A Spectrophotometric Analysis of the Absorption of Green Light Versus Red Light Absorption in Spinach Leaves
The goal of the experiment was to determine if green light had less ability to absorb than red light in spinach leaves. This was done by separating the photosynthetic pigments (chlorophyll a, chlorophyll b, carotene and xanthophylls) from one another using paper chromatography. The separated pigments were then analyzed for their absorption spectrum using a spectrographometer. When the data was graphed it clearly showed the higher rate of red light absorption over green light. These results along with previous research indicate the importance of red light in photosynthesis and the minor role green light plays.
The majority of life on Earth depends on photosynthesis for food and oxygen. Photosynthesis is the conversion of carbon dioxide and water into carbohydrates and oxygen using the sun's light energy (Campbell, 1996). This process consists of two parts the light reactions and the Calvin cycle (Campbell, 1996). During the light reactions is when the sun's energy is converted into ATP and NADPH, which is chemical energy (Campbell, 1996). This process occurs in the chloroplasts of plants cell. Within the chloroplasts are multiple photosynthetic pigments that absorb light from the sun (Campbell, 1996).
Photosynthetic pigments work by absorbing different wavelengths of light and reflecting others. These pigments are divided into two categories primary (chlorophyll) and accessory (carotenoids) pigments. Chlorophyll is then divided into three forms a, b, and c (Campbell, 1996). Chlorophyll a is the primary pigment used during photosynthesis (Campbell, 1996). This pigment is the only one that can directly participate in light reactions (Campbell, 1996). Chlorophyll a absorbs the wavelengths of 600 to 700nm (red and orange) along with 400 to 500nm (blue and violet) and reflects green wavelengths (Lewis, 2004). Chlorophyll b has only a slight difference in its structure that causes it to have a different absorption spectra (Campbell, 2004). The carotenoid involved with spinach leaf photosynthesis absorbs the wavelengths of 460 to 550nm (Lewis, 2004). The pigments are carotene and its oxidized derivative xanthophylls (Nishio, 2000). A wavelength is determined by measuring from the crest of one wave to the crest of the next wave. All the wavelengths possible are grouped in a range called the electromagnetic spectrum (Campbell, 1996). The range most important to life is from 380 to 750nm (Campbell, 1996). These wavelengths correspond to the wavelengths of visible light. Overall blue and red light works best, while green in least effective in the photosynthesis process (Nishio, 2000). The wavelengths that a pigment absorbs, absorption spectra, are determined using a spectrophotometer.
In order to obtain the photosynthetic pigment's absorption spectra the pigments are separated using paper chromatography. Paper chromatography is an analytical technique that separates a mixture based on the individual pigment's size, polarity and solubility (Lewis, 2004). The separation of the mixtures involves a stationary phase (the chromatography paper), which a mobile phase (solvent) moves up through. When the mixtures is applied to the paper and allowed to flow with the mobile phase, the different pigments move at different rates (Campbell, 1996). This means the pigments that absorb the strongest to the stationary phase (the chromatography paper) will move the slowest, while the weakest will move the fastest. The rate of the pigments movement will separate each pigment individually from the mixture (Maitland, 2002). This natural separation shows that each pigment is chemically different and plays different roles in photosynthesis (Maitland, 2002).
To analyze the separated pigments a spectrophotometer is used to obtain an absorbance spectrum. This spectrum is a graph that shows a pigment's...
References: Campbell, N.A., "Biology," New York: The Benjamin/Cummings Publishing Company, Inc., 1996, 182-200.
Karohl, D., "Principals of Biology I Laboratory," Lorain, Lorain County Community College, 2003, 65-71.
Lewis, R., "Life," Boston: McGraw-Hill , 2004, 97-114.
Nishio, J.N., "Why are higher plants green? Evolution of the higher plant photosynthetic pigment complement," Plant, Cell and Environment, 2000, 23, 539-5
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