Paper Chromatography Plant

Practical 4: Separating Plant Pigments in Leaves Using Ascending Paper
Chromatography
Introduction
Chromatography is a general term describing a variety of analytical methods for the
separation and identification of the compounds in a mixture.
Principle
The principle can either be adsorption or partition.
Adsorption: Between solid and liquid phases, whereby, the paper is the stationary
phase and the solvent used is the mobile phase.
Partition: The particles are distributed between liquid phases i.e., water is held in
pores of the filter paper and the solvent which passes through the paper. When the
mobile phase moves, the separation of the mixture takes place.
Aim: To distinguish the various pigments present in plant leaves.
Background
Plants carry out the process of photosynthesis, during which light energy from the sun
is converted into chemical energy (food). The capturing of light energy is carried out
by molecules known as pigments which are present within the plant cells. Pigments
are chemical compounds which are able to reflect only a particular range of
wavelengths of visible light. Leaves of plants primarily contain different types of
pigments within their tissues. In order to view and distinguish the primary four plant
pigments, a simple technique known as chromatography can be used.
Theory
Simple ascending paper chromatography can be used to separate a coloured
mixture. In paper chromatography, a mixture is ‘spotted’ onto a special type of highly
absorbent paper, and a solvent is then allowed to seep through the paper by capillary
action. The compounds in the mixture become separated as they dissolve in the
solvent and travel at different rates, depending on their relative affinity for the paper
(stationary phase) versus the solvent (mobile phase). Capillary action causes the
solvent to travel up a piece of filter paper.
Materials and Reagents

 Green leaves
 Mortar and pestle
 Methanol
 Capillary tube
 Pencil
 Measuring cylinder
 Beaker
 Whatman Number I Filter Paper
Procedure
1. Label a clean beaker and fill it one third way with the solvent provided.
2. Cut the green leaves into smaller pieces and add them to the mortar.
3. Accurately measure 10ml of methanol and add it into the mortar.
4. With the help of a pestle, grind the leaves into a smooth paste.
5. Cut the filter papers into appropriate sizes.
6. Using a pencil draw a line across the paper about 2cm from the lower edge.
Mark approximately at the centre of the line.
7. Using a capillary tube, apply a small sample of the paste to the spot previously
marked.
8. Allow the spot to dry completely. Then repeat step 6 above to concentrate the
spot.
9. Place an anchor at the top of the beaker to hold the filter paper in place and
ensure that it stays upright throughout the process.
10. Place the filter paper in an upright position so that the line with the pigment is
near the solvent. Do not let the pigment line directly touch the solvent. Allow
the solvent to rise up the filter paper to meet the pigment line.
11. Allow the chromatograms to develop until the solvent is about 1 cm away
from the outside edge of the paper.
12. Remove the filter paper from the beaker and allow it to dry.
13. For better results, repeat step 10 to 12.
14. Using a pencil, mark the solvent front.
15. Outline each spot in pencil and mark the centre of the spot.
Note: Include the chromatogram (or a copy in your report).

Expected Result
The dried paper strip displays four different bands. The pigments can be distinguished
with the help of colors.
1. The Carotene pigment is observed at the topmost as an orange band.

2. Below this, a yellowish band appears which indicates the pigment xanthophyll.
3. The third band appearing dark green indicates chlorophyll-a pigment.
4. The yellowish-green band present at the bottom is the chlorophyll b pigment.
Questions
1. What is the length of the run from the original position?
2. Calculate the retention factor (Rf) for each of the separated pigments.
Note: The retention factor (Rf), is the ratio of the distance a compound travels to the
distance the solvent travels, both measured from the origin.
Rf = Distance from the origin to the centre of spot

Distance from the origin to solvent front
3. Account for Rf values of each of the pigments in terms of their structure.

4. Explain the importance of retention factor (Rf).
5. Discuss each of the separated pigments:
a) Carotene
b) Xanthopyll
c) Chrolorophyll b
d) Chlororophyll a
6. What is phycobilin?
7. Why is it necessary to add the solvent to the chromatography bottle well before
starting the experiment?

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Practical 4: Separating Plant Pigments in Leaves Using Ascending Paper

Aim: To distinguish the various pigments present in plant leaves.

Materials and Reagents

Note: Include the chromatogram (or a copy in your report).

1. The Carotene pigment is observed at the topmost as an orange band.

Rf = Distance from the origin to the centre of spot

3. Account for Rf values of each of the pigments in terms of their structure.

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