SBL 1023

TECHNIQUE IN BIOLOGY AND

BIOCHEMISTRY LABORATORY

Lab 6: Plant Physiology

AYU ILYANA BT ZULKIFLI

STUDENT`S NAME
( E20161014077)
LAB 6 : PLANT PHYSIOLOGY
EXPERIMENT`S NO
(Paper Chromatography)

DATE/DAY/PLACE 19/12/2017 /TUESDAY/ B2-L3-MP 13

LECTURE`S NAME Professor Madya Dr. Shakinaz Binti Desa

PLANT PHYSIOLOGY
 Paper chromatography

INTRODUCTION

A pigment is a molecule that absorbs light. White light contains all of the different
colours of the visual spectrum. This can be observed in a simple rainbow during a rain storm
or by using a prism that splits white light into its various colours.

In plants, there are two categories of pigments used for photosynthesis: primary
pigments and accessory pigments. The chlorophylls are the primary pigments of
photosynthesis, with two types called chlorophyll a and chlorophyll b. The chlorophylls are
green pigment molecules that absorb blue, red, orange, yellow, etc., but reflects green light.
On the other hand, the accessory pigments are red, yellow or orange – they absorb all of the
other colours.

In this experiment, we use paper chromatography to separate the plant pigments from
a plant using a hydrophobic solvent. Chromatography (“colour” “measure”) is a technique
that allows us to separate different molecules from a mixture based on differences in
solubility. Some compounds do not like to dissolve in water. These are called hydrophobic
(“water” “fearing”) compounds. On the other hand, some molecules are hydrophilic (“water”
“loving”), meaning they like to dissolve in water. Chromatography is a method of separating
and isolating molecules based on their level of hydrophobic or hydrophilic properties. In
paper chromatography, we create a “molecular race track” in which molecules move through
a piece of filter paper, carried along by a wave of liquid solvent. Those pigment molecules
that have the highest solubility in the liquid solvent used will be “carried along” through the
paper the fastest. Those pigments that are least soluble in the solvent will move more slowly
or not at all. The various plant pigments have differing degrees of hydrophobicity. Therefore,
if we use a liquid solvent that is hydrophobic, different plant pigments will move at differing
rates through the piece of paper as the liquid solvent is absorbed upward. In this way,
individual pigments can be separated into bands on the filter paper.

PURPOSE
The paper chromatography were used to separate pigments and calculate RF values
using plant pigment chromatography, describe a technique to determine the photosynthetic
rate, compare photosynthetic rates at different light intensities using controlled experiments,
as well as explain why rate of photosynthesis varies under different environmental conditions.

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HYPOTHESIS
If pigments are separated, then RF values can be determined.

METHODOLOGY

1. The chloroplast extract has been dropped on the prepared chromatography paper used
the microbiurette or a pin head as the dropper,
2. The extract was dropped at about 1.0 cm from the pointing end of the paper. The drop
was dried with a hair dryer and repeated the process for 3-4 times until one small dot
of thick pigment available.
3. The paper strip was attached at the cork stopper used a pin. Then, the strip was placed
vertically and straight into the test tube which contained solvent.
4. Let the solvent moved and removed the paper before the solvent front reached the top
of the chromatography paper.
5. The last range of the solvent has been marked with pencil.

Figure 1

Figure 2

RESULTS

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Green spinach
Red spinach

Figure 2: Result of the experiment from chloroplast green spinach and red spinach

This result show that green spinach has mainly green pigment that is chlorophyll b and has
also a pigment of carotene. While, red spinach has all pigment except xanthophyll. This is
because we use small dot of specimen, so the colour was not obvious. We should use large
dot of the specimen, so that the colour result was obvious

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 Distance Distance Name of
Description
Pigment solvent front compound Rf value pigment (use
of Colour
travelled (cm) travelled (cm) appendix a)
2.5/6.4=
1 Light red 6.4 2.5 Chlorophyll b
0.391
Pale yellow 6.2/6.4 =
2 6.4 6.2 Carotene
orange 0.969
Table 1: green spinach

Distance Distance Name of

Description
Pigment solvent front compound Rf value pigment (use
of Colour
travelled (cm) travelled (cm) appendix a)
3.0/7.2 =
1 Yellow green 7.2 3.0 Chlorophyll b
0.417
4.9/7.2 =
2 Blue green 7.2 4.9 Chlorophyll a
0.681
Pale yellow 6.8/7.2=
3 7.2 6.8 Carotene
orange 0.944
Table 2: red spinach

CALCULATION
To find RF value of each pigment observed, this formula was used:

DISCUSSIONS
There are several pigment that can be found in a spinach that is Chlorophyll, Carotenoids,
Pheophytins and Xanthophyll. Chlorophyll is the green pigment in most plants that is
associated with photosynthesis. The pigment absorbs all coloured light except for the green
band, which it reflects to give spinach its characteristic leaf and stem colour. "Chlorophyll a"
is a strong blue-green colour and primarily responsible for photosynthesis, while "chlorophyll
b" is a supporting photosynthetic pigment.

