Biology 43: Plant Physiology

Laboratory Activity VI

Isolating Photosynthetic Pigments

Krizea Marie Kiamco Duron

BS Biology IV

krizeakduron@su.edu.ph

Abstract

The objective of this experiment is to extract the photosynthetic pigments from

Kangkong (Ipomea aquatica) by disrupting the membranes, thereby releasing the pigments. The

method employed in developing the pigments is through the use of thin layer chromatography.

The results were not very satisfactory since in the solvent, the spot did not run towards the other

end of the silica gel strip thus, chlorophyll and carotenoids were not clearly isolated and

observed. The two major pigments in plants are chlorophyll and carotenoids.

Introduction

The efficiency of plants at capturing light for photosynthesis varies. One means by which a

plant can increase its photosynthetic productivity is to use a variety of pigments to harvest a

wider range of light wavelengths. Pigments are chemical compounds that reflect only certain

wavelengths of visible light. There are three basic groups of pigments. Chlorophylls are greenish

pigments that contain a porphyrin ring around which electrons freely migrate. Carotenoids are

typically yellow, orange, or red pigments that contain two 6-carbon rings connected by a

hydrocarbon chain and do not directly transfer light energy to the reaction center and electron

transport chain.

Many of the colors associated with higher plants (green leaves in the spring and summer,

yellow or red leaves in the fall, the orange color of carrots, some colors in flower petals) are due

to the presence of pigment molecules. The major photosynthetic pigments of higher plants can be

divided into two groups, the chlorophylls and the carotenoids. Both types of pigments are

present in the subcellular organelles called chloroplasts, where they are bound to proteins in the

thylakoids, the photochemically active photosynthetic biomembranes. Intact pigment-protein

complexes, which are held together by weak, noncovalent bonds, have been isolated from

chloroplasts and characterized b~( polyacrylamide gel electrophoresis and isoelectric focusing.

The pigments are released in a protein-free form by grinding plant tissue in solvents such as

acetone, methanol or hexane. Since the chlorophylls and the carotenoids are readily soluble in

organic solvents, they are classified biochemically as lipids.

Chromatography is one technique that can be used to separate and identify a variety of

compounds. The separation of the components within the mixture is dependent on their different

affinities for a stationary phase (the silica) and a moving phase (the solvent). The migration

distance of a specific compound, a pigment in our case, is dependent on its affinity for the

solvent (a hydrophobic mixture of acetone, chloroform, and ether) versus its affinity for the silica

gel (hydrophilic). Under a specific set of conditions, the distance moved is a characteristic that

may be used to help identify the compound. The ratio of the distance traveled by a compound to

that of the solvent front is known as the Rf value.

The purpose of this lab experiment was to separate plant pigments using paper

chromatography, and to measure the rate of photosynthesis in isolated chloroplasts. Because of

capillary action the solvent moves up the paper causing the pigments to become visible at certain

distances.

The substances visible on the paper are called pigments. Chlorophyll a is the main

pigment that makes up about 75% of the pigmentation in plants. Chlorophyll b makes up about

25% of the pigmentation. And carotenes and xanthophylls are accessory pigments that make up

the rest of the pigmentation. Carotene is the most soluble of the pigments and as a result will be

carried the farthest by the solvent. The paper will display a spectrum of the pigments found in the

spinach leaves. Using the formula Rf one can determine the relationship between the distance the

solvent traveled to the distance the pigment traveled.

Materials and Methods

In this study, ethanol extraction was employed. We removed the petioles from the leaves

and weighed about 5g of our kangkong leaves. We used a blender to liquefy our leaves and

centrifuged for 2 minutes. We mounted a small portion on a microscope slide with 10% NaCl.

The appearance of the chloroplast was observed. We also put it under UV light to observe

autofluorescence. For the proper isolation and separation of pigments, we used thin layer

chromatography.

Figure 1. Preparation of our liquid extract. Image taken before it was centrifuged.

Figure 2. Adding a small portion of the extract on the glass slide to be observed under

the microscope to observe and describe chlorophyll appearance.

Figure 3. Letting the spot run thru the silica gel strip to isolate different pigments.

Figure 4. Shining UV to observe autoflouorescence.

