EXP NO.

06

Name: Boddu.Harshitha
Student id: 2021UCS0090

Topic: Determination of leaf chlorophyll content

Aim:
a) The aim of this experiment is to extract the total chlorophyll content
from select leaves, determine the absorption spectra of chlorophyll, and
find the relative concentration of chlorophyll a and b.
b) To separate leaf pigments using paper chromatography.
c) To visualize chlorophyll fluorescence.

Background:
Chlorophyll is the green pigment found in the leaves of aerial part of
plants such as young stem, fruits and sepals of flower. Chemically it
consists of a tetrapyrrole ring with a central magnesium ion. It has a
long hydrophobic phytol chain in its structure. Six different types of
chlorophyll are known viz, chl-a, b, c, d, e and bacteriophyll. They are
found to be distributed in the plant kingdom; of these chl-a and b are
found in all higher green plants. The difference between these two
chlorophyll molecules is that chl-a has a methyl group and chl-b has a
formyl group. The ratio of chl-a to chl-b in higher plants is
approximately 3:1. Chl-a is the primary pigment while chl-b is the
accessory pigment that collects energy and passes it on to chl-a.
Chlorophyll absorbs light mainly in the red (650 – 700 nm) and the
blue - violet (400 – 500 nm) regions of the visible spectrum. Green
light (~550 nm) is not absorbed but reflected giving chlorophyll its
characteristic color.

Methods:

System: Fresh leaves (spinach or any plant species of

the region).
Reagents: 80% acetone
Equipment: Spectrophotometer, centrifuge, mortar
and pestle Glassware/Plastic ware: Centrifuge tube
Procedure:

1. Weigh 100 mg of fresh leaves after removing the midrib.

2. Grind the sample with the help of mortar and pestle using
5 mL of 80% acetone.
3. Centrifuge the mixture for 5 minutes at 5000 rpm.
4. Transfer the supernatant to a clean screw cap graduated test
tube.
5. Repeat the centrifugation is the supernatant is not clear.
6. Make up the volume to 5 mL by adding 80% acetone.
7. Shake the extract properly and scan the sample for its
absorption spectra in the range between 400 to 700 nm.
8. Care must be taken to ensure that a blank is maintained.

Observation/Data:

1. Record the absorption spectra of chlorophyll and identify the

wavelengths at which the absorption is maximum.
2. Based on a set of simultaneous equations arrived by Arnon
(1949) for pure chlorophyll a and b, calculate the amount of
chlorophyll a and b in the given sample.

Chlorophyll a (mg/g tissue) = [12.7 (A663) - 2.69 ×

(A645)] × V/(1000 ×W) Chlorophyll b (mg/g tissue) =

[22.9 (A645) - 4.68 × (A663)] × V/(1000 × W)

Total Chlorophyll (mg/g tissue) = [20.2 (A645) + 8.02 ×

(A663)] × V/(1000 ×W) Where,

A = Absorbance at specific wave length
V = Volume of chlorophyll extracted in 80%
acetone.
W = Fresh weight of the leaf (in grams) tissue taken for the
extraction.

Table 1: Concentration of chlorophyll a, b and total in the

selected leaf sample
Sample A 645 nm A 663 nm Chl a (mg/g Chl b (mg/g Total Chl
leaf tissue) leaf tissue) (mg/g)
1
2
3
4
Inference:
The primary goal of this experiment was to evaluate the total chlorophyll
content within specific leaves and to analyse the absorption spectra of
chlorophyll. Additionally, the
experiment aimed to ascertain the relative concentrations of chlorophyll a
and chlorophyll b in these leaves. The obtained results provide valuable
insights, indicating that the absorption spectra data can be utilized to
quantify the overall chlorophyll content in the leaves. This
information is crucial for understanding plant photosynthetic activity and
overall health.
Furthermore, determining the proportions of chlorophyll a and b sheds
light on the pigment composition within the leaves, aiding researchers in
studying plant adaptation strategies and environmental responses. In
summary, this experiment's findings enhance our understanding of
photosynthesis and plant pigment composition, impacting agriculture,
ecology, and plant physiology.

