BIO 703

APPLIED PLANT PHYSIOLOGY

SEMSTER 1 2020
PRACTICAL 3
SPATIAL SEPARATION OF
DIFFERENT PHOTOSYNTHETIC
PIGMENT THROUGH PAPER
CHROMATOGRAPHY

NAME: SANJANI SINGH

ID: 2018000909

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AIM

 To distinguish and study the various pigments present in plants through

the process of paper chromatography.

INTRODUCTION

As producers in the food chain, algae and plants produce their own food by a
process known as photosynthesis. In this process of photosynthesis, plants
convert the suns energy into chemical energy that is stored in the bonds of the
glucose molecule which fuels the metabolic processes of the cells. For
photosynthesis to transform light energy into chemical energy, pigment
molecules absorb light to power chemical reactions.

Plant pigments are macromolecules produced by the plant and these absorb
specified wavelengths of visible light to provide energy required for
photosynthesis. Chlorophyll is necessary for photosynthesis, but accessory
pigments collect and transfer energy to chlorophyll. Plants contain different
pigments. Some include carotenoids, xanthophyll, betalains, phaeophytin a and
b and chlorophyll a and b.

The process of chromatography separates molecules because of different

solubilities of the molecules in a selected solvent. In paper chromatography,
paper marked with unknown such as plant extract is placed in a developing
chamber with a specified solvent. The solvent carries the dissolved pigments as
it moves up the paper. The pigments are carried at different rates because they
are not equally soluble. A pigment that is most soluble will travel the greatest
distance and a pigment that is less soluble will move a shorter distance.

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MATERIAL

 4 different types of Leaves (Bougainvillea, Lavender, Casta Cuernavaca,

Hibiscus)
 Acetone
 Pestle mortar
 Measuring cylinder
 Petroleum ether
 Methyl alcohol
 Paper
 Test tube
 Buchner funnel
 Separating funnel
 UV lamp
 Ethyl ether
 KOH

METHOD

1. 4 different types of Leaves (Bougainvillea, Lavender, Casta Cuernavaca,

Hibiscus) was collected from outside.
2. 1g of fresh leaf tissue from four different plants was taken and boiled for
a few minutes to kill the cells.
3. The leaf material was removed.
4. The excess water was blotted off using a blotting paper.
5. It was then grinded in a mortar with about 1mg fine grained quartz sand.
For several successive 10-15mL portion 50:1 petroleum ether: methyl
alcohol solution was added.
6. It was grinded very thoroughly until few millimeters of solvent was left.
7. 5ml of this pigment was decanted into a test tube.
8. A strip of filter paper was cut to a slightly lesser diameter than the tube ad
inserted inside it. Chlorophyll b was absorbed strongly than a, carotenes
were not absorbed and was collected at the top of the strip as the solvent
evaporated.
9. Positions of chlorophyll a and b were marked.
10.The carotenoids were identified and the chromatogram was mounted.

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OBSERVATION

 The dried paper strip displays 4 different bands. Discrete pigments can
be distinguished with the help of colors.

Different types of pigment present in 4 types of leaves collected:

A. Name: Bougainvillea leaf Pigment: chlorophyll b, xanthophyll

Color: yellowish Green

B. Name: Lavender, foliage Pigment: anthocyanin, chlorophyll b, xanthophyll

Color: Purplish green

C. Name: Casta Cuernavaca (dalo leaf) Pigment: Chlorophyll b, xanthophyll

Color: yellowish Green

D. Name: Hibiscus Pigment: chlorophyll b, xanthophyll

Color: yellowish Green

 Chlorophyll b gives green color.

 Xanthophyll gives yellow color in young plants used in this practical
 Anthocyanin gives purple color.

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RESULT

STRIP 1

a) 1st solute 2.4cm (pigment) (green chlorophyll b)

b) 2nd solute 2.6cm (pigment) (Yellow pigment xanthophyll)
Solvent travelled= 6. 7cm

A. RF value= Distance travelled by pigment

Distance travelled by solvent
= 2.4
6.7

= 0.36(green chlorophyll b) (least distance covered)

B. RF value= Distance travelled by solute

Distance travelled by solvent

= 2.6

6.7
= 0.39 (Yellow pigment xanthophyll)

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STRIP 2

a. 1st solute 4.8cm (pigment) (green chlorophyll b)

b. 2nd solute 6.5cm (pigment) (Yellow pigment xanthophyll)
Solvent travelled= 8.4 cm

a. RF value= Distance travelled by solute

Distance travelled by solvent

= 4.8

8.4

= 0.57(green chlorophyll b)

b. RF value= Distance travelled by solute

Distance travelled by solvent

= 6.5
8.4
= 0.77 (Yellow pigment xanthophyll)

STRIP 3

a. 1st solute 5.7cm (pigment) (Yellow pigment xanthophyll)

