Biology HL Internal Assessment

The effect of different concentrations of ethanol on the rate of diffusion of betanin

from Beta vulgaris?

School name: **********************

Candidate name: Agnes *********
Candidate number: *********

Candidate number: ******-006

School name: ******

Candidate name: Agnes *******
Candidate number: ******-***

Candidate number: ******-006

Research question:
What is the effect of different concentrations of ethanol on the rate of diffusion of
betanin from Beta vulgaris?

Background information:
Diffusion is a very important biological process. It is a process resulting from
random motion of molecules by which there is a net flow of matter from a region of high
concentration to a region of low concentration. 1 Rate can be calculated using photon
diffusion equation2:

Visible spectrophotometer is a device which measures the amount of light that sample
absorbs. In spectrophotometer beam of light is split into two beams with equal wavelengths.
One of them passes through a cuvette containing solution being study. The second one goes
through a cuvette congaing just only solvent. The intensities are then recorded and compared.
The results are usually present as absorbance. It could be defined by the Beer-Lambert Law,
which states the formula for absorbance as follow3:
,
where:
A is absorbance.
e is the molar absorbtivity (L mol-1 cm-1).
b is a path length of a cuvette containing solution.
c is a concentration of solvent (mol L-1).
Betanin is a red pigment present in beetroots. It has many functions, but the most
popular is as a food and beverage dye. It degrades when exposed to light, heat and oxygen.

1
2
3

http://www.britannica.com/EBchecked/topic/163068/diffusion
http://en.wikipedia.org/wiki/Photon_diffusion_equation
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm

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The colour of betanin depends on pH, in low acidic solutions pH, it has dark red colour
which lightest with the increase of pH and finally degrades in alkaline solution, giving
yellow-brown colour.
Ethanol is ethyl alcohol with chemical structure C2H5OH. It is used both in chemical
industry as a solvent and additive to gasoline and is the ingredient of alcoholic beverages.
Pure ethanol is colourless and inflammable liquid. Posses hydroscopic properties and easily
absorbs water from the air. Alcohol affects nervous system and may cause permanently
damage to the organism. 4

Hypothesis:
According to the background information, with the increase of concentration of
ethanol, the percentage damage of partially permeable membrane will also increase which
will result in bigger amount of red pigment released. With the increase of ethanol
concentration, pH of the solution will increase, this cause the change of betanin. As stated
above the more acidic environment, pigment changes its colour into darker red. However, I
can predict that in a solution with 0% ethanol concentration, there will be no red pigment
excretion, because of neutral pH of water.

Variables:
Independent:

Percentage concentration of ethanol in solution. The con will be as follows:

0%, 25%, 50%, 75% and 100%.

Dependent:

Rate of diffusion. The diffusion will be calculated by the photon diffusion

equation using the absorbance results obtained from visible spectrophotometer.

Controlled:

One type of beetroot. Only the middle of the beetroot is used to have the most
similar sample possible.

http://www.britannica.com/EBchecked/topic/194354/ethyl-alcohol

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The same size of beetroot. All of the samples will be cut into cubes with 1cm
length.

The same size and type of cuvettes. This will eliminate a source of error from
the visible spectrophotometer results of absorbance.

Once prepared solution for all trials. This will result in the same %
concentration of ethanol for all samples and eliminate systematic mistake.

One source of ethanol. Ethanol from one bottle.

The same source of distilled water.

Careful timing. All samples will be put into test tubes for exactly one hour.

The same temperature of all substrates. Beetroots, distilled water and ethanol
left for night in laboratory.

The same laboratory conditions. No change in room temperature, light and so

on.

Apparatus and materials:

Visible Spectrophotometer.
25cm3 pipette with accuracy 0.05cm3.
5cm3 pipette with accuracy 0.1cm3
Knife.
12 the same type cuvettes.
1 litre of distilled water.
1 litre of 100% ethanol.
30 the same type test tubes with lids.
Stopper.
Marker.
Ruler with accuracy 0.1mm.
5 beakers with lid and volume at least 300cm3.
Beaker with volume 150cm3.
8 the same type of beetroots.
Rubber gloves.
Tweezers.

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Methodology:
I.

