This document describes a biology project on paper chromatography submitted by Abhay Panchal of Class XI B. The project investigates the separation of chlorophyll pigments from spinach leaves using paper chromatography. It provides details on the theory, materials, procedure, advantages and disadvantages of the paper chromatography technique. The student expresses thanks to their teacher and principal for guidance and for the opportunity to conduct this research project.

1. “BIOLOGY PROJECT”
SESSION:- 2016-2017
TOPIC:- PAPER CHROMATOGRAPHY
SUBMITTED TO:- SUBMITTED BY:-
MRS.ATIYAAZHAR ABHAYPANCHAL
Class-XIB

2. CERTIFICATE
This is hereby to certify that the original and genuine
investigation work has been carried out to investigate
about the subject matter and the related data
collection and investigation has been completed solely,
sincerely and satisfactorily by ABHAY PANCHAL of
CLASS XI B, COLUMBIA CONVENT SCHOOL,INDORE
regarding his project title “INVESTIGATORY PROJECT ON
PAPER CHROMATOGRAPHY” .
SIGNATURE OF SIGNATURE OF
BIOLOGY TEACHER PRINCIPAL

3. ACKNOWLEDGEMENT
In the accomplishment of this project successfully,
many people have best owned open me there blessing
and the heart pledged support, this time I am utilizing
to thank all the people who have been concerned with
project.
I would like to express my special thanks of
gratitude to my teacher MRS.ATIYA AZHAR as well as
our principal MRS.NALINI PAUL who gave me the golden
opportunity to do this wonderful project on the topic
PAPER CHROMATOGRAPHY, which also helped me in
doing a lot of Research and I came to know about so
many new things I am really thankful to them.
Secondly I would also like to thank my parents and
friends who helped me a lot in finalizing this project
within the limited time frame.

4. Conclusion
Light could have affected the nature of chlorophyll
thus led to inaccurate results. I would recommend not
taking out the pigment until the last minute when it is
needed in order to minimize the effect of light; * An
alternative technique (thin layer chromatography)
mentioned in the preparation paper can be employed:
instead of absorptive paper, a glass of plastic slide
(or aluminium sheet) coated with silica gel is used. The
gel is spotted and then the slide or aluminium sheet is
placed vertically in a beaker or Coplin jar containing
solvent at the bottom; * Further experiment can
employ pigments from different plants. Chlorophyll a
is of universal occurrence in all photosynthesizing
plants but we can compare the different pigments in
different plants. Because different pigments absorb
light of different wavelengths, by comparing the
occurrence of different pigments we can deduce the
plants.

5. Precaution
 Spinach leaves should be fresh and green.
 The loading spot should be 2-3 cm away
from the tip of the notch.
 While hanging the strips in the
chromatography chamber, the loading spot
should remain about 1 cm above the
solvent level.

6. Real Lab Procedure
 Take a few freshly plucked green spinach leaves.
 Using scissors cut the spinach leaves into small pieces and let
them fall into the mortar.
 Take a measuring cylinder that contains 5ml of acetone and
pour it into the mortar.
 Grind the spinach leaves using the mortar and pestle.
 Take a strip of filter paper having a narrow notch at one end
of the strip.
 Draw a horizontal line with a pencil about 2-3 cm away from
the tip of the notch.
 Put a drop of the pigment extract in the middle of the line
with the help of a capillary tube.
 Allow the drop to dry and repeat till four or five drops are
placed on the paper.
 Take the chromatographic chamber and pour ether acetone
solvent in it.
 Fold one end of the filter paper strip and staple it.
 Using a thread, hang the filter paper strip in the
chromatographic chamber.
 The loading spot should remain about 1cm above the solvent
level.
 Leave the chromatographic chamber undisturbed for some time.
 When the solvent rises about 3/4th up the strip,
remove the strip carefully and let it dry.