Solvent travelled= 8.3 cm

RF value= Distance travelled by solute

Distance travelled by solvent

= 5.7
8.3
= 0.69(Yellow pigment xanthophyll)

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STRIP 4

a. 1st solute 6.5cm (pigment) (green chlorophyll b)

b. 2nd solute 7.2cm (pigment) (Yellow pigment xanthophyll)
Solvent travelled= 8.9cm

a. RF value= Distance travelled by solute

Distance travelled by solvent

= 6.5
8.9
=0.73(green chlorophyll b)

b. RF value= Distance travelled by solute

Distance travelled by solvent

= 7.2
8.9
= 0.80 (Yellow pigment xanthophyll) (most distance covered)

 Xanthophyll is most soluble and travelled the most distance

whereas chlorophyll b is least soluble and covered the least
distance.

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 DISCUSSION
1. Very briefly describe the principle which govern the separation of
chloroplast pigments by paper chromatography?
ANS
The principle involved is partition chromatography where the substances
are distributed or partitioned between liquid phases. One phase is water
which is held in the pores of the filter paper used and the other is the
mobile phase which moves the paper. The compounds in the mixture get
separated due to differences in their affinity towards water (in stationary
phase) and mobile phase solvents during the movement of mobile phase
under the capillary action of pores in the water.

2. What other pigments are found in higher plant besides the chloroplast
pigments?
 Carotenoids
 Xanthophyll
 Anthocyanins
 Flavonoids
 Betalains

3. Why is chlorophyll “a” present universally in higher plants and other

photosynthesizing organisms?
ANS
Chlorophyll a is responsible for absorbing light from orange-red and
violet areas of the electromagnetic spectrum. It transfers energy to the
reaction center and donates 2 excited electrons to the electron transport
chain. Primary role of chlorophyll is as a primary electron donor in the
electron transport chain. It functions in transporting energy to reaction
center where P680 and P700 are located. (in photosynthesizing
organisms). It participates mainly in oxygenic photosynthesis in which
oxygen is the by product of the process. All oxygenic photosynthetic
organisms have this including almost all plants and bacteria.

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 4. Why do the chlorophylls appear green? or for that factors carotene
yellow?
ANS
 Chlorophyll absorbs certain wavelength of light within the visible light
spectrum. It shows light in red (long wavelength) and the blue (short
wavelength) regions of the visible light spectrum. Green light is not
absorbed but reflected, making chlorophyll appear green.
 Carotene appears yellow because they absorb ultraviolet, violet and blue
light and scatter orange or red light and in low concentrations yellow
light.

DISCUSSION
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A yellowish band appears which indicated the pigment xanthophyll was present.
The green band present indicated the presence of chlorophyll b pigment. The
yellow xanthophyll is most soluble and covered the most distance (0.80)
whereas y chlorophyll b was the least soluble and covered the least distance
(0.36). The 2 pigments observed in this practical were xanthophyll ad
chlorophyll b.

Paper chromatography is a method of separating the components of a mixture.

During the procedure, the mixture will be separated into its individual
components allowing the individual parts to be identified. It is at technique that
involves placing a small dot or line of sample solution onto a strip of
chromatography paper. The paper is then placed in a jar containing a shallow
layer of solvent and sealed. As the solvent rises through the paper it meets the
sample mixture which starts to travel up the paper with the solvent.

Some plant pigments present

 Carotene-an orange pigment

 Xanthophyll- a yellow pigment
 Chlorophyll a – a blue green pigment
 Chlorophyll b- a green pigment
 Phaeophytin a - a gray brown pigment
 Phaeophytin b- a yellow brown pigment
 Anthocyanin- a purple-green pigment
 Betalains- a red pigment

The Rf value is defined as the ratio of the distance moved by the solute and the
distance covered by the solvent along the paper. It indicates how soluble the
particular pigment is in the solvent by how the pigment moves on the paper.
Small Rf values tend to indicate larger, less soluble pigments while the high
soluble pigments have a Rf value near to one.

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CONCLUSION

After conducting this experiment, it can be said that the students were able to
distinguish and study the various pigments present in plants through the process
of paper chromatography. Paper chromatography proved to be an accurate
method of separating and observing various colors of leaves collected. The
pigments dissolved in the solvent and migrated upward. The colors were
observed and their migration distances were measured and recorded. The Rf
value of each pigment was determined by dividing pigment migration by the
migration of the solvent. it was concluded that 2 pigments were found
xanthophyll and chlorophyll b. The yellow xanthophyll is most soluble and
covered the most distance (0.80) whereas y chlorophyll b was the least soluble
and covered the least distance (0.36).

REFERENCE

 Bio 703 lab handout practical 3

 https:// www.biologyjunction.com/chromatography of
 https://en.m.wikipeda.org> photosynthetic pigments.
 https://ucmp.berkeley.edu> photosynthetic pigments.
 https://www.khansacademy > photosynthetic pigments

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