Results

The results basically were not satisfactory since the spot did not run through the

silica gel strip. Perhaps not enough extract were dropped into the strip. But for my results, I

borrowed Patrik’s reults so that I can conclude and discuss further the different types of

pigments. But first, here is a photograph of the chloroplast under the microscope.

Figure 5. Chloroplast of Kangkong under the light microscope.

Figure 6. Silica gel strip partly soaked in the developer petroleum ether. No running of

pigments were observed.

For my discussions and conclusions, I’d like to present in my results my classmate

Patrik’s experiment where he got the ideal result for this experiment.

Figure 7. Replicate 1. Silica gel strip showing the isolated pigments © Patrik Narouzi

Figure 8.Replicate 2. Silica gel strip showing the isolated pigments. © Patrik Narouzi

Table 1. Showing the different Rf values of the specific pigments isolated.

Plant

Kangkong

Pigment Rf Value Color of the spot or

band

Replicate 1 Chlorophyll c 1cm/6.5cm= 0.15cm Green

Carotenoids

(violaxanthin)

2cm/6.5cm= 0.31cm Yellowish/ Orange

Replicate 2 Chlorophyll b 3.5cm/6.5cm= 0.53 Green

Carotenoids (neoxanthin) 0.5cm/ 6.5cm= 0.08 Orange/ Brown

Figure 9. The two strips under UV shine. Parts encircled in yellow autofluoresced under

UV shine. ©Patrik Narouzi

Discussion

There are four different pigment groups present in leaves of photosynthesizing plants.

Chlorophyll a and b impart the green color that one associates with plant leaves. Carotenoids,

which are yellow pigments, are also present in leaves but are usually masked by the chlorophylls.

It is only in the fall when the chlorophylls are degraded faster than the carotenoids that the

yellow color becomes visible to us. The chlorophyll and carotenoid contents of plants can vary

markedly with its age, or depend on environmental factors such as light intensity or quality

during growth. The pale green appearance of a willow tree in early spring is markedly different

from its olive-green of late summer. The intense dark green of "shade adapted" plants differs

from the lacy green colors one sees at the top of a forest canopy.

Carotenoids and chlorophylls are found in the chloroplasts and are associated with the

thylakoids, the internal membrane network of these organelles. It is now established that all

chlorophylls are organized as discrete chlorophyll-protein complexes within the lipid matrix of

the photosynthetic membrane.

Conclusion

Leaves contain usually 7 different carotenoids (neoxanthin, violaxanthin, lutein,

zeaxanthin, antheraxanthin, β-carotene and α-carotene) that function in light harvesting.

Although most plant leaves appear green to our eyes, several pigments of different color are

usually present in the chloroplasts. Chlorophylls a and b provide the green color and absorb the

light energy needed for photosynthesis. However, other accessory pigments, such as yellow

xanthophylls and orange carotenes are also present in the chloroplasts and collect additional light

energy for photosynthesis.

Acknowledgement

In the conduct of this study, I’d like to thank first, the God Almighty for giving us the drive, the

good health, and the spirit to perform the experiment.

I’d also like to thank the following individuals and institutions who helped us in this

activity:

To Professor Hilconida Calumpong for being our instructor and giving us the proper

instructions fit for our experiment.

To Sir Ian Canlas for helping us set up our developer and also for helping us in handling

the apparatuses we used.

To my classmate, Patrik Narouzi, for sharing with me his results and thoughts regarding

why our experiment failed to get the ideal results.

To the Stock room of the the Biology Department for providing us with the materials

needed for this experiment.

And of course, to our parents, for their never-ending financial support.

References

Edwards-Knox Central School Science Department -

http://ekcsk12.org/science/lelab/chromatographylab.html.

Tomkins, S. P. and Miller, M. B. (1994). A rapid extraction and fast separation of leaf pigments

using thin layer chromatography. School Science review75 (273), 69 - 72.

CSU Stanislaus Chemistry http://wwwchem.csustan.edu/chem1102H/Green_Pigments.html.

http://sps.k12.ar.us/massengale/lab_4_plant_pigments.htm

http://www.ualr.edu/~biology/botany/pigmentlab.html

http://gened.emc.maricopa.edu/bio/bio181/BIOBK/BioBookPS.html