NAME: ABDUL RAHMAN BIN MOHD YUSOF ZAKI

NRIC: 930228-02-6153
SID: 2011892538
CLASS: 12M10
LECTURER`S NAME: MADAM RITA ROHAIZAH SOHARI
PARTNERS`S NAME: AKRAM MAHMUD

Title : The eIIect oI temperature on membranes
Objectives : To investigate the eIIect oI temperature such as 5C, 28C, 35C, 45C,
55C and 65C on beetroot`s (Beta vulgaris sp. membrane structure
Problem statement: Does temperature aIIect the structure oI membrane?
Introduction :
Plasma membrane controls over the passage oI substances across it. Some
substances, particularly those that dissolve very easily in lipids, simply pass through the
membrane in a process oI diIIusion as though the membrane was not there. Other very small
molecules, such as the gases oxygen and carbon dioxide as well as water itselI, also pass
Ireely in and out oI cells through the membrane.

Facilitated diIIusion involves proteins in the membrane which allow only speciIic
substances to move through passively down their concentration gradient. They may simply be
channel proteins which Iorms pores through the membrane.


Beetroot 8 one oI the many cultivated varieties oI beets (Beta vulgaris and arguably
the most commonly encountered variety in North America, Central America and Britain. In
Malaysia, there is an Indian shop in BrickIields selling beetroot. Beetroot is a rich source oI
potent antioxidants and nutrients, including magnesium, sodium, potassium and vitamin C,
and betaine, which is important Ior
cardiovascular health. It Iunctions by acting
with other nutrients to reduce the concentration
oI homocysteine, a homologue oI the naturally
occurring amino acid cysteine, which can be
harmIul to blood vessels and thus contribute to
the development oI heart disease, stroke,
and peripheral vascular disease. Betaine
Iunctions in conjunction with S-
adenosylmethionine, Iolic acid, and
vitamins B
6
and B
12
to carry out this Iunction.
A betaine in chemistry is any neutral chemical
compound with a positively
charged cationic Iunctional group such as an quaternary ammonium or phosphonium cation
(generally: onium ions which bears no hydrogen atom and with a negatively charged
Iunctional group such as a carboxylate group which may not be adjacent to the cationic site.
Betanin, or Beetroot Red, is a red glycosidic Iood dye obtained Irom beets;
its aglycone, obtained by hydrolyzing away
the glucose molecule, is betanin. As a Iood additive, its E
number is E162. Betanin degrades when subjected to light,
heat, and oxygen; thereIore, it is used in Irozen products,
products with short shelI liIe, or products sold in dry
state.
|1|
Betanin can survive pasteurization when in
products with high sugar content. Its sensitivity to oxygen
is highest in products with high content oI water and/or
containing metal cations
(e.g. iron and copper; antioxidants like ascorbic acid and
sequestrants can slow this process down, together with
suitable packaging. In dry Iorm betanin is stable in
presence oI oxygen. Normally, the betanin cannot pass
through the membranes oI beetroot but they leak out when the beetroot is cooked.
A spectrophotometer is a photometer (a device Ior measuring light intensity that
can measure intensity as a Iunction oI the light source wavelength. Important Ieatures oI
spectrophotometers are spectral bandwidth and linear range oI absorption or reIlectance
measurement. A
spectrophotometer
is commonly used
Ior the
measurement oI
transmittance or
reIlectance oI
solutions,
transparent or
opaque solids, such
as polished glass,
or gases. However they can also be designed to measure the diIIusivity on any oI the listed
light ranges that usually cover around 200nm - 2500nm using diIIerent controls
and calibrations. Within these ranges oI light, calibrations are needed on the machine using
standards that vary in type depending on the wavelength oI the photometric determination.
The use oI spectrophotometers spans various scientiIic Iields, such as physics, materials
science, chemistry, biochemistry, and molecular
biology. They are widely used in many
industries including semiconductors, laser and
optical manuIacturing, printing and Iorensic
examination as well in laboratories Ior the study
oI chemical substances. Ultimately, a
spectrophotometer is able to determine,
depending on the control or calibration, what
substances are present in a target and exactly
how much through calculations oI observed
wavelengths.

In scientiIic Iields the word generally reIers to the device that measures
the absorbance oI particular wavelengths oI light by a speciIic solution. This device is most
commonly used to determine the concentration oI a known solute in a given solution by the
application oI the Beer-Lambert law, which states that the concentration oI a solute is
proportional to the absorbance.