Experiment 2: Separation of leaf pigments using paper chromatography

Background:

The principle of chromatography involves the separation of molecules

based on their solubilities in a given solvent. In paper chromatography,
paper marked with a plant extract (such as the chlorophyll extract), is placed
in a developing chamber with a specified solvent. The solvent carries the
dissolved pigments as it moves up the paper. The movement is facilitated
by capillary action. The different leaf pigments (including chlorophyll,
carotenoids, xanthophylls etc) are carried at different rates because of the
differences in their solubilities. A pigment that is the most soluble will travel
the greatest distance and that which is least soluble will move the shortest
distance.

The distance the pigment travels is unique for that pigment in the set
conditions and can be indicated by its Retention Factor (Rf).

Rf = distance travelled by pigment

distance travelled by the solvent front
The band derived in paper
chromatography contain the
pigments in the leaf extract. The bands can
be cut apart, and placed in alcohol (or any
non-polar
solvent), to elute out the pigment. Each
pigment can then be examined for their
absorbance characteristics.

Material required:

Spinach leaves (or any other convenient material

as well) Test tubes
Chromatography paper
Solvent (9:1 petroleum ether : acetone)
Capillary tubes
Pestle and mortar
Acetone (80 %)
Pencil
Scale
Plastic wrap

Procedure:

1. Cut a piece of Whatman #1 filter paper or chromatography paper of the dimension

of 2 cm X 6 or 8 cm long (these can be altered to suit the dimension of the
developing chamber).
2. With a pencil lightly make a line 1.5 to 2 cm from the bottom edge of the paper.
Also mark the mid-point of the line. Make a sharp pointed cut of the paper at the
bottom end.
3. Select 2 or 3 spinach leaves and grind it a small volume of acetone (2 or 3 ml) to
make a thick paste.
4. Using a capillary, apply the extract so made in (3) on the chromatography paper (at
the mid-point marked on the paper). Allow the extract to dry and repeat the
application process until enough extract has been applied.
5. Depending on the volume of the developing chamber (here a 100 ml beaker), add
20 ml of solvent (petroleum ether:acetone in the ratio of 9:1) and cover it with a
lid.
6. Carefully place the chromatography paper with the pointed end dipping into the
solvent and ensuring that the paper stays firm on one side of the beaker. Close the
beaker with a lid.
7. After the run is over (usually between 20 and 30 min), remove the chromatography
paper and observe for the separation of the various pigments.
8. Mark the solvent end, with a pencil mark. Trace the center point of each of the
pigment using a pencil. Encircle the pigment spots.
9. Measure the distance from the starting line to solvent end (this is the distance
travelled by the solvent). Now measure the distance from the starting line to each
of the separated pigment centers. This is the distance travelled by the respective
pigment.
10. Calculate the Retention factor of each of the pigment.
Observation/Data:

Sl. No. Pigment (colour) Retention Factor

1
2
3
4
5
6

Q1. What is the inference with respect to the separation obtained?

Answer: The separation of leaf pigments using paper chromatography yields

valuable insights into the composition and characteristics of pigments within plant
leaves. This process allows for a detailed analysis through several key inferences.

Firstly, paper chromatography facilitates the identification of pigments present in the

leaf extract. By comparing the colors and migration distances, known pigments like
chlorophyll a, chlorophyll b, carotenes, and xanthophylls can be recognized.

The determination of Rf values is another critical inference. These values provide

quantitative data on pigment mobility, with high Rf values indicating pigments that are
more soluble and move further up the paper, and low Rf values representing less
soluble pigments closer to the baseline.

Relative abundance is gauged by the intensity of pigment bands or spots. Darker, more
prominent spots signify higher concentrations of their corresponding pigments.

