Adam Hazem Nahar

Arab Inter National Academy.

Nizar Samey Amery
2023 SI trail.

BIOLOGY HIGHER LEVEL

SCIENTIFIC INVESTIGATION

Analyzing the effect of various ethanol concentrations on the plasma membrane permeability
of beta vulgaris.

Word count: 3266

JAN,2024
1-Research question:

Examining the impact of different concentrations of ethanol ( C 2 H 5 OH ¿ / (0, 0.2, 0.4, 0.6,
0.8 mol) on the permeability of the plasma membrane of beta-vulgaris, assessed quantitively using a
spectrophotometer.

2-Introduction:

The objected aim of this investigation is to observe how varying concentrations of ethanol can
cause denaturation of beta-vulgaris’s cell membrane. The study specifically focuses on understanding
ethanol’s effect on the membrane’s permeability, and its ability to function. The experiment will
present different concentration of ethanol in the following order (0 mol, 0.2 mol, 0.4 mol, 0.6 mol, 0.8
mol, 1mol), allowing us to study Beta-Vulgaris’s cell membrane. The dark reddish color of beta-
vulgaris is caused by pigment named “Betanin”, which is stored in the cell’s vacuole, once ethanol
interacts with beta-vulgaris, its cell membrane will start to denature, causing the pigment to diffuse
and escape into the solution, which will be measured quantitively using a spectrophotometer on the
wavelength of 534 nm.

3-Background Research:

Ethanol is a clear, colorless liquid that possesses bactericidal activity, and is rapidly absorbed
by the body from the gastrointestinal tract. High doses of ethanol have several hazardous effects on
the body. One example could be how it can deplete endogenous
antioxidants in the liver. Ethanol can furthermore cause
respiratory collapse, hypothermia, hypertension or alcoholic
poisoning. Regardless of the various alarming consequences
brought by its consumption. Ethanol is still used as a
preservative in pharmaceutical preservation and as a topical
disinfectant, used to treat a variety of conditions such as
bacterial infections, skin irritation, or methanol poisoning. The
solvent also poses the ability to disrupt organic molecules, such
as the phospholipid of the cell membrane. It is also cheap, and
easy to obtain. Hence, it serves as a suitable independent Figure 1.1 chemical formula of ethanol.
variable for my experiment, making it an ideal choice for this investigative study. (6)

On the other hand of this investigation, beta

vulgaris cells possess a large central vacuole, that is
surrounded by a membrane called the tonoplast, these
vacuoles are filled with a pigment called Betalains
comprised of both betaxanthins (red colored) &
betacyanin (yellow colored), giving off beta-vulgaris’s
unique color. The cells of beta-vulgaris also obtains a

Figure 2.1-A diagram of a Beta-vulgaris L cell

 cell membrane that is composed of various organic molecules such as peripheral proteins, receptors,
and integral proteins, giving it a similar look to a mosaic, yet the membrane is notably made up of
around 50 % to 60 % of phospholipids, which are comprised of a hydrophilic heads entailing a head
group, a phosphate group, and a glycerol molecule, alongside a hydrophobic tail made of hydrocarbon
chains, giving it unique amphipathic properties. (1) Both the head and tail are bonded together by
ester bonds. Which When placed in water, the phospholipids will form a phospholipid bilayer, where
the tails are attracted to each other, and the heads are attracted to water. Giving it the ability to act as a
barrier, to control which molecules can pass through. The phospholipids can also flow past each other
laterally but can’t move vertically, making the cell membrane dynamic and fluid. Small, uncharged or
non-polar molecules can pass the cell membrane by passive transport through simple diffusion,
facilitated diffusion or osmosis, with the concentration gradient. While large, charged, polar
molecules pass via active transport through protein carriers that shape shift, or bulk transport. Against
the concentration gradient and requiring energy in the form of ATP. (2)

