WHITEFIELD GLOBAL SCHOOL

INVESTIGATORY PROJECT REPORT

2019-20
BIOLOGY

Project title:
Chromatography of Plant Pigments

Submitted To:

CENTRAL BOARD OF SECONDARY EDUCATION

By

Name of the student – Gagan K Gowda

Grade XII
Register No.

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 WHITEFIELD GLOBAL SCHOOL

DECLARATION
I hereby declare that the investigatory project work entitled Space science
development in India from small satellite to heavy falcon satellite
submitted to the Department of Science, Whitefield Global School is prepared
by me.

All the content is the result of my personal effort.

Place:

Date: Gagan K Gowda

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 WHITEFIELD GLOBAL SCHOOL

CERTIFICATE
This is to certify that the original and genuine investigatory project work carried
out to study the subject matter and the related data collection and experiment
has been completed solely and sincerely by Gagan, a candidate of Whitefield
Global School under my guidance and direction.

Signature of Head of Institution

Signature of the teacher Signature of External Examiner

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 ACKNOWLDEGEMENT

With high regards, I wish to express my gratitude and sincere thanks to my

research supervisor Dr. Gayatramma K, PGT BIOLOGY for her stimulating
guidance and constant encouragement throughout have been responsible for
the successful completion of this project.

My sincere thanks to Dr. Sarada Chandrashekaran, Academic Director, Ms.

Arpita Tripati, H.M. Secondary, and Ms. Sakila, Senior Coordinator for their
support in completion of this project.

It gives me immense pleasure to acknowledge my parents and Laboratory

assistants Ms. Tanuja and Ms. Latha for their support.

I am deeply grateful to all those who have helped me, directly or indirectly,
towards the completion of my investigatory project work.

Gagan K Gowda

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 INDEX

Sn.no Contents Pg.no

1. Introduction 6
2. Principle 7
3. Requirements 8
4. Procedure 9
5. Observations 12
6. Results 10
7. Conclusion 11
8. Bibliography 12

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INTRODUCTION
Some materials appear homogenous, but are actually a combination of substances. For
example, green plants contain a mixture of different pigments. In addition, the black ink in the
pens that are used in this experiment is a mixture of different colored materials. In many
instances, we can separate these materials by dissolving them in an appropriate liquid and
allowing them to move through an absorbent matrix, like paper. Chromatography is a method
used by scientists for separating organic and inorganic compounds so that they can be analyzed
and studied. By analyzing a compound, a scientist can figure out what makes up that
compound. Chromatography is a great physical method for observing mixtures and solvents.
The word chromatography means "color writing" which is a way that a chemist can test liquid
mixtures. While studying the coloring materials in plant life, a Russian botanist invented
chromatography in 1903. His name was M.S. Tswett. Chromatography is such an important
technique that two nobel prizes have been awarded to chromatographers. Over 60% of
chemical analysis worldwide is currently done with chromatography or a variation thereon.
Chromatography is used in many different ways. Some people use chromatography to find out
what is in a solid or a liquid. It is also used to determine what unknown substances are. The
Police, F.B.I., and other detectives use chromatography when trying to solve a crime. It is also
used to determine the presence of cocaine in urine, alcohol in blood, PCB's in fish, and lead in
water. Chromatography is used by many different people in many different ways.
Chromatography is based on differential migration. The solutes in a mobile phase go through a
stationary phase. Solutes with a greater affinity for the mobile phase will spend more time in
this phase than the solutes that prefer the stationary phase. As the solutes move through the
stationary phase they separate. This is called chromatographic development. How it works In all
chromatography there is a mobile phase and a stationary phase. The stationary phase is the
phase that doesn't move and the mobile phase is the phase that does move. The mobile phase
moves through the stationary phase picking up the compounds to be tested. As the mobile
phase continues to travel through the stationary phase it takes the compounds with it. At
different points in the stationary phase the different components of the compound are going to
be absorbed and are going to stop moving with the mobile phase. This is how the results of any
chromatography are gotten, from the point at which the different components of the
compound stop moving and separate from the other components. In paper and thin-layer
chromatography the mobile phase is the solvent. The stationary phase in paper
chromatography is the strip or piece of paper that is placed in the solvent. In thin-layer

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chromatography the stationary phase is the thin-layer cell. Both these kinds of chromatography
use capillary action to move the solvent through the stationary phase. What is the Retention
Factor, Rf ? The retention factor, Rf, is a quantitative indication of how far a particular
compound travels in a particular solvent. The Rf value is a good indicator of whether an
unknown compound and a known compound are similar, if not identical. If the Rf value for the
unknown compound is close or the same as the Rf value for the known compound then the two
compounds are most likely similar or identical. The retention factor, Rf, is defined as Rf =
distance the solute (D1) moves divided by the distance traveled by the solvent front (D2) Rf =
D1 / D2 where D1 = distance that color traveled, measured from center of the band of color to
the point where the food color was applied D2 = total distance that solvent traveled The
Different Types of Chromatography There are four main types of chromatography. These are
Liquid Chromatography, Gas Chromatography, Thin-Layer Chromatography and Paper
Chromatography. Liquid Chromatography is used in the world to test water samples to look for
pollution in lakes and rivers. It is used to analyze metal ions and organic compounds in
solutions. Liquid chromatography uses liquids which may incorporate hydrophilic, insoluble
molecules. Gas Chromatography is used in airports to detect bombs and is used is forensics in
many different ways. It is used to analyze fibers on a persons body and also analyze blood
found at a crime scene. In gas chromatography helium is used to move a gaseous mixture
through a column of absorbent material. Thin-layer Chromatography uses an absorbent
material on flat glass or plastic plates. This is a simple and rapid method to check the purity of
an organic compound. It is used to detect pesticide or insecticide residuesin food. Thin-layer
chromatography is also used in forensics to analyze the dye composition of fibers. Paper
Chromatography is one of the most common types of chromatography. It uses a strip of paper
as the stationary phase. Capillary action is used to pull the solvents up through the paper and
separate the solutes.

