Assessment of Beet Membrane Permeability with Ethanol

The assignment involves carrying out an experiment using cuvettes with different concentrations of ethanol (5%, 30%, and 70%) on a solution containing B. vulgaris, measuring absorbance to assess membrane permeability. The results are analyzed to determine the impact of ethanol concentration on beet membrane integrity.

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ETHANOL CONCERTRATION EFFECTS ON CELL MEMBRANE
NAME
Class Professor
School
City
3/28/2018

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OBJECTIVE OF THE EXPERIMENT
The objective of this experiment is to study the effect of different ethanol concentrations on cell
membrane integrity by using visible spectroscopy
METHODS
Requirements
The requirements are, three test tubes, test-tube rack, cuvettes, beetroot (Beta vulgaris),
spectrophotometer, detergent SDS 10%, small dowel rods, room temperature water, 3 replica,
corkborer with 4mm inside diameter,6 replicas, 70% ethanol, prepared 50% ethanol +10 ml
water, prepared 15ml of 70%ethanol+20ml water, distilled water.
Procedure
Cut B vulgaris into four equal pieces with the same diameter and length using a cork borer in a
size as to fit in a cuvette. Rinse each piece of B. vulgaris with tap water to remove the excess red
dye (pigment betalain) that resulted from cutting, number the test tubes from 1-4, mix the
contents in the test tubes after placing the contents in each cuvettes and name them for ten
minutes, warm the spectrophotometer for at least 20 minutes, place the cuvette with room
temperature water(used as the control experiment) in to the spectrophotometer with triangle on
the cuvette facing directly 10ml in front of the instrument and press the 0 ABS 100% and then
remove it from the instrument, repeat this for the other cuvette A,B and C and record the values
obtained, the values obtained are at an interval of 15 minutes, and values are recorded thrice for
precision to be compared to the replica.
RESULTS
The results of absorbance are obtained from the spectrophotometer with the time interval of 15
minutes as shown in the table below;
Cuvette Concentration Time in 15 minutes 30 minutes

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(%) minutes
0.00
A 30 0.00 0.90 1.8
B 50 0.01 1.5 0.3
C 70 0.08 2.1 4.2
Getting the concentrations
15ml of 70%ethanol +20ml water
70%of ethanol =70ml ethanol dissolved in 100ml of water
100ml of water contains 70ml of ethanol
15ml of water will contain?
(15×70) ÷100=10.5ml
Hence 10.5ml of ethanol can be dissolved in 15 ml of water
Concentration of the whole solution
15ml of 70% of ethanol+20ml of water
(10.5×100) ÷35=30%
Molar concentration
10.5÷35=0.3M
Concentration of 50% ethanol+10ml of water
(50×100) ÷100= 50%
70% ethanol=70%
Calculation of absorbance of a substance in each cuvette is based on knowing the transmittance
which will build on the absorbance (Ultee, A. 2002). To calculate transmittance, we use the
following formula:
T=I/IO

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T represent transmittance
I represent incident.
IO represents incident intensity.
Calculating the different absorbance’s, we use the following formula.
A=-log (T)
Whereby T is the transmittance.
Calculating absorbance
Log-1.41=0.15
Log -7.94=0.9
Log -125.89=2.1 (Lambert, R. 2001).
Absorbance given correct to one decimal place
This means as the transmittance increases the absorbance decreases hence the transmittance is
inversely proportional to the absorbance since the increase in transmittance leads to a reduction
of absorbance and a decrease in transmittance leads to an increase in absorbance.
To determine the relationship between absorbance and concentration we need to look at
Slope of the curve (m) =Y2-Y1/X2-X1
X represents concentration
Y represents absorbance.
This consequently leads us to the Y=MX+B
Which derives another formula:
A=ϵLC
A represents the absorptivity

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From this we find that the absorption is inversely proportional to the transmittance and
concentration is directly proportional to the absorbance (Garcia, C. 1994).
DISCUSSION
The objective of the experiment is to study the effect of different ethanol concentration on the
cell membrane integrity by using visible spectroscopy. An increase in the concentration of
ethanol results to an increase in the permeability of the membrane hence high absorption results
to an increase in the color intensity of the solution, from the results obtained this proved. The
absorption by a substance in cuvette is determined by the concentration of the solution. This
means that high concentrated ethanol has high interaction between molecules leading to
increased absorbance by the solution hence the detection registered a higher absorption than the
less concentrated solution. The highly concentrated ethanol (Ethanol 70%) registered high
absorbance because there was increased interaction between molecules leading to large
permeability of the membrane hence absorption improved due to an increase in the concentration
of ethanol. Increase in the color intensity is directly proportional to the membrane permeability.
The cuvette with ethanol at a concentration of 30% has a slightly lower absorbance to the one
with the 70% concentration since its molecules have less interaction compared to that of 70%
concentration hence B. vulgaris is stronger in the 70% concentration.
The ethanol with the lower concentration (ethanol 5%) registers a low absorbance since there is
the least interaction between molecules leading to the lowest absorbance by the lowest
concentrated solution. The B .vulgaris is not strong compared to the ethanol with 30%
concentration.
Absorbance is directly proportional concentration. Absorption of solution in cuvette is used to
measure the damage on the cell membrane that directly affects the permeability of the cell. Other
points to note can be the shape of cuvette can affect light transmittance, the rate at which the beet
will be damaged is directly proportional to the absorbance and concentration and the darker the
color of the solution due to the damage of the beet the more the absorbance and the more the
concentration. In all this absorbance is directly proportional to the concentration of ethanol. The

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higher the concentration the higher the absorbance and the lower the concentration the lower the
absorbance.
Limitations
When carrying out the experiment the following limitations were encountered, ethanol was
flammable and could ignite, steam and hot water may cause burns, equally timing the time
allocated by both cuvettes.
Remedies
This was solved by putting the ethanol away from the sources of ignition, wearing suitable heat
proof gloves and opening the water baths carefully.

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References
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essential for action against the food-borne pathogen Bacillus cereus. Applied and environmental
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Lambert, R.J.W., Skandamis, P.N., Coote, P.J. and Nychas, G.J., 2001. A study of the minimum
inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol.
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Garcia-Ruiz, C., Morales, A., Ballesta, A., Rodes, J., Kaplowitz, N. and Fernández-Checa, J.C.,
1994. Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria
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(Beta vulgaris). Plant and Soil, 150(2), pp.177-191.
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Renwick, J.A.A., 2003. A saponin correlated with variable resistance of Barbarea vulgaris to the
diamondback moth Plutella xylostella. Journal of chemical ecology, 29(6), pp.1417-1433.
Ultee, A., Kets, E.P.W. and Smid, E.J., 1999. Mechanisms of action of carvacrol on the food-
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