Investigating the composite pigments in a plant leaf using Thin Layer Chromatography.

INTRODUCTION

According to Giddings (2020), chromatography is a technique for separating the components or

solutes of mixtures on the basis of the relative amounts each solute distributed between the

moving fluid stream called the mobile phase and a contiguous stationary phase. According to

Sofer G.K et al (1989) chromatography is generally recognized as the technique which allows

the highest degree of purification of biomolecules.

The mobile phase maybe a liquid or a gas while the stationary phase is either a solid or a liquid.

Living organisms have various structures and are composed of various chemical elements. These

different chemical molecules can be extracted and identified. Plants, especially green plants have

a variety of leaf components that are essential for the process of photosynthesis. Chromatography

is used to extract those components.

Thin layer chromatography being used for this investigation is based on the partitioning of the

mixture between the mobile phase and the stationary phase. The stationary phase used was

alumina and the mobile phase comprised of cyclohexane, petroleum ether, ethyl acetate, acetone

and methanol in the ratio 16:60:10:10:14. According to Jabiur (2010) the mobile phase flows

through the stationery phase and carries the components of the mixture with it and the stationery

phase remains fixed in place while the components of the sample are being carried through the

medium used. Thin layer chromatography is useful in this experiment because it allows analysis

of the smallest samples.

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There many pigments found in different plants and plant pigments give colour to the leaves and

flowers of plants. The life on Earth primarily relies on sunlight energy captured by plant

pigments to drive the process of photosynthesis. The main photosynthetic pigments in plant

leaves are chlorophylls a and b. The main carotenoids in green leaves of terrestrial plants are α-

and β-carotene, lutein, zeaxanthin, violaxanthin, antheraxanthin, and neoxanthin (Esteban et al.,

2015).

The experiment was carried out to study the composition of leaf extracts of different plants. The

main aim of the experiment was to separate the pigments of the leaf extracts of Morus alba,

Duranta sp, Cissus sp and Bougainvillea sp and identify them by using thin layer

chromatography. The purpose of this experiment was to be knowledgeable on the pigments

leaves have and if they are common amongst the selected plants.

The alternative hypothesis for this experiment being there are multiple pigments within plant

leaves that absorb different wavelengths of light and are common amongst different species of

plants and the null hypothesis being the pigments are not common amongst the plant species.

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MATERIALS AND METHODS

Students were divided into groups composed of four members each. Each group selected a plant

from samples of mulberry scientifically known as Morus alba, Cissus sp, Duranta sp and

bougainvillea sp for the experiment. They were weighed using a top pan balanced and 1.5 of

each sample was obtained as required. Prior to obtaining the measurements, the balance was

tared to zero to eliminate zero error. A mass of 0.5g of anhydrous magnesium sulphate and 1g of

acid washed sand were also added. The magnesium sulphate was added so that it acts as a drying

agent and increase the concentration of the pigments.

The acid washed sand was added to aid in crushing the leaves, break open their cells and allows

the organic compounds to dissolve in the solvent. A volume of 10mL of acetone was measured

using a measuring cylinder and the mixtures were dissolved in the10mL of acetone. The acetone

is used for it is an amphipathic substance, meaning it has both polar and non-polar hence it has

the capacity to dissolve both nonpolar and polar substance and the fact that it is less polar than

water it means it will have greater resolution between pigments on paper. Acetone is volatile.

The supernatants were transferred into a centrifuge tube and vortex. Vortexing was done to mix

the liquid. The mixtures were centrifuged at 2300 rpm for 10 minutes and collected into clean

bottles. This was done to eliminate any solid from the mixtures. Four labeled Eppendorf tubes

were filled with the four different extracts from the different groups. Four capillary tubes used to

draw the mixtures were also labelled. A chromatography solvent that compromised of

cyclohexane, petroleum ether, ethyl acetate, acetone and methanol in the ratio 16:60:10:10:14

was measured to a volume of 20mL and added into a chromatography development tank.

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The vessel was immediately closed to ensure that the solvent does not evaporate, since it is

volatile. The chromatography plate was placed on a clean surface with no contact with the

surface of the paper and special precautions where undertaken to avoid fingerprints on the plate.

