Moscow Institute of Physics and Technology

Methods of Chloroplast Isolation

General Biology
Lab Report
By Joy Al Haddad
B06-208
1. Purpose of the experiment: to isolate chloroplasts from green plant cells by
centrifugation: to detect peaks of light absorption using a spectrophotometer.

2. Tools and instruments:

● Lettuce leaf
● Mortar and pestle
● Filter cloth
● Test tubes with lids
● Alcohol
● Buffer I
● Buffer II
● Centrifuge
● Spectrophotometer

3. Theoretical introduction:
● Chloroplasts are organelles in plants which perform the function of
photosynthesis. These structures are mainly located in the palisade
parenchyma of the mesophyll cells in the leaf. The green color of these
structures in plants is due to the presence of a pigment called
chlorophyll which resides in the chloroplasts.
● The system consists of many liquid-filled flat sacs called thylakoids;
thylakoids form stacks, or granules, which are connected to each other
by lamellae (single granules). Each grain resembles a stack of coins,
and the lamella resembles a plate.
● The pigments of photosynthesis in higher plants are divided into two
classes: chlorophylls and carotenoids. The main purpose of pigments
is to absorb light energy, then convert it into chemical energy. Pigments
are located on the membranes of chloroplasts (thylakoids), and
chloroplasts in the cell are usually oriented in such a way that the
membranes are at right angles to the light source (for maximum light
absorption).
● Carotenoids are yellow, orange, red or brown pigments that are highly
absorbent in the blue-purple area. They are called auxiliary pigments
because they transfer the light energy absorbed by them to chlorophyll.
● In addition to their function as auxiliary pigments, carotenoids protect
chlorophylls from excess light and from oxidation by oxygen formed
during photosynthesis.
● Chlorophylls absorb mainly red and blue-violet light, green light is
reflected by them, which gives plants a specific green color if it is not
masked by other pigments
● Since chlorophyll contains a Magnesium ion, its deficiency leads to a
decrease in the formation of chlorophyll and includes a long yellowing
of the leaves of the plant.
● There are four types of chlorophyll found in nature: a, b, c, d.
Chlorophylls a and b contain higher plants and green algae, diatoms
contain a and c, red algae contain a and d.
● Different chlorophylls have different side chains attached to their
heads, which leads to a change in their absorption spectra, increasing
the wavelength range of the absorbed light.

● Intact chloroplasts are the best source for studying the processes like
carbon assimilation, electron flow and phosphorylation. The
concentration of the chlorophyll molecules can be estimated from the
isolated chloroplasts. DNA and RNA of chloroplasts can also be
extracted.
● Photosystems are functional and structural units of protein complexes
involved in photosynthesis. Together they carry out the primary
photochemistry of photosynthesis: the absorption of light and the
transfer of energy and electrons.
● There exists 2 types of photosystems: photosystem I and photosystem
II is that PS I tends to absorb light of longer wavelengths > 680nm,
whereas PS II absorbs light of shorter wavelengths <680 nm.
 ● Spectrophotometry is a method to measure how much a chemical
substance absorbs light by measuring the intensity of light (amount of
photons) as a beam of light passes through a sample solution.
● Depending on the range of wavelength of light source, the
spectrophotometer classifies it into two different types: UV-visible
spectrophotometer, uses light over the ultraviolet range (185 - 400
nm) and visible range (400 - 700 nm) of electromagnetic radiation
spectrum. And IR spectrophotometer, that uses light over the infrared
range (700 - 15000 nm) of electromagnetic radiation spectrum.
4. Experimental progress:
● Finely chop the leaves of the lettuce, place them in a mortar;
● Grind the leaves in a cooled mortar with the addition of 6 ml of buffer I;
● Filter the resulting solution into tube 1;
● Centrifuge tube 1 for 5 minutes at a speed of 1000 rpm;
● Pour the supernatant into tube 2;
● Centrifugate the supernatant of the test tube 2 at a speed of 4200rpm
for 5 minutes;
● Drain the supernatant from the test tube 2;
● Dissolve the precipitate by adding 8 ml of buffer II to the precipitate,
stir thoroughly;
● Perform spectrophotometry of the sample.
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5. Results and conclusion:

● The spectrometer graph indicates the presence of chlorophyll a, as it has a
pronounced absorption peak at 677 nm and less pronounced peaks at 484
nm and 437 nm. This is consistent with the absorption spectrum of
chlorophyll a, which typically shows peaks at these wavelengths.

● The resulting graph may differ from the absorption spectrum of pure
chlorophyll due to several factors. One possible reason is that the chlorophyll
sample being analyzed may not be pure, and could contain other pigments or
compounds that contribute to the overall absorption spectrum. Additionally,
environmental factors such as temperature, pH, and light intensity can also
affect the absorption spectrum of chlorophyll.

● It's also important to note that different species of plants may have variations
in their chlorophyll absorption spectra, so the specific characteristics of the
spectrometer graph can provide information about the type of chlorophyll
present and potentially about the plant species being studied.