Leaf Chromatography (Rita Faynerman)

Student prior knowledge: Prior to performing this activity, students should know that the chloroplasts of plants capture the energy of the sun and convert it to chemical energy that is stored in sugar and other organic molecules through a process called photosynthesis. All green parts of a plant, including green stems and unripened fruit, have chloroplasts, but the leaves are the major sites of photosynthesis in most plants. Chlorophyll, the green pigment that gives leaves their color, resides in the thylakoid membranes of the chloroplast. It is the light energy absorbed by chlorophyll that drives the synthesis of organic molecules in the chloroplast.

Furthermore, students should know that light is a form of electromagnetic radiation. Wavelengths of electromagnetic radiation range from less than a nanometer (for gamma rays) to more than a kilometer (for radio waves). This entire range of radiation is known as the electromagnetic spectrum. However, the segment that's most important to life is the narrow band from 380 nm to 750 nm in wavelenth. This radiation is known as visible light because it can be detected as various colors by the human eye. Visible light is the radiation that drives photosynthesis.

Students should also know that that substances that absorb visible light are known as pigments. Different pigments absorb light of different wavelengths, and the wavelengths (colors) that are absorbed disappear. Thus, we see green when we look at a leaf because chlorophyll absorbs violet-blue and red light while transmitting and reflecting green light.

Explanation: The absorption spectrum of chlorophyll a alone underestimates the effectiveness of certain wavelengths in driving photosynthesis. This is because accessory pigments with different absorption spectra are also photosynthetically important in chloroplasts and broaden the spectrum of colors that can be used in photosynthesis. Thus, in addition to chlorophyll a, which absorbs violet-blue and red light, chloroplasts also contain the accessory pigments chlorophyll b and carotenoids (carotene and xanthophyll). A slight structural difference between chlorophyll a and chlorophyll b causes the two pigments to absorb at slightly different wavelengths in the red and blue parts of the spectrum. As a result, chlorophyll a is blue green and chlorophyll b is olive green. Carotenoids are other accessory pigments that are various shades of yellow and orange because they absorb violet and blue-green light. Besides broadening the spectrum of light that can drive photosynthesis, carotenoids also serve a photoprotective function: they absorb or dissipate excessive light energy that would otherwise damage chlorophyll or interact with oxygen, forming free radicals (reactive oxidative molecules) that are dangerous to the cell.

Paper chromatography is a process that uses special filter paper to separate and identify different substances in a mixture (chromatography means "to write with color"). The substances in the mixture dissolve in the alcohol (the solvent) and move up the paper. Because heavier substances travel up the paper more slowly than do lighter substances, which travel more quickly, paper chromatography results in a separation of the substancse in the mixture. The heavier substances remain toward the bottom of the paper, while the lighter substances travel to the top (polarity and solubility of the pigments also play a role). In leaf chromatography, the different sized pigments become separated as the solvent moves up the paper. A sample completed chromatogram is shown below:

The paper chromatogram reveals the presence of 4 different pigments in the leaf, each a different size and color.

Chlorophyll a = blue green

Chlorophyll b = olive green

Xanthophyll = yellow

Carotene = orange yellow

Different leaves will contain different amounts and types of pigment. Have students perform leaf chromatography on a variety of leaves of their choosing. Oak and maple leaves are especially good for this activity because they contain a large amount of xanthophyll and carotene pigment.

Questions & Answers:

1. Why are chlorophyll a & b green?

Chlorophyll a & b are green because they reflect green light.

2. Why do leaves change colors in the fall?

Leaves turn different colors in the fall because chlorophyll a & b are not being made. As these pigments dissipate out of the leaves, the accessory pigments become visible.

3. How could you predict the color a tree's leaves will turn in the fall?

You could predict the fall color of a leaf by separating out the pigments found in the leaf. The fall color will be a combination of the accessory pigment colors.

Applications to Everyday Life:

1. Color Change in Leaves: The presence of accessory pigments in green leaves explains why leaves change color in the fall. When chlorophyll a and b are no longer being made by the plant, the accessory pigments, which can range in color from yellow to orange to red, become visible.

2. Phytochemicals: Carotenoids, which play a photoprotective role in photosynthesis, also have a photoprotective function in the human eye. These molecules, often found in health food products, are valued as "phytochemicals", compounds with antioxidant properties. Plants can synthesize all the antioxidants they require, but humans and other animals must obtain some of them from their diets.

3. Color Vision: The retina of the human eye has two types of photoreceptive cells, the rods and cones. Rods are much more sensitive than cones, but cannot discern colors. As a result, scenery viewed under low light conditions appears only in black and white. The cones are stimulated by higher light intensities and are sensitive to either red, green, or blue light. A red stop signal produces light in the 700 nm wavelength range and causes the red cones to send a signal to the brain that we interpret as the color red.

Photographs:

https://picasaweb.google.com/109476389628005518561/November292011#5680350656330549522

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