The cell membrane can be disrupted by ethanol, which

when added, forms hydrogen bonds with the lipids in the
phospholipid bilayer, leading to disruption in the alignment of the
cell membrane and the appearance of gaps, which will allow the
release of beta-vulgaris’s pigment to leak into the alcohol solvent,
causing the solution to turn red. The membrane’s permeability can
be investigated by measuring the concentration of beta vulgaris’s
pigments in the alcohol solvent using a spectrophotometer, which
functions by emitting a beam of light (typically in the visible
spectrum) under a specific wavelength that is complementary to
Figure 3.1- A diagram of how ethanol
that of the solution. (3) The beam is then partially absorbed by the disrupts the cell membrane.
provided sample solution. A detector, measures how much light passes through, granting the
concentration of the solution. The more the substance absorbs light, the higher its concentration. The
spectrophotometer is a great tool to measure absorbance, allowing us to obtain the concentration of
various solutions. (4)

In this study, my hypothesis revolved around the prediction, that as the concentration of
ethanol mixed in the solution raises, the permeability of beta-vulgaris’s cell membrane increases, this
is because ethanol has the ability to disrupt the fluidity and arrangement of the phospholipid
molecules, creating more gaps in the cells membrane, leading to the release of a higher amount of
pigment into the alcoholic solution, meaning that an increase in absorbance would be observed.

4-Variables:

I) Independent variable:
The various solutions with increasing concentrations of ethanol (C 2 H 5 OH ¿ , (0 mols, 0.2
mols, 0.4 mols, 0.6 mols, 0.8 mols.)

II) Dependent variable:

the absorbance of the beta-vulgaris and ethanol solution. Measured using a spectrophotometer
and noted on a sheet of paper.

Controlled variables How they controlled? Why are they controlled?

 A timer was set to 20 mins to control the amount It’s important to control time in this experiment, as
The Time of the experiment of time the beta-vulgaris pieces interacted with the longer the pieces of beta vulgaris are placed in
the ethanol solution the tubes, the more pigment would leak out,
the pieces of beta-vulgaris were produced by Every species of beetroot has a specific amount of
The Species, size and puncturing the same plant with a cork borer betanin inside the vacuole, therefore using various
amount of beta-vulgaris in multiple times, then cutting them to 5cm long, species may affect the membranes permeability.
each beaker creating multiple specimens under the same Additionally, the size and surface area of the
sample. Furthermore, each solution contained 5 cylinders greatly affect the amount of time required
pieces of the prepared specimens. for the pigment to leak.
The tubes were let to sit in a beaker, over a hot Different temperatures may affect the kinetic
The Temperature of the plate, under the same temperature. (room temp) energy withing particles, causing the cell membrane
tubes to be destabilized. lowering the accuracy of the
retrieved data.
The type of The specimens were all tested using the same Whenever using a spectrophotometer, the
Spectrophotometer spectrophotometer, under the wavelength of wavelength set must be controlled, as changing the
534.5 nm (visible light, light green) wavelength may yield different absorbance results.
The size of the crevette The size of the curvet of each specimen was Using different sizes of crevettes for each sample
3 may result in lower accuracy of the retrieved
2 cm
results.
5-Material and apparatus:
 25 x graduated cylinders of Beta-  Beaker (250mL± 0.12 mL)
vulgaris.  5x graduated cylinders (100 mL, ± 0.05 cm
)
 Scalpel
 Spectrophotometer (at 534.5 nm)
 Forceps
 5x Cuvettes (2 cm3 ¿
 Cutting board
 Ruler, (± 0.05 cm)  Timer
 Tongs  Thermometer
 Distilled water  Cork Borer (8mm, ± 0.05 mm)
 Pipette  Paper towels
 Ethanol (C 2 H 5 OH ¿ ,
6-experiment design:

Figure 4 The experiment was held in the biology laboratory; the picture depicts the 5 graduated cylinders that were used. to produce the
solutions, with the 5 beakers that contained the solutions, and the specimens of beta vulgaris used.