The separation efficiency of chromatography is evaluated by the clarity of pigment

separation. Well-separated pigments with distinct bands or spots indicate efficient
separation, while overlapping or poorly separated pigments may require adjustments
to chromatography conditions.

Furthermore, the composition of photosynthetic pigments, such as chlorophylls, can

be deduced. This is particularly relevant for understanding the roles of pigments in
processes like photosynthesis.

Lastly, these inferences hold various research implications, from assessing plant health
to studying the impact of environmental factors on pigment composition. In summary,
paper chromatography of leaf pigments is a powerful analytical tool, offering a
comprehensive understanding of pigment composition and behaviour in plant tissues,
thus advancing research in fields such as botany, biology, and environmental science.

Q2. What are the pigments that were separated?

Answer: The specific pigments that are separated in a paper chromatography

experiment of leaf pigments can vary depending on the type of plant species used and
the solvent system employed. However, in typical green plant leaves, you can expect to
find the following major pigments:

1. Chlorophyll a: This is the primary pigment responsible for capturing light energy
during photosynthesis. Chlorophyll a appears blue-green and is usually the most
abundant pigment in green plant tissues.

2. Chlorophyll b: Chlorophyll b complements chlorophyll a in capturing light energy and

expanding the range of light wavelengths that can be utilized in photosynthesis. It
appears more yellow-green in color.

3. Carotenes: Carotenes are orange pigments, including beta-carotene and others,

that are part of the carotenoid group. They play a role in photoprotection, as well as
assisting in photosynthesis by capturing light energy in different spectral regions.

4. Xanthophylls: Xanthophylls are yellow pigments also within the carotenoid

group. They help in photoprotection by dissipating excess light energy as heat,
preventing damage to chlorophyll molecules.

In a paper chromatography experiment, these pigments can be separated based on

their differential solubilities in the chosen solvent system, their affinity for the paper,
and their migration rates. The resulting chromatogram will typically show distinct
bands or spots corresponding to each of these pigments, allowing for their
identification and analysis based on factors like migration distances (Rf values) and
color characteristics.
Inference:
The objective of Experiment 2, which involves the separation of leaf pigments using paper
chromatography, is to elucidate the various pigments present within a leaf sample and to
separate them based on their different rates of migration through the chromatography
paper. By analysing the results, one can infer the composition of pigments in the leaves,
potentially identifying chlorophylls (a and b), carotenoids, and other accessory pigments.
This information is critical for understanding the photosynthetic apparatus of plants and
their adaptation to varying environmental conditions. Furthermore, the experiment aids in
teaching fundamental principles of chromatography and pigment analysis, contributing to
a broader comprehension of plant biology and analytical techniques in science education.

Experiment 3: To visualize chlorophyll fluorescence

Chlorophyll in plant leaves absorb red light and pass the energy on to other parts of the
plant, hence leaves look green. But when extracted in solution, the chlorophyll molecules
are unable to share their excited energy with neighbouring molecules and hence release the
excited energy in the form of light. This is called fluorescence.

Grinding the spinach leaves disrupts the normal structure of the leaf cells and releases
chlorophyll. Normally, chlorophyll would absorb light energy that excites an electron which
is then transferred to an acceptor molecule. However, when the leaves are extracted, the
excited electron is not transferred anywhere and the energy is released again as light, i.e. an
electron gets excited to a higher energy level by a photon of light and then releases it again
when the energy is not transferred anywhere else. Under normal lighting conditions, the
solution appears green because the chlorophyll is absorbing red light from all directions.
Under darkened conditions, the chlorophyll is absorbing red light from the torch and then
releasing it again (which is perceived as red).
Material required

Chlorophyll extract (prepared for experiment 1 above), a glass vial, Torch or lamp - Dark
room/corner, Trans-illuminator