Prepare solutions with 0%, 25%, 50%, 75% and 100% ethanol concentration. Cover
the beakers to prevent water loss by evaporation and water gain from air, due to
ethanol hydroscopic properties. Proportions of ethanol and distilled water are written
in Table 1, below the instruction.

II.

From the middle of beetroots, cut 6 chips with volume 1 cm3. Leave the rest to prevent
water loss from the rest of the samples, which will have to leave for more than an
hour. Do not touch the chips with naked hands, the oil from palms may affect the rate
of diffusion. Wear rubber gloves.

III.

To 150 cm3 beaker pour 100cm3 of distilled water and put there all six samples for 10
minutes. In this way we will get rid of betanin excreted as a result of cell surface
membrane damage.

IV.

Using tweezers place each chip in one test tube and add 5cm3 of 0% ethanol
concentrated solution. Leave it for an hour to allow diffusion to occur.

V.

Calibrate visible spectrophotometer.

VI.

Take out the chips and pour solutions from test tubes into cuvettes. To the next 6
cuvettes pour 10 cm3 of 0% medium. This is needed to conduct the experiment by
spectrophotometer.

VII.

Before placing cuvettes in spectrophotometer, shake them to ensure that betanin

concentration is the same in the whole solution.

VIII.
IX.

Using the visible spectrophotometer calculate absorbance.

Repeat the experiment for 25%, 50%, 75% and 100% ethanol concentrated solutions.

Table 1.
Volume of ethanol and volume of distilled water needed to produce different
concentrations of medium.
The volume
The volume
The volume
Concentratio of distilled
of medium
of ethanol used
n of medium
water used
produced
/
/
100%
0
40
40
75%
10
30
40
50%
20
20
40
25%
30
10
40
0%
40
0
40

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Safety rules:

Wear lab coat and glasses to protect yourself.

Use knife and ethanol very carefully.

Data collection:
Qualitative data:

Samples with higher percentage concentration of ethanol, so with lower pH had a very
dark red colour. In 100% solution there was red-violet colour, which brighter with the
increase of pH, in 50% solution the colour was red. I could also observe that in a solution
consisting just only of distilled water, there was no change in colour.

Quantitative data:

After conducting the experiment , I obtained the following data. Results does not have
unit because as mentioned in background information, absorbance is the ratio between the
ratio of radiation falling to the radiation passing through the sample.5

http://en.wikipedia.org/wiki/Absorbance

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Table 2.
The results from visible spectrophotometer presenting absorbance at =575nm of light in a
solution of betanin from Beta vulgaris and different concentrations of ethanol.
Ethanol concentration / %
Sample
0%

25%

50%

75%

100%

0.032

0.062

0.256

0.787

1.210

0.035

0.099

0.286

0.845

1.002

0.043

0.067

0.281

0.703

0.984

0.031

0.062

0.243

0.794

1.307

0.022

0.092

0.246

0.794

1.294

0.027

0.063

0.273

0.842

0.968

0.025

0.078

0.284

0.749

0.992

0.041

0.083

0.293

0.821

1.246

0.040

0.073

0.289

0.801

1.293

0.023

0.075

0.276

0.805

1.086

Data processing:

Mean:
Mean is the average of outcomes obtained from all trials for one type of
solution. It will be very useful in drawing conclusions. Considering the average value
for each type of medium, we will be able to see the pattern the relationship between
the concentration of ethanol and the rate of diffusion. The formula is:

,
where

are the respective values of absorbance from one kind of

solution.
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Table 3.
The mean value of betanin absorbance in different ethanol
concentrations.
Ethanol concentration /
Mean value of
%
absorbance
1.138
100
75

0.794

50

0.273

25

0.075

0.032

Standard deviation:
Standard deviation is a statistical tool which helps to determine the uncertainty of
obtained results. The dictionary gives such a definition a measure of dispersion in a
frequency distribution, equal to the square root of the mean of the squares of the
deviations from the arithmetic mean of the distribution6. Therefore the formula is:
,
where

is the value of betanin absorbance for each trial for one ethanol concentrated

solution.
Table 4.
The standard deviation of betanin absorbance in different
ethanol concentrations.
Ethanol concentration
Standard deviation of
/%
betanin absorbance
100
0.145