Hypothesis :
The higher the temperature increases, the higher the percentage absorbance oI
spectrophotometer
Null hypothesis:
There is no diIIerent colouration between colour change and percentage absorbance as the
temperature is increasing.
Variables :
a) manipulated: Temperature oI water bath
b) responding: The percentage oI absorbance oI spectrophotometer
c) fixed : The diameter and length oI beetroot, type oI solution beetroot is put into
(distilled water, type oI beetroot, and size oI boiling tube.
Apparatus and Material:
A raw beetroot, 1cm diameter cork borer, tissue paper, white tile, kniIe, metre rule,
beaker about 250 cm3, Iorceps, stirrer, water bath at 5C, 28C, 35C, 45C, 55C and 65C,
a boiling tube rack, 6 boiling tubes, thermometer (one per water bath, spectrophotometer,
cuvettes, stopwatch, distilled water, 5cm3 measuring cylinder, marker pen and sticky labels


Method :
1. A raw beetroot is obtained and was cut into sections using the 1cm diameter cork
borer. The sections were put at the white tile and then were cut by the kniIe into 6 pieces
and were measured 1cm oI length each. Always be careIul when using the cork borer as it
can hurt the users hand and also be careIul not to pressure the cork borer as the sections oI
the beetroot can spill out juice that can stain clothes very badly.

2. The 250 cm3 beaker was Iilled with 200cm3 oI distilled water and the 6 pieces oI
beetroot is placed into the beaker by using the Iorceps. The solution is stirred by stirrer to
wash away the excess dye Irom the membrane oI beetroot which is damaged when the
pieces were cut.

3. 6 boiling tubes were labelled by using the marker pen and sticky labels as 5C, 28C,
35C, 45C, 55C and 65C and 5cm3 oI distilled water is measured using the 5cm3
measuring cylinder and was poured into each boiling tube. All the boiling tubes were
placed into the water baths at 5C, 28C, 35C, 45C, 55C and 65C Ior 5 minutes Ior
the incubation oI the distilled water. AIter 5 minutes, the 6 pieces oI beetroot were taken
out by Iorceps and were rinsed by tissue paper and were placed into each oI the boiling
tubes. By using the stopwatch, the beetroot pieces were leIt Ior 30 minutes in the water
baths.

4. AIter 30 minutes, careIully, the boiling tubes were taken out n by using Iorceps, the
beetroot pieces were taken out and thrown away. Then, the boiling tubes were shaken to
disperse the dye oI the beetroot completely in the solution and were placed back into the
test tube rack.

5. The spectrophotometer was switched on and was set to read absorbance. The
spectrophotometer was set to use 500nm wavelength. Using the 5cm3 measuring
cylinder, 3cm3 oI distilled water is measured and was placed into the cuvette. The cuvette
was placed into the spectrophotometer and the cuvette was made sure that the light
shining through the smooth sides. CareIully, do not let the smooth sides were touched by
hands and always wipe the smooth sides beIore it was places into the spectrophotometer.

6. The spectrophotometer was adjusted to read the zero absorbance Ior clear water. The
settings did not need to be altered again during the experiment.

7. 3cm3 oI the dye solution oI temperature 5C was placed into a spectrophotometer
cuvette and a reading Ior absorbency was taken. The readings Ior all the temperatures
were repeated.

8. The results oI the experiment were recorded and were presented in an appropriate
way.

9. Any trends or patterns were identiIied in the results.


Data :



Temperature/C Absorbance/AU
5 0.023
28 0.040
35 0.077
45 0.128
55 0.457
65 0.528

1
e
m
p
e
r
a
t
u
r
e
]

C

ercentage Absorbance]AU
Graph of 1emperature]C aga|nst ercentage
Absorbance]AU
Discussion :
Analysis oI data
The percentage absorbance increases as temperature increases Irom 5C to 65C. This
result shows that the dye concentration is increasing when the temperature increases.
Heat aIIects the structure oI plasma membrane. As the temperature increase, due to
the molecules vibrating with more kinetic energy, and at higher Irequencies and amplitudes,
the Van der Waals Iorces will be overcome between molecules. This could cause the
phospholipid bilayer to move apart, and Ior the cholesterol molecules to bind weaker with the
Iatty acid chains oI the phospholipids. Also, the increased kinetic energy oI the protein
molecules will cause the hydrogen bonds to break, causing a change in the shape oI the 3D
tertiary structure oI the proteins. This could cause holes in the membrane to appear. In
addition to this, the higher temperatures will cause the phospholipid bilayers to become more
Iluid, and thereIore become more permeable. ThereIore, the betanin can pass through the
membranes oI beetroot according to the holes that the membrane has created. As a result, the
solution becomes more stain with red colour as net movement oI betanin into the solution is
high.

