triplicate trials were obtained. This is also reflected by the small standard deviation on graph 1. The uncertainty of the concentration varied with the concentration. Although it seems likely that there is a positive correlation between the rate of diffusion, according to graph 1, neither the 0% data nor the linear regression line pass through the origin of the data. This shows that both systematic and random errors were present. Since this experiment dealt with small pieces of beetroots and total of 2.5 cm3 of solution per trial, small systematic errors led to large uncertainties. For instance, an extra drop of ethanol can change the percent concentration to a great extent. Major errors could have been reduced if bigger beet root samples with greater amount of ethanol solutions were used.

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IB Bio logy HL Name: Yoojin Lee Candidate Nu mber: 002213-067

Limitations and Improvements: Limitations

Improvements

The beetroot pieces were not identical. Since the beetroot used for the experiment was very small, many strands could not be prepared. Although only middle pieces were used, it does not guarantee that every piece is the same. Even though the beetroot pieces were different to a small extent, they could add high uncertainties, because only small amount of solutions were used.

Prepare more strands of beetroot using the cork borer and use only one middle part from each strand so that all of the pieces are approximately the same.

The beetroot pieces were not completely washed after they were cut. There was no way to be sure that the pigments on the surface of the cut beetroot were washed away by observing with naked eyes. Even if there were a method to check, it is impossible to check all of the pieces simultaneously. Due to the time constraints, the beetroot pieces were washed for 30 minutes.

The beetroot pieces should be left longer. It will be faster if magnetic stirrer was used in the process. However, a very small magnet has to be used to prevent damages on beetroot pieces.

When mixing ethanol and water with micropipette, the solution formed bubbles that might have served as obstacles to measure the exact absorbance of the solution.

Place the micro-pipette tip inside the water and transfer ethanol slowly to make the solutions homogenize smoothly.

Table 7 shows the limitations and the improvements

Bibliography: Pucher, George, Lawrence Curtis, and Herbert Vickery. “The Red Pigment of the Root of the Beet (beta Vulgaris).” The Journal of Biological Chemistry.http://www.jbc.org/content/123/1/61.full.pdf (accessed March 14, 2010). “Beer's Law.” Sheffield Hallam University.http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm (acces sed March 14, 2010). “Ultraviolet–visible spectroscopy.” Wikipedia, the free encyclopedia.http://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscop y (accessed March 14, 2010).

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Beetroot experiment