75

0.042

50

0.018

25

0.013

0.008

http://dictionary.reference.com/browse/standard+deviation

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Rate of diffusion:
The aim if investigation is to check the correlation between the concentration of
ethanol in medium and the rate of diffusion. Therefore to make any observation, values for
rate of diffusion must be calculated. To do this, photon diffusion equation will be used:

At the beginning, the value of absorbance for all ethanol concentrations is equal to zero.
Our final values for absorbance are those obtained from visible spectrophotometer.
Table 5.
The rate of diffusion in different ethanol concentrations.
Ethanol
Initial
Final

concentration
absorbance absorbance Absorbance
/%
1.138
1.138
100
0

t / h

rate of
reaction / h-1

1.138

75

0.794

0.794

0.794

50

0.273

0.273

0.273

25

0.075

0.075

0.075

0.032

0.032

0.032

ANOVA Test:

ANOVA Test is the statistical test used to determine the difference between means of
multiple independent groups, which in this experiment are different rates of reaction. The
purpose of this test is to check the validity of null hypothesis, which is :

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where is the value of group mean and k is number of groups7.
The null hypothesis states that means of all groups are equal and by conducting ANOVA test,
the hypothesis can be rejected or confirmed.

Picture 1.
Picture represents the results obtained from ANOVA test calculated in Excel.

From the picture 1 we can easily notice that in this case with critical value of
0.05, F value equal to 507.368 is significantly greater than F critical value equal to
2.579. What is more, the P value is very small, much below 0.05, what reflects ratio
between F and F crit. This means that we can reject null hypothesis and therefore
conclude that there is huge difference between groups.

https://statistics.laerd.com/statistical-guides/one-way-anova-statistical-guide.php

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Uncertainties:
Calculating uncertainties is very important for analyzing experimental data.
We have already calculated mean and standard deviation, but what we also need is the
uncertainties involving mediums.

1) Uncertainty

of producing medium is the

Uncertainty of producing medium

2) Total

percentage

uncertainty

Total percentage uncertainty

3) Absolute uncertainty

Table 6.
Values of absolute uncertainty for different concentrations
of ethanol
Ethanol concentration Absolute uncertainty
/%
/%
100

2.4

75

1.8

50

1.2

25

0.6

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Data presentation:

Now, as we collected and processed all data and calculated uncertainties, we can
present them together in one table and then translate into graph.
Table 7.
Correlation between different ethanol concentration and the rate of
reaction with standard deviation.
Ethanol concentration with
Rate of reaction with standard
absolute uncertainty / %
deviation / h-1

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By observing the graph, we can notice that it is not the best method to represent data,
due to the very small values of data, which are invisible on the graph. The example is the
standard deviation values for the rate of betanin diffusion from Beta vulgaris. Uncertainties in
size of 0.003 are not possible to be displayed on graph.
However despite that technical problem, the value of

is high, proving that

the regression line drawn is the best-fit line and therefore represents the trend with the highest
precision. Slope equal to 0.0117 means that with the increase of ethanol concentration in
medium by one percent, the rate of diffusion will increase by

What is more, to obtain the most accurate results, slope uncertainty should be
calculated. The best method to obtain this value is to draw maximum and minimum gradient
lines. Their slopes will indicate the boundaries, between which best-fit line gradient should
be.

Table 8.
Values needed to draw maximum and minimum gradient lines, considering
values for 0% and 100% ethanol concentrated mediums.
Maximum gradient line
X value / %

Y value /

Minimum gradient line

X value / %

Y value /

0.0

-0.040

-0.024

102.4

1.283

97.6

0.993

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Conclusion and evaluation:

Having my knowledge and going on background information, I can conclude that the
experimental results are reasonable and justifiable, which is confirmed by high