A 1.5 cm line was drawn with a pencil from the bottom of the plate. A pencil was used for it

does not have dyes like a pen hence would not affect the experiment’s results. 2 cm intervals

markings were done from the left edge of the plate. Marked capillary tubes for each extract were

used to carefully apply incremental amounts of the extracts along the 2cm markings on the

chromatography plate.

The chromatography plate was carefully inserted in the development tank and covered with a

glass plate. All the edges of the chromatography plate were inserted all at once so that the results

obtained are accurate. The chromatography plate was observed up until the solvent had covered

80% of the chromatography plate. It was then removed and a line using a pencil was carefully

drawn across the solvent wetting front before the chromatogram got dry. The center of the

darkest region of each spot was marked using a pencil, and the distances from the origin to each

of the dark spot were measured using a ruler of accuracy 0.1 and recorded in a table. The colors

of the spots were also recorded. The retention factors (Rf values) were calculated as followed;

Distance travelled by the analyte

Rf=
Distance travelled by the solvent

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Results

Table 1; Different pigments and their Rf values for the four-leaf extracts

Plant Specie Pigments colour RF values Suggested pigment

1. Bougainvillea Yellow 0.95 Carotene
sp Yellow 0.16 Xanthophyll
green 0.21 Chlorophyll b
olive green 0.30 Pheophytin b
Blue green 0.56 Chlorophyll a
2. Cissus sp Green 0.22 Chlorophyll b
Light Yellow 0.17 Neoxanthin
Yellow 0.98 Carotene
Blue green 0.46 Chlorophyll a
3. Duranta sp Blue Green 0.19 Chlorophyll a
Yellow 0.94 Carotene
Green 0.14 Pheophytin b
Green 0.10 Chlorophyll b
Light Yellow 0.11 Neoxanthin
4. Morus alba sp Yellow 0.97 Carotene
Green 0.27 Chlorophyll a
Green 0.21 Chlorophyll b
Yellow 0.16 Xanthophyll
Table 1 shows that the pigments identified on the plant leaves are carotene, chlorophyll a,

chlorophyll b, neoxanthin, pheophytin and xanthophyll. All of the extracts had carotene,

chlorophyll a and chlorophyll b common. Carotene had the highest Rf value across all the leaves

extracts.

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Table 2; Information used to help identify the pigments

Pigment Rf range colour

Carotene 0.89-0.98 Yellow
Xanthophyll 0.15-0.35 yellow
Chlorophyll a 0.55-0.65 Blue green
Chlorophyll b 0.42-0.49 Green
Pheophytin b 0.32-0.40 Olive green
Neoxanthin 0.04-0.28 Light yellow
The Rf values are given in ranges because the components of the solvent mixture evaporate at

different rates, so the solvent mixture used might be in slightly different proportions to the other

times this chromatography was done.

Sampled calculation

Distance travelled by solvent = 63mm

Using sample from Duranta which travelled 62mm

Distance travelled by the analyte

Rf=
Distance travelled by the solvent

= 62mm/63mm

= 0.984126984

=0.98 to 2 significant figures

= 0.98

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Discussion

After collection and analysis, the results supported the notion that there are multiple pigments in

plant leaves and where relatively common amongst the plants used for the experiment. The

results contained enough evidence to reject the null hypothesis that suggested the plant pigments

are not common hence taking the alternative hypothesis that suggests that the multiple pigments

obtained are common amongst the plant species. The pigments found helps the science

community in knowing the importance of the pigments in photosynthesis.

When grinding, the acid washed sand was used to help crush the leaves by providing a rough

surface for grinding, and it broke open the cells of the leaves to release their cell contents. The

anhydrous Magnesium Sulphate was added so as to remove water from the leaves hence

exposing the cell contents. The supernatant was placed in a centrifuge vortex so as to separate

the solid in the mixture and the liquid. The results showed a series of pigments that where

common amongst the four sampled plants. The most common ones being chlorophyll a,

chlorophyll b and carotene.