Chlorophyll often hides the other pigments present in leaves. In Autumn, chlorophyll breaks
down, allowing xanthophyll and carotene, and newly made anthocyanin, to show their colors.
The mix of pigments in a leaf may be separated into bands of color by the technique of paper
chromatography. Chromatography involves the separation of mixtures into individual
components. Chromatography means “color writing.” With this technique the components of a
mixture in a liquid medium are separated. The separation takes place by absorption and
capillarity. The paper holds the substances by absorption; capillarity pulls the substances up the
paper at different rates. Pigments are separated on the paper and show up as colored streaks.
The pattern of separated components on the paper is called a chromatogram.

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MATERIALS REQUIRED
 Safety goggles

Chromatography solvent (92 parts Petroleum ether to 8 parts acetone)

Chromatography paper (or filter paper) about 1 cm x 15 cm

Ethyl alcohol

Fresh spinach

Test tube

Test tube rack

Scissors and Ruler

Fresh leaves of plants

Glass stirring rod

Paper clip

Cork (to fit test tube)

Mortar and pestle

Sand (optional)

10-ml Graduated cylinder

PROCEDURE

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Leaves should be grouped by kind (maple, etc.) and color. Work with a spinach leaf and with
one or more other types. CAUTION: Chromatography solvents are flammable and toxic. Have
no open flames; maintain good ventilation; avoid inhaling fumes.

1. Cut a strip of filter paper or chromatography paper so that it just fits inside a 15-cm (or
larger) test tube. Cut a point at one end. Draw a faint pencil line as shown in figure 1. Bend a
paper clip and attach it to a cork stopper. Attach the paper strip so that it hangs inside the tube,
as shown. The sides of the strip should not touch the glass.

2. Tear a spinach leaf into pieces about the size of a postage stamp. Put them into a mortar
along with a pinch or two of sand to help with grinding. Add about 5 ml ethyl alcohol to the leaf
pieces. Crush leaves with the pestle, using a circular motion, until the mixture is finely ground.
The liquid in which the leaf pigments are now for paper chromatography dissolved is called
the pigment extract.

3. Use a glass rod to touch a drop of the pigment extract to the center of the pencil line on the
paper strip. Let it dry. Repeat as many as 20 times, to build up the pigment spot. NOTE: You
must let the dot dry after each drop is added. The drying keeps the pigment dot from spreading
out too much.

4. Pour 5 ml chromatography solvent into the test tube. Fit the paper and cork assembly inside.
Adjust it so that the paper point just touches the solvent (but not the sides of the tube). The
pigment dot must be above the level of the solvent. Watch the solvent rise up the paper,
carrying and separating the pigments as it goes. At the instant the solvent reaches the top,
remove the paper and let it dry. Observe the bands of pigment. The order, from the top, should
be carotenes (orange), xanthophylls (yellow), chlorophyll a (yellow-green), chlorophyll b (blue-
green), and anthocyanin (red). Identify and label the pigment bands on the dry strip. Write the
species of leaf on the strip as well.
Record the species, external color, and chromatogram pigments in the DATA TABLE of your
report sheet.

5. Each pigment has an Rf value, the speed at which it moves over the paper compared with the
speed of the solvent.

Rf = Distance moved by the pigment / Distance moved by the solvent

Measure the distance in cm from the starting point (pencil line) to the center of each pigment
band. Then measure the entire distance traveled by the solvent. Remember, the starting point
for the solvent is also the pencil line and the ending point for the solvent is the top edge of the
paper. Do the required divisions and record your Rf values in the DATA TABLE of your report
sheet.

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6. Wash the mortar and pestle thoroughly, using a little alcohol to remove any remaining
pigment.

7. Repeat steps 1 through 6 for each species.

OBSERVATIONS

The results of the experiment are shown in a chart and a graph.

Ratio Traveled
Color of Ink Distance each
Distance Solvent (Distance color moved
(listed in Color Traveled
Traveled (mm) divided by distance solvent
order) (mm)
moved)

Yellow 70 mm 111 mm .63

Pink 82 mm 111 mm .74

Red 101 mm 111 mm .91

Purple 110 mm 111 mm .99

Blue 111 mm 111 mm 1.0

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PICTORIAL REPRESENTATION OF EXPERIMENT

CONCLUSIONS

The proposed hypothesis was correct. The paper chromatography did show that black ink could
be separated into various colors. The black ink gets its color from a mixture of various colored

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inks blended together. The first color of ink to appear on the filter paper was yellow followed by
pink, red, purple then blue. The colors separated the way they did because of the differences in
their molecular characteristics, specifically, their solubility in water and their rate of absorption
by the paper. The most soluble and readily absorbed ink color was the yellow. The least soluble
and least absorbable ink color was the blue.

BIBLIOGRAPHY

 The Britain Encyclopedia

 www.biologyjuntion.com

 www.google.com

 www.wikipedia.com

 www.studyadda.com

 CBSE CLASS XII LAB MANUAL

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