During the experiment I confirmed that my hypothesis was correct, because as we could
notice with the increase of ethanol concentration in medium there was an increase in release
of betanin from Beta vulgaris. This resulted in greater absorbance measured by visible
spectrophotometer and higher rate of diffusion.
As stated in background information, with the increase of alcohol concentration, there
was a colour change, resulting in more and more darker reddish solution. This is the effect of
more serious damage of partially permeable cell membrane and therefore bigger amount of
red pigment secretion.
In the graph 2, representing the correlation between the rate of diffusion of betanin
from Beta vulgaris and the concentration of ethanol in medium with maximum and minimum
gradient lines, we could see that best-fit lines slope value is between the boundaries set by
the maximum and minimum gradient lines slopes. This determines the gradient uncertainty
and proves that the trend line was plotted with precision.
Moreover, from the graph 2 we could easily notice that the best-fit line is much below
maximum and minimum gradient lines and this led me to think if drawing straight line was
the best idea. I have noticed that in low ethanol concentrations the rate of diffusion increases
very slowly and in alcohol concentrations above 50%, the rate of reaction increases more
significantly. The possible cause of such trend was the fact that at the beginning, alcohol
concentrations were too low, resulting in relatively small change in pH (pH was slightly
acidic), which was not able to denature proteins in partially permeable membrane and
therefore much smaller amount of pigment was obtained. On the other hand, in mediums with
high ethanol concentration, pH was very low. The outcomes are change in protein structure
denaturalisation and bigger amount of betanin secretion. All of these information spurred me
to draw trend line one more time, this time not straight. The result I obtained surprised me,
because the

really proved that the increase of the rate of diffusion is not directly

proportional to the increase of ethanol concentration. This outcome also validates my theory
about the influence of medium pH on the amount of betanin released and therefore on the rate
of diffusion, what is presented on the graph 3 below.
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Another tool proving the high accuracy of experiment outcomes was small value of
standard deviation, which in cases of 0% and 25% alcohol concentration were so small that
were not taken into consideration.
However the value of

is not equal to 1, which means that despite my hard effort to

keep variables as much controlled as possible, both systematic and random errors were
present.
First of all, even if the source of Beta vulgaris was the same, there is no assurance the
all vegetables were taken from ground in the same day and stored in identical conditions.
This might highly affect the amount of red pigment present in beetroots. There was no method
to check that. To minimise the error I used just chips cut from the middle of vegetables, from
where I thought the conditions are least affected. However this could be improved by buying
Beta vulgaris from farmer we know or taking vegetables from the ground by ourselves and
conducting the experiment in a short period of time.
Secondly, during cutting chips, betanin was released. I tries to get read of it by placing
the samples in distilled water for 10 minutes, but it does not mean that pigment from the
surface area was get rid of to the same extent in all samples. Good idea is to keep beetroot
pieces in distilled water for longer period of time and then before putting samples to test
tubes, drying them with paper tower to get rid of water on the surface, which after adding
medium, might change its concentration. It seems insignificant, however we have to
remember that experimenting on such small samples, even slight uncertainties are a huge
source of error.
What is more, I cut my beetroot cubes using ruler and knife. This caused another
uncertainty, because by operating with naked eye there is a huge chance for human mistakes.
To avoid that I suggest using special equipment which will cut perfectly similar Beta vulgaris
cube chips.
Ethanol and all medium should always be kept in closed containers. This will prevent
not only water loss to the atmosphere by evaporation, but also water gain from air by
hydroscopic ethanol.Finally, a huge limitation is small size of Beta vulgaris chips. It might
be beneficial to use bigger samples.

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Bibliography:
Books:
1. Atkins P., de Paula J., 2006. Physical chemistry for the Life Sciences, UK, Oxford,

Oxford University Press.

2. Greenwood T. , Allan R., 2009. Senior Biology 2, UK, Edinburgh, Biozone.
3. Roberts M., Reiss M., 2000. Advanced Biology, UK, Cheltenham, Nelson.

Webs:
1. Britannica, Diffusion. Available on:

http://www.britannica.com/EBchecked/topic/163068/diffusion. [Accessed
06.09.2013].
2. Britannica, Ethyl alcohol. Available on:

http://www.britannica.com/EBchecked/topic/194354/ethyl-alcohol. [Accessed
08.09.2013].
3. Dictionary.com, Standard deviation. Available on:

http://dictionary.reference.com/browse/standard+deviation. [Accessed 11.09.2013].

4. Sheffield Hallam University Homepage, Beers Law. Available on:

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm. [Accessed
10.09.2013].
5. Wikipedia, Absorbance. Available on: http://en.wikipedia.org/wiki/Absorbance.

[Accessed 10.09.2013].
6. Wikipedia, Photon diffusion equation. Available on:

http://en.wikipedia.org/wiki/Photon_diffusion_equation. [Accessed 06.09.2013].

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