[생명과학/실험] Separation and Identification of Plant Pigments by Paper Chromatography

Date: April 5, 2011

 

Subject: to learn chromatography and identify plant pigments of forceps by chromatography

 

Abstract:

Chromatography is the technique for the separation of mixtures which exist in small amount. It is based on the affinity of the sample with stationary phase and mobile phase. If the sample has a high affinity with stationary phase, it doesn`t travel far, but if the sample has a high affinity with mobile phase, it travels far. There are variety kinds of chromatography such as column chromatography, paper chromatography, gas chromatography, liquid chromatography, etc.

Paper chromatogaphy is a kind of thin layer chromatography and commonly used for separating and identifying pigments. It uses a cellulose chromatography paper which is polar, and non-polar solvent such as acetone. The sample is placed upper 2cm of the bottom of the paper and the end of the paper is dipped in solvent; but the sample isn't wet directly. The paper absorbing solvent, pigments are seperated on the paper by the affinity with the paper and solvent. Rf value presents the affinity of the sample with the solvent and the degree of exclusion the paper.

Plant pigments are the colored substance in the plant. They are produced by plant and there are two big categories of plant pigments; carotenoids and chlorophylls. Chlorophyll a and b are green pigments and they are very important in photosynthesis because they functions in Photosystem I and II. The molecule of chlorophyll has some O, N and Mg2+, so the molecule is polar. In addition, because chlorophyll b has one more O, chlorophyll b is more polar than chlorophyll a. Carotenoids do photosynthesis with blue light and they protect chlorophyll from photodamage. There are two kinds of carotenoids, carotene and xanthophyll. Carotene consists of only carbon and hydrogen, but xanthophyll has also oxygen with carbon skeleton, so xanthophyll is more polar than non-polar carotene. 

Materials:

tips

forceps

beakers

scissors

spinach

chromatography paper

acetone

mortar&pestle

petroleum ether / acetone solvent (9:1)

Procedure:

1. Grind 2~3 leaves of spinach with 5ml acetone.

2. Using a tip, streak the leaf pigment extract on a pencil line previously drawn 1cm from the edge of the paper (the darker your band of pigments, the better the results of your experiment will be.)

3. Using forceps, carefully loser the loaded paper cylinder into the solvent (petroleum ether and acetone) , and quickly cover the beakers tightly with clean wrap.

4. Allow the chromatography to proceed until the solvent front has reached to within 3cm of the top of the paper.

5. Remove the paper from the beaker, and allow it to dry.

Results:

1. Sketch the chromatography paper and label the color of the various bands.

2. Calculate the Rf values of each band.

Pigment	Distance traveled by compound	Rf value	Color
chlorophyll a	4cm	0.83	emerald green
chlorophyll b	3.4cm	0.71	olive green
carotene	4.7cm	0.98	yellow
xanthophyll	4.3cm	0.90	lemon yellow
Distance traveled by the solvent front: 4.8cm

DIscussion:

1. List the photosynthetic pigments in low polarity based on molecular structure.

Chlorophyll a and b have some polar molecules such as O, N and Mg+, so the molecules are polar. In addition, because chlorophyll b has one more O, chlorophyll b is more polar than chlorophyll a. Carotene consist of only carbon and hydrogen, so it is purely non-polar. However, xanthophyll has also oxygen with hydrocarbon skeleton, so xanthophyll is more polar than non-polar carotene. Therefore, the list of pigments in low polarity based on molecular structure is like this: carotene, xanthophyll, chlorophyll a, and chlorophyll b.

In this paper chromatography, stationary phase is polar cellulose paper and mobile phase is non-polar mixture of petroleum ether and acetone. Therefore, polar pigments do not travel far from loading line, but non-polar pigments travel far. According to the result, carotene travels the most far, then xanthophyll, chlorophyll a, and chlorophyll b. This is the same as the list based on molecular structure.

2. Discuss the relations between Rf value and polarity among the four pigments.

The Rf value means the affinity of pigments with the solvent and the degree of exclusion the paper. If pigment has a high affinity with nonpolar petroleum ether and acetone solvent and low affinity with polar cellulose paper, pigment travels far from loading line. On the other hand, if pigment has a low affinity with the solvent and high affinity with the paper, pigment does not travels far. Therefore, the Rf value presents the nonpolarity of the pigments. The polarity of pigments increases in this order: carotene, xanthophyll, chlorophyll a, and chlorophyll b, based on Rf value.

Reference:

http://en.wikipedia.org/wiki/Chromatography

http://www.scienceall.com/dictionary/dictionary.sca?todo=scienceTermsView&classid=&articleid=255769&bbsid=619&popissue=

 

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