Method

1. Take 2 or 3 ml of the chlorophyll extract in a glass vial and firm it with a screw cap.
2. Place the glass vial with the extract on a uv transilluminator with proper dark shield.
Care must be taken to ensure that the shield is firmly in place.
3. Switch on the trans-illuminator and observe the fluorescence given out by the
chlorophyll.
Observation/Data:

Inference:
Experiment 3, focusing on the visualization of chlorophyll fluorescence, aims to provide
insights into the photosynthetic activity and health of plants. By observing chlorophyll
fluorescence, researchers can infer the efficiency of photosynthesis and the plant's
response to environmental stressors. High fluorescence levels may indicate stress or
damage, while lower fluorescence suggests healthier photosynthetic activity. This
experiment serves as a valuable tool for assessing plant vitality, which can have
applications in agriculture, ecology, and environmental monitoring. Additionally, it aids in
the study of plant physiology and provides practical knowledge for assessing the impact of
various factors on photosynthesis, making it a crucial experiment in plant science research
and education.
Take home questions:

1. What is the absorbance spectra of chlorophyll?

Answer: The absorbance spectra of chlorophyll refers to the

wavelengths of light that chlorophyll molecules can absorb.
Chlorophyll is a pigment found in the
chloroplasts of plant cells and is responsible for capturing light energy
during photosynthesis. There are several types of chlorophyll, with
the two most common forms in plants being chlorophyll-a and
chlorophyll-b.

The absorbance spectra of chlorophyll-a and chlorophyll-b typically

show peaks in the blue and red regions of the electromagnetic
spectrum. Chlorophyll-a primarily absorbs light in the blue (around
430-450 nanometers) and red (around 640-680 nanometers)
wavelengths, while chlorophyll-b primarily absorbs in the blue
(around 640-660 nanometers) and red-orange (around 610-640
nanometers) wavelengths.

The absorbance spectra are essential for photosynthesis because

they determine which wavelengths of light chlorophyll molecules
can capture and convert into chemical energy. This captured energy
is then used to drive the photosynthetic process, where carbon
dioxide and water are converted into glucose and oxygen.

The specific absorbance peaks can vary slightly depending on factors

such as the type of chlorophyll, environmental conditions, and the
presence of accessory
pigments. Scientists use spectrophotometry to measure these
absorbance spectra and study the light-absorbing properties of
chlorophyll molecules.

2. Plants growing in shade or under a canopy of trees are generally

weak and at times also may die. From the point of view of
chlorophyll, what do you think is happening?

Answer: Plants growing in shade or under a canopy of trees often

exhibit characteristics such as elongated stems, smaller leaves, and
reduced growth compared to plants in full sunlight. This
phenomenon can be attributed to several factors, including the
availability of light and its impact on chlorophyll function:

1. Reduced Light Intensity: In shaded or canopy-covered

environments, the intensity of light reaching the plants is
significantly lower than in full sunlight. Chlorophyll molecules
require sufficient light energy to perform photosynthesis
effectively. When light is limited, the rate of photosynthesis
decreases, which can lead to slower growth and weaker plants.
 2. Altered Light Quality: Light quality, or the spectrum of light,
also changes in shaded areas. Light under a canopy tends to be
skewed towards the blue and red wavelengths, as green
wavelengths are absorbed by the
chlorophyll in the upper canopy leaves. This altered light spectrum
can affect the efficiency of chlorophyll in capturing light energy.

3. Light Competition: Plants growing in the shade often have

to compete with neighbouring plants for the limited available light.
This competition can further reduce the amount of light each
plant receives, exacerbating the challenges faced by chlorophyll in
these shaded conditions.

4. Chlorophyll Adaptations: Some plant species have evolved

adaptations to low-light conditions. They may have higher
chlorophyll concentrations,
increased chloroplast density in their cells, or other mechanisms to
enhance
light capture and photosynthetic efficiency under low-light conditions.
However, even with these adaptations, the growth of shade-tolerant
plants may still be slower compared to plants in full sunlight.