Table 2: shows plant leaves and the number of pigments each have.

Plant specie Number of pigments present

Durant sp 5
Morus alba 4
Cissus sp 4
Bougainvillea sp 5

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When a beam of light falls on a pigment, certain wavelengths are absorbed, the remaining

wavelengths are scattered and make the pigment appear coloured to us as started by (Sadava et

al.,2014). If we plot a graph of light absorbed against wavelength, it results in an absorption

spectrum of that pigment. In contrast to the absorption spectrum, an action spectrum is a plot of

the rate of photosynthesis carried out by an organism against the wavelengths of light to which it

is exposed.

Comparison of the two graphs will determine the components responsible for photosynthesis,

which will reveal chlorophyll being the component with a high rate. Other studies suggest to

determine which component is responsible for absorption of light, a white light is exposed to a

sample containing chlorophyll, blue and red light will be absorbed to show presence of

chlorophyll.

The different pigments have different roles in the leaf, Carotene assists with photosynthesis and

helps protect structures in the leaf (Anderson,1996), neoxanthin helps protect the leaf from photo

oxidative stress, xanthophyll helps protects the leaf from excess amount of sunlight and

chlorophyll is the green pigment that helps absorb light during photosynthesis. Chlorophyll is a

molecule made by magnesium atom in a porphyrin ring, it has two types which are chlorophyll a

and chlorophyll b both separated by their composite side chains. Pheophytin serves as the first

electron carrier intermediate in the electron transfer pathway of photosystem in plants.

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The distance travelled by the analyte was influenced by polarity, adsorption and hydrogen bonds.

Some studies may deny that for example, Kirchner (1973) argue that the factors that are involved

in the separation of the pigments are solubility, size of particles and their attractiveness to the

paper. Temperature also affects the speed at which the solvent is taken up by capillary action the

higher the temperature the higher the speed. The most absorbent molecule will form analytes

closer to the origin (Forgacs,2001) and in this case neoxanthin was the most absorbent.

The errors that were most likely during the experiment were as follows, the students contacted

the chromatography plate using their finger prints, not adding enough dots on the

chromatography paper and poorly placing the plate in the developing tank. The experiment could

be limit by factors such as that some of the capillary tubes might be swapped during exchange,

which means that a similar sample might be added twice hence affecting the result. These errors

where minimized with the help of the demonstrator showing the students the proper technique to

use to avoid such instances.

There are some potential limits to the effectiveness of the chromatographic technique. One limit

is that it cannot differentiate between some isomers and enantiomers. Another limit could be that

in order to identify some compounds, their Rf values should be known beforehand when a beam

of light falls on a pigment, certain wavelengths are absorbed, the remaining wavelengths are

scattered and make the pigment appear coloured to us (Sadava et al ,2014). Also, column

chromatography could have been used for it produces accurate results more than thin layer

chromatography.

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Conclusion

Thin Layer Chromatography has proved to be a successful method of separating and comparing

pigments. Through the chromatography method the Rf values of the components were

calculated, the plant leaves pigments were identified and it was found out that most of the plant

leaves contain almost identical photosynthetic components.

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References

1. Jabiur, M. (2016). Mobile phase chromatography. Daffodil University

https://pt.slideshare.net/jabirrahaman/mobile-phase-in-chromatography
2. Kircher, K. (1973). Thin Layer chromatography quantitative analysis (Volume 82). CRC

Press

3. Sadava, D., Heller, H., Hills, D., Berenbaum, M. (2014). Life; The Science of Biology (10th
ED.). Macmillan Publisher
4. Sofer G.K et al (1989). Process Chromatography, A Practical guide. Academic Press

5. Giddings, J.C., and Keller, R. A. (2020). Chromatography. Encyclopedia Britannica. The

Magazine of Separation Science

6. Forgacs, E., and Cserhati, T. (2001). Chromatography in environment protection (1st ed.).

CRC Press

7. Esteban R, Moran J.F, Becerril J.F, García-Plazaola J.I., (2015). Versatility of carotenoids:

an integrated view on diversity, evolution, functional roles and environmental interactions.

Frontier Media SA

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