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Separation of plant pigments by paper chromatography

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Office of the Provost Separation of Pigments by Paper Chromatography All living organisms require energy for their metabolic chemical processes. The ultimate source of this energy is the sun. Photosynthetic organisms, including plants, protists single-celled organismsand blue-green algae cyanobacteriaconvert light energy into the chemical energy of sugars, which can be used to power metabolism.

During photosynthesis, molecules referred to as pigments due to the wavelength, thus color, they reflect are used to capture light energy. Four primary pigments of green plants can easily be separated and identified using a technique called paper chromatography.

These pigments include two greenish pigments called chlorophylls and two yellowish pigments called carotenoids. Pigments are separated according to differences in their relative solubilities. In order to extract these pigments from the thylakoid membranes of the chloroplasts, the organelles in which photosynthesis occurs, fresh, ground or torn leaves preferably spinach may be soaked in acetone or concentrated alcohol.

Pigments are then "painted" onto strips of chromatography paper with V-shaped tips using a small, hollow glass tube or a small paintbrush.

  • Visible light, or white light, is made up of the colors of the rainbow;
  • Instead, the energy is released as heat and light in a process called fluorescence;
  • The V-shaped tip of the paper is placed in the chromatography solvent and acts as a wick to draw the solvent up the paper, separating pigments according to their relative solubility and molecular weights;
  • The yellowish band appearing below it indicates the xanthophylls;
  • The extent to which any particular component moves up the paper is dependent not only on its solubility in propanone but also on its attraction for the cellulose in the chromatography paper;
  • The yellow carotene spot with a higher RF value tends to move up the paper the furthest.

For best results, allow the line of pigments to dry, then repeat the process until a dark green line of pigments is evident about six times is sufficient to achieve a dark pigment line. Next, chromatography solvent is used to separate the mixture of pigments painted on the paper. In the experiment pictured at left, the solvent used was comprised of nine parts petroleum ether and one part acetone. A small amount of this solvent is added to a large test tube and capped with a rubber stopper.

Materials required

Note that chromatography solvent is highly volatile and flammable. The V-shaped tip of the paper is placed in the chromatography solvent and acts as a wick to draw the solvent up the paper, separating pigments according to their relative solubility and molecular weights.

The paper is allowed to remain in the solvent until the uppermost pigment band nears the top of the paper.

  • Teaching notes This experiment works very well providing care is taken over preparing the spot on the chromatography paper;
  • The yellow carotene spot with a higher RF value tends to move up the paper the furthest;
  • Ensure the propanone level is below the spot;
  • Instead, the energy is released as heat and light in a process called fluorescence;
  • This photograph shows the four main pigments separated from green plants using paper chromatography.

This photograph shows the four main pigments separated from green plants using paper chromatography. The primary pigments in green plants are chlorophylls, represented by chlorophyll a and b, which appear green.

  • The third from above dark green band represents chlorophyll a;
  • Take a measuring cylinder that contains 5ml of acetone and pour it into the mortar;
  • Because the pigments have been isolated from the thylakoid membranes of the chloroplasts, the energy cannot be used for photosynthesis;
  • Add five more drops of solution, letting each one dry before putting on the next.

Visible light, or white light, is made up of the colors of the rainbow. Some of these colors are absorbed "used" by pigments and others are reflected.

Separation of photosynthetic pigments by paper chromatography.

Pigments appear the color of the reflected light, so the chlorophyll pigments do not use the green portion of the spectrum. The other two pigments are types of carotenoids, which appear yellow, orange, or brown.

The top band of pigments in the separation are carotenoids called carotenes, most likely beta-carotene, and appear yellowish-orange. The second type of carotenoid separated in the experiment are xanthophylls, which appear bright yellowish and are most likely lutein. The "loading line" is the location of the original pigment line painted on the paper. A second experiment using the chloroplast pigment extract obtained using the methods described above can be easily done.

Green-colored pigment extract is added into a test tube. When a light is shone on the extract, pigment molecules absorb energy. Because the pigments have been isolated from the thylakoid membranes of the chloroplasts, the energy cannot be used for photosynthesis.

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Instead, the energy is released as heat and light in a process called fluorescence. The pigments flouresce at a lower energy level than what they absorb, so the chlorophylls flouresce a red color red has the lowest amount of energy of any of the colors in the visible spectrum.

Flourescence of the pigment extract is shown in the photo.