7. MATERIAL REQUIRED

9. Theory
Before going into detail, let’s understand the role
of pigments in plants.
Photosynthetic plants convert light energy from the
sun to chemical food energy. During
photosynthesis, molecules referred to as pigments
are used to capture light energy. Pigments are
chemical compounds which reflect only certain
wavelengths of visible light. Plant leaves contain
four primary pigments: chlorophyll a (dark green),
chlorophyll b (yellowish-green), xanthophylls (yellow)
and carotenoids (orange).
To separate and visualize the four primary pigments
of green plants, we can use a simple technique
called chromatography.

11. Pigment & Polarity
Paper chromatography is one method for testing
the purity of compounds and identifying substances.
Paper chromatography is a useful technique because
it is relatively quick and requires only small
quantities of material. Separations in paper
chromatography involve the same principles as those
in thin layer chromatography, as it is a type of thin
layer chromatography. In paper chromatography,
substances are distributed between a stationary
phase and a mobile phase. The stationary phase is
the water trapped between the cellulose fibers of
the paper. The mobile phase is a developing solution
that travels up the stationary phase, carrying the
samples with it. Components of the sample will
separate readily according to how strongly they
adsorb onto the stationary phase versus how readily
they dissolve in the mobile phase.

14. TYPES OF PAPER CHROMATOGRAPHY
 Descending Paper Chromatography-In this type,
development of the chromatogram is done by
allowing the solvent to travel down the paper.
 Ascending Paper Chromatography-Here the
solvent travels up the chromatographic paper.
Both Descending and Ascending Paper
Chromatography are used for the separation of
organic and inorganic substances.
 Ascending-Descending Paper Chromatography-It
is the hybrid of both of the above techniques.
The upper part of Ascending Chromatography
can be folded over a rod in order to allow the
paper to become Descending after crossing the
rod.
 Radial Paper Chromatography-It is also called
Circular Chromatography. Here a circular filter
paper is taken and the sample is deposited at
the center of the paper. After drying the spot,
the filter paper is tied horizontally on a Petri
dish containing solvent, so that the wick of the

15. paper is dipped in the solvent. The solvent rises
through the wick and the components are
separated into concentric circles.
 Two-Dimensional Paper Chromatography-In this
technique a square or rectangular paper is used.
Here the sample is applied to one of the
corners and development is performed at right
angle to the direction of the first run.

16. BIBLIOGRAPHY
 LAB MANUAL OF 11 CLASS.
 WWW.GOOGLE.COM

17. CONTENT
 INTRODUCTION
 DEFINATION OF PAPER CHROMATOGRAPHY
 HISTORY OF PAPER CHROMATOGRAPHY
 PRINCIPLE OF PAPER CHROMATOGRAPHY
 TYPES OF PAPER CHROMATOGRAPHY
 THEORY
 PIGMENT & POLARITY
 MATERIAL REQUIRED
 PROCEDURE
 RF VALUE
 FACTORS AFFECTING RF VALUE
 ADVANTAGES & DISADVANTAGES OF
PAPER CHROMATOGRAPHY
 CONCLUSION
 PRECAUTIONS
 BIBLIOGRAPHY

18. ADVANTAGES OF PAPER
CHROMATOGRAPHY
1. One of the major advantages of paper
chromatography is the sensitivity with
which compounds can be located after
separation. Amounts as little as 0.1
microgram (one micrograms is one
millionth of a gram) of some compounds
can be detected with routine reagents.
2. The operation time is small.

19. DISADVANTAGES OF PAPER
CHROMATOGRAPHY
1. Paper chromatographic techniques can
not be used in separation of
volatile substances such as hydrocarbons
and volatile fatty acids.
2. The lower limit for the detection of
most compounds is 1-5 microgram.

20. HISTORY
The discovery of paper chromatography in
1943 by Martin and Synge provided, for the
first time, the means of surveying
constituents of plants and for their
separation and identification. There was an
explosion of activity in this field after 1945.