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 Next, Carotenoids are especially useful to humans since they are broken down in our
bodies to become Vitamin A, an essential nutrient for health and survival. They are typically
yellow-orange pigments that give carrots their characteristic colour. Beta carotene is the
primary carotenoid pigment found in spinach.

Besides, Pheophytins pigments similar to chlorophyll in structure, pheophytins are

actually decomposition products. They are "spent" chlorophyll that has lost an ion but which
remains in the leaf and continues to lend the leaf its colour. The ion lost comes from the
magnesium component.

Furthermore, when carotenoids become oxidized, or take on an oxygen molecule, they are
known as xanthophyll. The name changes since their structure has changed. These pigments
are still yellowish in colour but not reddish or oranges the way that carotenoids often are.

Discussion from question

1. The developing solvent mixture is prepared fresh before use?

The developing solvent mixture comprises of a pure solvent but more often it is a
mixture of two or more solvents in specified proportions. In case solvents are mixed
and stored for long periods there could be loss of volatile component which will alter
the mixing proportions.

2. Is it important to keep the dye spots (leaf extract) above the solvent level?

It is important to keep the dye spots above the solvent level because if the dye spots
of submerged in the solvent, then the spots would dissolve into the solvent
preventing them from separating out and no measurements observations could be
made.

3. Is it necessary to cover the test tube during the paper development?

During the chromatogram development chamber is covered. This is essential as the

environment inside the chamber should remain saturated with the solvent vapour.
Development times can vary from about an hour to several hours and a saturated
environment prevents losses due to evaporation

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 4. It is important to stop the chromatogram before the solvent front reaches the top of
your chromatography paper?

It is important to stop it because you cannot determine the Rf value unless you can
measure the distance from the start to the front of solvent, you have to be able to see
where it stops even if you let it go as high as you want.

5. Is it important to mark the solvent level on the chromatography paper when you
remove it from the test tube?

It is important to mark the solvent level on the chromatography paper when you
remove it from the petri dish because so the point at which the solvent stopped could
be noted in case the solvent kept advancing when removed.

6. Which of the pigments migrated the farthest and why the separation of pigments
occur as it did? How does paper chromatography work?

Carotene (orange) because it was the most soluble in the solvent. Paper
chromatography as the layer of adsorbent is known as the stationary phase. After the
sample has been applied on the plate, a solvent or solvent mixture (known as the
mobile phase) is drawn up the plate via capillary action. Because different analytes
ascend the plate at different rates, separation is achieved.

7. Explain what would happen to your chromatogram if you let it run too long?

Therefore we would not be able to calculate the Rf values without a measured

solvent front

CONCLUSION

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 The proposed hypothesis was correct. If pigments are separated, then RF values can
be determined. RF value was determined by distance travelled by compound divide by
distance travelled by solvent. Thus, from RF value we can determine the affinity of the
solute to the solvent. Greater RF value, greater affinity of solute to the solvent

Besides, the paper chromatography did show that from a dot of chloroplast extract
could be separated into various colours of pigment. The chloroplast contain mixture of
various pigment together. The first colour of pigment to appear on the filter paper was pale
yellow. The colours separated 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 chloroplast was the pale yellow green in colour. The least
soluble and least absorbable of chloroplast pigment was blue in colour.

REFERENCE

1) Scienceteacherresources, Plant pigment Chromatography. Retrieved from

https://www.depts.ttu.edu/ciser/science-teacher-resources/traveling-
lab/curriculum/plants/Plant_Pigment_Chromatography.pdf on 27/12//2017

2) Elinavanuska, 2017, Chromatography of Different Coloured Leaves: Lab Report. Retrieved

from https://elinavanuska.wordpress.com/investigations/chromatography-of-different-
colored-leaves-lab-report/ on 27/12/2017