Time Action to be done

First 5-10 mins Preparing the specimens
10- 15 mins Preparing the solutions
 20 min Placing the specimens in the
ethanol solution.
5-10 mins Filtering the specimens
5-10 mins Extracting samples and
adding them to the cuvettes
10 – 15 mins Using the spectrophotometer
to obtain the absorbance

The above table was created before the experiment for me to be able to manage my time efficiently, and have a
clear Idea and Plan of how the experiment should take place.

7-Procedure:

1. Obtain 5 pieces of relatively the same size of beta-vulgaris using a cork borer, then further cut
them into 30 smaller pieces using a scalpel.
2. Use a paper towel to dry and clean any pigments released by the 25 x specimens.
3. Produce 5 solution of increasing concentrations of ethanol, using distilled water to dilute
them. According to the table 1.1

Note - volume of each graduated cylinders is 100 mL.

Percentage of ethanol Num of mols of Volume of ethanol Volume of distilled water
in the solutions ethanol mL mL
0% 0 0 100
20% 0.2 20 80
40% 0.4 40 60
60% 0.6 60 40
80% 0.8 80 20

4. Place 5 pieces of the specimens in each graduated cylinder and leave them for exactly 20 min
as set by the timer. Then filter out the samples from the ethanol solution.
5. Extract samples from all the graduated cylinder using the pipette, then fill and prepare the
cuvettes making sure that no leaks can occur.
6. Finally, Place the cuvettes in spectrophotometer and set the wavelength to 534.5 nm (green
light) to obtain the absorbance of each solution, making sure to take notes of the data on a
paper.

8-Risk assessment and Consideration:

Hazard Hazard Reason for Precaution & Emergency protocol Degree of

Category Description Hazardousness prevention hazardousness
Equipment Spectrophotometer Electrical Make sure to keep your Turn off the source of
(Electrical) equipment may hands dry, and any electricity immediately,
cause an electrical liquids away when and ask for help from any MODO-
shock or dealing with electrical of the staff and teachers RATE
equipment equipment
damage
Chemical Ethanol Flammable, Keep away from any Wash away any ethanol
inhalation or contact with eyes, mouth that may have entered to
consumption may or skin. Prevent spillages your eyes with cold water.
cause irritation. or unnecessary leaks, In case of a fire, use a fire HIGH
and make sure to keep extinguisher. And make
away from all types of sure to seek medical
 flames. attention if needed
Equipment Scalpel Sharp blade may Handle the tool with care In a result of an injury,
cause injuries if and always under make sure to wash, and
misused. supervision. And always disinfect the cut. Make MODO-
wear the appropriate lab sure to seek medical RATE
safety equipment. attention if the injury is too
serious.
Equipment Glassware Broken glass may Always handle all glass In case one of the glass
cause severe ware with care, making wares breaks, make sure to
injuries. sure to follow the lab clean it up to prevent
rules, and always wear others from injuring
the appropriate lab safety themselves. And if an Low
equipment injury occurs make sure to
wash and disinfect it
properly. seek medical
attention if the injury is too
serious.

I - Ethical consideration:

As per the IBs ethical values, this experiment had no effect or harm made on
any form of animals, as it was implemented on an ethical plant subject.

II - Environmental consideration:

The experiment was designed and set up with the environment in its perspective, the used
ethanol solutions will be fully diluted and disposed of appropriately to prevent any harm.
Furthermore, the used beta-vulgaris specimens will be carefully discarded, sealed within a designated
hazardous bag to prevent its consumption and ingestion by animals after it is disposed of, While the
left-over Beta-vulgaris L, will be repurposed and used as a biofertilizer for the school’s garden,
contributing to sustainability rather than it being wasted, given its biodegradable nature.

9- Data and Analysis:

I ended up producing tables and statistics that conveyed the portrayed data that using
Microsoft excel and google sheets to better showcase the data.