Limitation and ways to overcome it
First oI all, we cannot obtain a precise cut oI 6 pieces oI beetroot with 1cm length and
1cm diameter. There are slight diIIerences in mass oI all the pieces where it supposed to be
the same mass. To overcome this problem, we need to slice the pieces using sharp kniIe and
weight the pieces beIore the experiment is conducted.
The dye oI the pieces were not washed perIectly, it was washed in distilled water Ior 5
minutes as it was supposed to be washed and leIt overnight to wash away the excess dye.
These conditions aIIect the results oI the percentage absorbance oI the solution. To overcome
this problem, we need to wash the pieces in a beaker oI distilled water and leIt it overnight
Ior the excess dye to be washed oII Irom the plasma membrane.
The readings oI the percentage absorbance were not precise. This is because only the
readings were taken only once. ThereIore, we cannot obtain the average value oI the
percentage absorbance. To overcome this problem, we need to take multiple readings at least
5 readings and obtain the average values.

Precaution
1. When entering the laboratory, always wear a lab coat as it will protect us Irom
chemicals spillage.
2. CareIully cut the beetroot as it will stain the clothes and skin with red dye.
3. When using the kniIe, always careIul and not to play with it as it may hurt
someone.
4. Press the cork borer gently when using it as the sharp end oI it can injured
someone.
5. Always be careIul when using the water bath as it would scald the skin when the
water is hot and gives oII steam.

Further Work
This experiment is to investigate the eIIects oI alcohol concentration on plasma membrane.
The hypothesis oI this experiment is the higher the concentration oI alcohol, the higher the
percentage absorbance oI the spectrophotometer. The Iixed and responding variables are kept
constant but the manipulated variable is diIIerent. In this experiment, we use diIIerent
concentration oI ethanol such as 0.1mol, 0.5mol, 1mol, 2mol and 3mol and it is the
manipulated variables. The experiment was carried out as above experiment but using these
new variables, which is the manipulated variable. Each beetroot is placed in 5 diIIerent
labelled boiling tubes which contains diIIerent concentration oI ethanol. The beetroot pieces
were leIt in the boiling tubes about 30minutes and were taken out oI the boiling tubes and the
boiling tubes were shaken to disperse the dye. Then, one by one oI the solution was measured
by the spectrophotometer oI its percentage absorbance. The predicted results were the reading
oI the percentage absorbance oI the spectrophotometer increase as the concentration oI
alcohol increase. This is because lipid is soluble in ethanol. Cell membranes are made
(mostly oI lipids. Ethanol was shown to "Iluidize" the membrane and dissolve the
phospholipids. This is because it has both polar and non-polar ends. The aIIinity oI ethanol
Ior water and Iat, based on its hydrogen bonding ability, altered the Iluidity oI the membrane.
This changes the ability oI membrane embedded proteins to Iunction (they are denatured due
to altered hydrogen bonding and interact. The membrane`s integrity diminishes and there are
more gaps within the membrane Ior the betanin pigments to diIIuse to the outside oI the cell
and into the solution. The higher the ethanol concentration the more pigment leakage.
Conclusion :
As the temperature increase, the percentage absorbance increase which showing that
the dye concentration increase. When temperature increase, the protein molecules embedded
in the plasma membranes beginning to change their shape and cannot Iunction properly as
usual. ThereIore, betanin can diIIuse easily across the plasma membrane into the solution.
During increase oI temperature, the kinetic energy oI the betanin also increases
rapidly as the particles in betanin vibrate vigorously. ThereIore, the net movement oI the
betanin into the solution is high which results in the red colour intensity oI the solution to be
higher as temperature increase.

Reference :

1. Fullick, A. (2010. /excel AS Biology. London: Pearson Education Limited.
2. Wikipedia, the Iree encyclopaedia
3. Other biology report samples