5. Stress and Survival: In extreme cases of prolonged shading,

plants may experience stress and reduced vitality, which can make
them more susceptible to diseases and herbivores. This can lead to
weakened plants that may
ultimately die if they cannot compete and survive in the shaded
environment.

In summary, the challenges faced by plants growing in shade or

under a canopy of trees are largely related to the limitations imposed
on chlorophyll by reduced light availability and altered light quality.
Chlorophyll is less effective in
capturing light energy under these conditions, which can result in
weaker and slower-growing plants, and in some cases, even death.
However, it's important to note that some plant species have
evolved to thrive in lowlight conditions and have adapted their
chlorophyll and photosynthetic processes accordingly.

3. If plants did not have chlorophyll b, what could be the

consequences for the plant, especially under conditions of
high irradiation?

Answer: Chlorophyll-b is one of the two primary types of

chlorophyll pigments found in plants, with the other being
chlorophyll-a. Chlorophyll-b plays a specific role in photosynthesis,
and its absence could have several consequences for a
plant, especially under conditions of high irradiation (intense light).
Here are some potential consequences:

1. Reduced Light Absorption: Chlorophyll-b primarily absorbs

light in the blue (around 640-660 nanometers) and red-orange
(around 610-640 nanometers)
wavelengths. Without chlorophyll-b, the plant would have a reduced
capacity to
capture light energy in these specific regions of the spectrum. This
can limit the plant's overall ability to absorb and convert light
energy for photosynthesis.

2. Lower Photosynthetic Efficiency: Photosynthesis relies on

the absorption of light energy by chlorophyll molecules, which is
then used to convert carbon
dioxide and water into glucose and oxygen. Chlorophyll-b helps
complement the light absorption spectrum of chlorophyll-a,
ensuring that a broader range of wavelengths is utilized. Without
chlorophyll-b, the plant may be less efficient at photosynthesis,
particularly in utilizing light energy from specific wavelengths.

3. Susceptibility to Photodamage: Under conditions of high

irradiation, such as intense sunlight, chlorophyll molecules can
become overexcited and lead to the production of harmful reactive
oxygen species. Chlorophyll-b can help dissipate excess energy,
reducing the risk of photodamage. In its absence, the plant may be
more susceptible to oxidative stress and photodamage, which can
harm cellular
structures and impair photosynthesis.

4. Limited Adaptation to Light Variability: Chlorophyll-b allows

plants to adapt to changing light conditions by expanding the range
of wavelengths they can absorb. Without chlorophyll-b, plants may
have a reduced ability to adapt to fluctuations in light quality and
intensity, which can be especially problematic in
environments with varying light conditions.

5. Impaired Shade Tolerance: Chlorophyll-b contributes to a

plant's ability to capture light in the shade, complementing the
action of chlorophyll-a. In the
absence of chlorophyll-b, plants may have reduced shade tolerance
and may struggle to photosynthesize efficiently in low-light
conditions.

6. Altered Pigment Composition: Without chlorophyll-b,

plants may need to rely more heavily on chlorophyll-a and other
pigments. This could lead to
alterations in the overall pigment composition of the plant, which may
impact its appearance and ability to absorb and utilize light energy.

In summary, the absence of chlorophyll-b could result in reduced

photosynthetic efficiency, increased susceptibility to photodamage,
and limited adaptability to changing light conditions, especially
under conditions of high irradiation. However, it's important to note
that some plants, like certain algae and photosynthetic
bacteria, use different combinations of pigments and are adapted to
thrive without chlorophyll-b, so they have evolved alternative
mechanisms to capture and utilize
light energy effectively.
4. What is the principle of a spectrophotometer?

Ans: - The principle of a spectrophotometer is based on the

measurement of the
intensity of light absorbed or transmitted by a sample solution as a
function of the wavelength of light. It allows for the quantitative
analysis of substances by measuring the amount of light absorbed or
transmitted by a sample