The effect of light intensity on the amount of chlorophyll in “Cicer arietinum”
Protochlorophyllide
Chlophyllide
Chlorophyll b Chlorophyll a
Chlorophyll[3] is a green compound found in leaves and green stems of
plants. Initially, it was assumed that chlorophyll was a single compound
but in 1864 Stokes showed by spectroscopy that chlorophyll was a mixture.
If dried leaves are powdered and digested with ethanol, after concentration
of the solvent, 'crystalline' chlorophyll is obtained, but if ether or
aqueous acetone is used instead of ethanol, the product is 'amorphous'
chlorophyll.
In 1912, Willstatter et al. (1) showed that chlorophyll was a mixture
of two compounds, chlorophyll-a and chlorophyll-b:
[pic]
Chlorophyll-a (C55H72MgN4O5, mol. wt.: 893.49). The methyl group marked
with an asterisk is replaced by an aldehyde in chlorophyll-b (C55H70MgN4O6,
mol. wt.: 906.51).
The two components were separated by shaking a light petroleum
solution of chlorophyll with aqueous methanol: chlorophyll-a remains in the
light petroleum but chlorophyll-b is transferred into the aqueous methanol.
Cholorophyll-a is a bluish-black solid and cholorophyll-b is a dark green
solid, both giving a green solution in organic solutions. In natural
chlorophyll there is a ratio of 3 to 1 (of a to b) of the two components.
The intense green colour of chlorophyll is due to its strong
absorbencies in the red and blue regions of the spectrum, shown in fig. 1.
(2) Because of these absorbencies the light it reflects and transmits
appears green.
[pic]
Fig. 1 - The uv/visible adsorption spectrum for chlorophyll.
Due to the green colour of chlorophyll, it has many uses as dyes and
pigments. It is used in colouring soaps, oils, waxes and confectionary.
Chlorophyll's most important use, however, is in nature, in
photosynthesis. It is capable of channelling the energy of sunlight into
chemical energy through the process of photosynthesis. In this process the
energy absorbed by chlorophyll transforms carbon dioxide and water into
carbohydrates and oxygen:
CO2 + H2O [pic](CH2O) + O2
Note: CH2O is the empirical formula of carbohydrates.
The chemical energy stored by photosynthesis in carbohydrates drives
biochemical reactions in nearly all living organisms.
In the photosynthetic reaction electrons are transferred from water to
carbon dioxide, that is carbon dioxide is reduced by water. Chlorophyll
assists this transfer as when chlorophyll absorbs light energy, an electron
in chlorophyll is excited from a lower energy state to a higher energy
state. In this higher energy state, this electron is more readily
transferred to another molecule. This starts a chain of electron-transfer
steps, which ends with an electron being transferred to carbon dioxide.
Meanwhile, the chlorophyll which gave up an electron can accept an electron
from another molecule. This is the end of a process which starts with the
removal of an electron from water. Thus, chlorophyll is at the centre of
the photosynthetic oxidation-reduction reaction between carbon dioxide and
water.
Treatment of cholorophyll-a with acid removes the magnesium ion
replacing it with two hydrogen atoms giving an olive-brown solid,
phaeophytin-a. Hydrolysis of this (reverse of esterification) splits off
phytol and gives phaeophorbide-a. Similar compounds are obtained if
chlorophyll-b is used.
[pic]
Chlorophyll can also be reacted with a base which yields a series of
phyllins, magnesium porphyrin compounds. Treatment of phyllins with acid
gives porphyrins.
[pic]
Now knowing all these factors affecting the synthesis and destruction
of chlorophyll I propose that the amount of chlorophyll in plant depends on
light intensity in the following way: with the increase of light intensity
the amount of chlorophyll increases, but then it starts decreasing because
light intensity exceed the point when there is more chlorophyll destructed
than formed.
[pic]
Variables.
Independent:
. Light intensity, lux
Constant:
. pH of soil
. water supply, ml
. temperature, to C
Dependent:
. length, cm
. amount of chlorophyll in gram of a plant, gram per gram
III. Method.
Apparatus:
. seeds of Cicer arietinum
. 28 plastic pots
. water
. scissors
. ruler (20 cm ( 0.05 cm)
. CaCO3
. soil (adopted for home plants)
. digital luxmeter (( 0.05 lux)
. test tubes
. H2SO4 (0.01 M solution)
. Pipette (5 cm3 ( 0.05 cm3)
. mortar and pestle
. burette
. ethanol (C2H5OH), 98%
. beakers
Firstly I went to the shop and bought germinated seeds of Cicer
arietinum. Then sorted seeds and chose the strongest ones. After that I
prepared soil for them and put them in it.
As the aim of this project is to investigate the dependence of mass of
chlorophyll in plants during different light intensities it was needed to
create those various conditions. Pots with seeds were placed into the
following places: in the wardrobe with doors (light intensity is o lux),
under the sink (light intensity is 20,5 lux), in the shell of bookcase
(light intensity is 27,5 lux), above the bookcase (light intensity is 89,5
lux), above the extractor (light intensity is 142 lux), beyond the curtains
(light intensity is 680 lux) and on the open sun (light intensity is 1220
lux). Light intensity was measured with the help of digital luxmeter. It
was measured four times each day: morning, midday, afternoon, evening.
During those four periods four measurements were done and the maximum value
was taken into consideration and written down. Those measurements lasted
for three weeks of the experiment as the whole time of the experiment was
three weeks. The luxmeter’s sensitive part was placed on the plants (so it
was just lying on them) in order to measure light intensity flowing
directly on plant bodies, then two minutes were left in order to get
stabilized value of light intensity and the same procedure was repeated.
All those actions were done in order to get more accurate results of light
intensity.
Growing plants were provided with the same amount of water (15 ml, once a
day in the morning) and they were situated in the same room temperature
(20o C), pH of soil was definitely the same because all the plants were put
in the same soil (special soil for room flowers).
After three weeks past the length of plants was measured with the help of
ruler. Firstly the plants were not cut, so their length had to be measured
while they were in the pots. The ruler was placed into the pot and plants
were carefully stretched on it. The action was repeated three times and
only maximum value was taken into consideration. After that plants were
cut. Then those already cut plants were put into the dark place and quickly
dried.
Titration.
I have chosen three plants from each light intensity group and measured
their weight. . In order to obtain the pigments, three plants were cut into
small pieces and placed in a mortar. Calcium carbonate was then added,
together with a little ethanol (2 cm3). The leaf was grinded using a pestle
until no large pieces of leaf tissue were left, and the remaining ethanol
was poured into the mortar (3 cm3). Then 1 ml of obtained solution was
placed into the test tube and this 1 ml of solution was then titrated
against 0.01 M solution of sulfuric acid, through the use of a pipette. The
titration was complete when the green solution turned dark olive-green[4].
This solution obtained from the first action was stored as the etalon for
the following ones. The settled olive-green coloring meant that all
chlorophyll had reacted with H2SO4. So the process of titration was
repeated 7 times for all light intensity groups.
The solution is titrated until the dark olive-green color because it is
known that when the reaction between chlorophyll and sulfuric acid happens,
chlorophyll turns into phaeophetin which has grey color (see table 1),
therefore when the solution is olive-green, than the reaction has
succeeded. But while searching in the internet and books I found out that
there are several opinions about the color of phaeophytin – in the book
written by Viktorov it is ssaid to have grey color, but in the internet
link http://www.ch.ic.ac.uk/local/projects/steer/chloro.htm it is said to
have brown olive-green color. Also I made chromatography in order to
investigate the color of phaeophytin and the result was that it has grey
