Candidate Name :Gina Yoojin LeeCandidate Number :Date of Practical :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, from beetroot cubes placed in water, measured using visible spectrophotometer?

Introduction:

Visible spectrophotometer 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

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 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.

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=∆ AbsorbanceTime

/h−1

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

Variables:Variables Description Method of Measuring

Independent Ethanol Concentration (%) 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.

Dependant Rate of diffusion of betanin pigments from beetroot cells

Rate of diffusion is represented by the change of absorbance in an hour. Absorbance was measured using the visible spectrophotometer at λ max 480.5nm. Only one cuvette was used for each trial to reduce systematic errors.

Controlled 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 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.

Time An hour was given for all trials for diffusion. Trials were simultaneously stopped by taking out beetroot pieces at the same time.

Temperature Experiment was conducted in the lab at a constant room temperature, which is 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

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/% Volume of diluted ethanol, V1/cm3

Volume of distilled water, V2/cm3

Total Volume,V/cm3

100 2.5 0.0 2.5

80 2.0 0.5 2.5

60 1.5 1.0 2.5

40 1.0 1.5 2.5

20 0.5 2.0 2.5

0 0.0 2.5 2.5

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.

Data Collection and Processing:

Diluted Ethanol Concentration,

c/%

Absorbance at λ Max 480.5nmMean ± SD(b)

1 2 3 Mean(a)

100 0.937 0.933 0.978 0.949 0.949 ± 0.020

80 0.902 0.854 0.905 0.887 0.887 ± 0.023

60 0.403 0.443 0.436 0.427 0.427 ± 0.017

40 0.070 0.068 0.082 0.073 0.073 ± 0.006

20 0.035 0.029 0.038 0.034 0.034 ± 0.004

0 0.043 0.029 0.045 0.039 0.039 ± 0.007

Quantitative DataTable 3 shows mean absorbance of triplicate trials at λ max 480.5nm.

(a) Mean: average of triplicate trials for each set.(b) SD: standard deviation for triplicate trials.

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.

Data Processing

Calculation of 100% ethanol concentration for the mean of triplicate trials.

Mean (x) = Trial1+Trial 2+Trial 3

3

= 0.937+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 0.887 ± 0.023

60 0.427 ± 0.017

40 0.073 ± 0.006

20 0.034 ± 0.004

0 0.039 ± 0.007

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

Data Presentation:

0 10 20 30 40 50 60 70 80 90 1000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

f(x) = 0.0106614285714286 x − 0.131571428571429R² = 0.873882308935025

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

Ethanol Concentration, c/%

Rat

e of

Diff

usio

n, r

/hr-

1

Graph 2 shows the rate of diffusion at λ max 480.5nm(a) 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)

(b)

Uncertainties:

Diluting 100% ethanol% uncertainty for volume use

Total % uncertainty, %

Volume of 100% ethanol using 25cm3 pipette,

(ΔV = ± 0.03)/cm3

% uncertainty in volume, %

Volume of distilled water using 25cm3

pipette,(ΔV = ± 0.03)/cm3

% uncertainty in volume, %

25 (0.03/25)x100 = 0.12 25 (0.03/25)

x100 = 0.120.12 + 0.12 =

0.24

Table 5 shows the percent uncertainty for diluting ethanol.

EthanolConcentration, (c = ± 0.24)/%

% uncertainty for volume use

Total % uncertainty, %

Concentration with %

uncertainty, %

Concentration with absolute

uncertainty, %(a)Volume of ethanol using micropipette,(ΔV = ± 0.006)/cm3

% uncertainty in volume, %

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

% uncertainty in volume, %

100 2.5(0.006/0.5)

x5x100= 6

0.0 0.0 0.24 + 6 + 0 = 6.24 100±6.24 100±6.24

80 2.0(0.006/0.5) x4x100

= 4.80.5

(0.006/0.5) x100= 1.2

0.24 + 4.8 + 1.2 = 6.24 80±6.24 80±4.99

60 1.5(0.006/0.5) x3x100

= 3.61.0

(0.006/0.5) x2x100

= 2.4

0.24 + 3.6 + 2.4 =6.24 60±6.24 60±3.74

40 1.0(0.006/0.5) x2x100

= 2.41.5

(0.006/0.5) x3x100

= 3.6

0.24 + 2.4 + 3.6= 6.24 40±6.24 40±2.50

20 0.5(0.006/0.5)

x100= 1.2

2.0(0.006/0.5) x4x100

= 4.8

0.24 + 1.2 + 4.8 = 6.24 20±6.24 20±1.25

0 0.0 0.0 2.5(0.006/0.5)

x5x100= 6

0.24 + 0 + 6 = 6.24 0±6.24 0±0

Uncertainty for volume use Table 6 shows percent uncertainty and absolute uncertainty for volume use for different ethanol concentrations

(a) Absolute Concentration Calculation: 6.24100

× EthanolConcentration

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 vertical standard deviation on graph 2. 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 2, 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 cm3of 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.

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.

Limitations and Improvements:Table 7 shows the limitations and the improvements