A. Qualitative Data:

When observing the specimens of Beta-Vulgaris L that were submerged in the acholic
solution, it was evident that the higher the concentration of ethanol, the darker the shade of
red present in the solutions, while at lower concentrations the color was seamlessly paler and
transparent. After extracting the beta-vulgaris L specimens, it was observed that the samples
that were present at higher concentrations lost more pigments than the ones that were at lower
concentrations, this was due to the bleeding effect caused by the ethanol.

B. Qualitative Data:

Table:
Percentage of ethanol Concentration of Absorbance
 % (C 2 H 5 OH ¿ ethanol (C 2 H 5 OH ¿
0 0 mol 0.064 Au
20 0.2 mol 0.235 Au
40 0.4 mol 0.422 Au
60 0.6 mol 0.412 Au
80 0.8 mol 0.397 Au

Scatter plot/ graph:

Data information Calculation

Standard deviation 0.155
of absorbance
Relative standard 0.155
deviation. RSD= × 100=50.7 %
0.306
Range 0.358
Mean value of 0.306
absorbance.
The R value. 0.7358
Equation of the line y= 0.4215x+0.1374
of best fit.
C. Analysis:

The primary cause of this experiment was to determine if there was a correlation
between the concentration of ethanol and the permeability of the beta vulgaris L’s cell
membrane, the experiment yielded a set of absorbance using a spectrophotometer,
representing the degrees of pigments leaked in the various solutions. The hypothesis that I had
chosen in the beginning of the test. Was proven correct, as the degree of pigment leaked was
observed in larger quantities as the concentration of ethanol increased. This becomes evident
when comparing my lowest concentration of ethanol (0%) which yielded (0.064 Au) with
my highest concentration of ethanol (80%) which yielded (0.397 Au).

By calculating the relative standard deviation, it becomes evident that the set of data
yielded by the experiment are of a moderate degree of variability. Suggesting that the
absorbance measurements are noticeably spread around the mean. This also suggests that the
experiment was moderately accurate yet still exhibits some variability. One main factor that
might have contributed to this variability, is the wavelength that was experimented on the
beginning. After much more intensive examining it was clear that the usage of the green
wavelength was a pivotal mistake. As it affected the spectrophotometers’ ability to test the
concentration of the provided cuvettes. Leading to some inaccurate data. Such as the
concentration yielded of the last solution.

The line of best fit further back my hypothesis, indicating a positive slope, which
suggests that the concentration of ethanol solution exhibits a moderately strong correlation
with the absorbance yielded for each specimen. Furthermore, while searching online. I found
 a past study conducted on 2021 by a biology teacher by the name of Praewow Winmoon at
the international school of St Andrews. Who also yielded similar data. His experiment did a
similar approach to mine, yet different as it was based on measuring the transmission of light,
which is the quantity of light that passed through the solution while I had measured
absorbance which is the quantity of light that was absorbed by the solution. His research
yielded a strong negative correlation between transmission of light and ethanol concentration,
suggesting that there is a positive correlation between the concentration of ethanol and the
absorbance. Further supporting my hypothesis. (5)

10-Conclusion:

By undergoing this experiment, it was possible to identify, how ethanol catalyzes the rupture of the
cell membrane, leading to the release of the Betalains pigments. I observed that The higher the
concentrations of the ethanol solution, the greater the amount of Pigment leaked as more gaps would
start to form and the structure of the membranes would be disrupted, resulting in a change in the
phospholipid bilayer’s characteristic of being selectively permeable. Consequently, an increase in the
concentration of ethanol yielded a rise in the degree of permeability of the cell membrane as more
pigments are being leaked out into the solution. The study made by Praewow Winmoon, also
exhibited the same conclusion. Both experiments provided data that essentially agrees that higher
concentrations of ethanol result in lower transmission of light which results in higher absorbance,
concluding that ethanol can directly affect a cells permeability. (5)

11-Evaluation:

