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SBL 1023




NO MATRIC: E20161015640




Experiment 6: Plant Physiology


A pigment is a molecule that absorbs light. White light contains all of the different
colors 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 colors.

In plant, there are two categories of pigment that used for photosynthesis which are
primary and accessory pigment. 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, orange or that absorb all of the other
colors. Plant pigments include a variety of different kinds of molecules,
including porphyrins, carotenoids, and anthocyanins. All biological pigments selectively
absorb certain wavelengths of light while reflecting others. The light that is absorbed may be
used by the plant to power chemical reactions, while the reflected wavelengths of light
determine the color the pigment appears to the eye.

Paper chromatography is used in this experiment to separate the plant pigments from
a plant using a hydrophobic solvents which is acetone and ethanol. Paper chromatography is
a technique used to separate a mixture into its component molecules and identifying color
mixture. It allows to separate different molecules from a mixture based on their solubility.
Components of the sample will separate readily according to their level of hydrophobic or
hydrophilic properties. Those pigments molecules that have the highest solubility in liquid
solvent will travel fastest through the paper. While those pigments molecule that have least
solubility in liquid solvent will move more slowly through the paper. In this way, individual
pigments can be separated into bands of color on the filter paper.


1. To identify the pigment used in two types of spinach leaves.

2. To determine the Rf values for each pigment.

1. Test tube
2. Cork stopper
3. Pin
4. Strip of filter paper
5. Solvent
6. Spinach leaves
7. Green leaves
8. mortar


1. Pin head is used as the dropper then the chloroplast extract is dropped on the prepared
chromatography paper.
2. The extract is dropped at about 1.0 cm from the pointing end of the paper. The paper
is dried with hair dryer and the process is repeated for 3-4 times until one small dot of
thick pigment available.
3. The paper strip is attached at the cork stopper using a pin. The strip is placed
vertically and straight into the test tube which contained solvent.
4. Let the solvent move and remove the paper before the solvent front reaches the top of
chromatography paper.
5. The last range of the solvent is marked with the pencil

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑

Rf =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

Green spinach

Color Green
pigment Chlorophyll a
Rf value
Rf green pigment =

= 0.76
Red spinach

Color Green and purple

pigment Anthocyanin
Rf value
Rf green pigment =

= 0.87

Rf purple pigment =

= 0.25


There are two type of pigment observe in this experiment which is chlorophyll and
anthocyanin. Chlorophyll is any of several related green pigments found
in cyanobacteria and the chloroplasts of algae and plants. Chlorophyll is essential
in photosynthesis, allowing plants to absorb energy from light. Chlorophylls absorb light
most strongly in the blue portion of the electromagnetic spectrum as well as the red portion.
Conversely, it is a poor absorber of green and near-green portions of the spectrum, which it
reflects, producing the green color of chlorophyll-containing tissues. Two types of
chlorophyll exist in the photosystems of green plants is chlorophyll a and b. Anthocyanins are
blue, red, or purple pigments found in plants, especially flowers, fruits, and tubers. In acidic
condition, anthocyanin appears as red pigment while blue pigment anthocyanin exists in
alkaline conditions.
During doing this experiment, we have to make sure that the developing solvent
mixture is prepared fresh before use because. The developing liquid phase 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. Beside that, 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 or observations could be made. 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.

It is important to mark the solvent level on the chromatography paper when want to
remove it from the test tube because the point at which the solvent stopped could be noted in
case the solvent kept advancing when removed.Chlorophyll a migrated farthest than the
anthocyanin because it absorb all the color except for the green color. That is why it appears
as green color. The wavelength of the green color about 565 nm. Anthocyanin absorb green
color the most and for this experiment, it appears as red in color. The wavelength is about
740 nm. Therefore, green color can travel the farthest compared to red color.If the
chromatogram is develop too long, the solutes and solvent would travel too far and possibly
run of the plate. Therefore we would not be able to calculate the Rf values without a
measured solvent front


Paper chromatography proved to be an accurate method of separating and observing

the various colors of plant pigments. The pigments dissolved in the solvent and migrated
upward. The colors were observed and their migration distances measured & recorded.
The Rf value of each pigment was determined by dividing its migration by the migration of
the solvent. It was determined that 2 pigments were present on the filter paper which is
chlorophyll a, and anthocyanin. Chlorophyll a can travel farthest compared to anthocyanin
according to their wavelength.

 Wikipedia. Plant Physiology. 14 August 2017. From


 Course Hero. Chromatography. 20 Januari 2018. From


 course Hero. Chromatography. 20 Januari 2018. From


 lab.training.com. paper chromatography. 5 December 2014. From.


 laboratory manual, Dr Shakinaz Desa, Faculty Science and Mathematics.


Based on this experiment, I have been learn on how to determine the retention factor of plant
extract by separating the extract based on their pigment. Every pigment has different color. In
a strip of paper there are various colors we can see like a rainbow. Beside that, I learn on how
the color is occur because pigment is a material that changes the color of reflected or
transmitted light as the result of wavelength selective absorption.