STRENGTHS
Controlling the time proved to be a very important addition, as it allowed me to focus on the experiment, and provide a
clear set of data that required a specific set of time. It also limited other external factors from affecting the absorbance.
Controlling the temperature was proven to be essential for this experiment, because As temperature increases, its proven
that the permeability of cell membrane. This is because high temperatures can denature proteins in the mosaic membrane
by damaging their bonds in the tertiary stage. While at low temperatures, the membrane will have lower permeability, as
the phospholipids will exhibit lower temperatures which will cause the lipids to become much more rigid, decreasing the
membranes permeability. Which is why it is important for the temperature to be controlled.
The size of the set of tools used were very effective as they allowed me to measure accurately, having all the specimens
treated by the same set of tools allowed for a limiting of possible error. An example of this would be how all the solutions
were extracted into similar cuvettes and were tested on by the same spectrophotometer.
Washing, cleaning and then drying the specimens of beta vulgaris L, proved helpful as it had eliminated the possibility of
other factors affecting the amount of pigment released, such as the already released pigments which were caused by the
cutting of the beetroot.

The limitation Type of error Effect Possible improvements

The experiment None Not Having a 1 mol concentration of
lacked a 1 mol ethanol, limited the experiment from
concentration being completed fully. This one last
solution of ethanol. solution could’ve changed my overall
data which would’ve certainly yielded
another different or similar conclusion.
Washing the beta- Systematic error Although I had washed, cleaned and then
vulgaris L dried the specimens before initiating with
specimens properly the experiment. The spectrophotometer
still sensed some pigment in the solution
A possible improvement could be
the addition of this solution into
the experiment. Next time the
experiment should be complete,
allowing it to yield a full
conclusion.
Cutting the beta vulgaris
specimens then washing them
twice and drying them twice,
should eliminate the chance of
 of the 0 mol concentration. Suggesting
that still some pigment managed to leak
out because of the cutting.
Incorrect usage of the Random error The spectrophotometer was used
spectrophotometer incorrectly, as it was placed on the light
green wavelength while the correct
placement should have been on the blue
wavelength. This may have contributed
Time error Random error When removing the specimens from the
pigment, I wasted some time, as. I had to
extract every solution and placing them in
the cuvettes individually. Which may
have affected the number of pigments
leaked.
some pigments leaking out.
Doing more past research and
determining the complementary
color for the specimens is much
more effective. As it would limit
the ability of external factors to
affect my experiment.
Creating a schedule where every
specimen is placed after a set of
time, may create a time gap that
allows me to extract the solutions
into the spectrophotometer.

12-Bibliogoraphy:

1) Publishing, E. V. WongAxolotl Academica, et al. “Book: Cells - Molecules and Mechanisms (Wong).” Biology
LibreTexts, 30 Dec. 2022, bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Book%3A_Cells_-
_Molecules_and_Mechanisms_(Wong). (1)
2) “Brent Cornell.” BioNinja, www.ib.bioninja.com.au/standard-level/topic-1-cell-biology/13-membrane-structure/.
Accessed 13 Jan. 2024. (2)
3) University of Birmingham. “Biology A Level Revision Resource: Testing Cell Membrane Permeability.” University
of Birmingham, www.birmingham.ac.uk/teachers/study-resources/stem/biology/stem-legacy-membranes.aspx.
Accessed 13 Jan. 2024. (3)
4) Patil, Mahesh. “Advantages and Disadvantages of Spectrophotometry.” Advantages and Disadvantages of
Colorimetry, chrominfo.blogspot.com/2019/09/advantages-and-disadvantages-of_11.html. Accessed 13 Jan.
2024. (4)
5) Winmoon, Praewow. “The Effect of Different Ethanol Concentrations on the Cell Membrane Permeability in Beetroots.” St
Andrews International School, 2019. http://www.ijsred.com/volume4/issue5/IJSRED-V4I5P33.pdf (5)
6) “Ethanol.” National Center for Biotechnology Information. PubChem Compound Database, U.S. National Library of
Medicine, 2020, pubchem.ncbi.nlm.nih.gov/compound/Ethanol. (6)