A Spectrophotometric Analysis of the Absorption of

Green Light Versus Red Light Absorption in Spinach LeavesA 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 light absorption versus wavelengths (Lewis, 2004). The spectrophotometer quantitatively shows what fraction of light is passing through a given solution (Nishio, 2000). A spectrophotometer works by shining a light of a known wavelength through a liquid sample. The light that passes through is measured with a light meter (photometer). The greater the amount of pigment present, the more light will be absorbed, leaving less light to be detected by the photometer (Campbell, 1996).
It is the goal of this experiment to show that spinach has less ability to absorb green light than red light. The experiment will conclude that green lights has a low absorbency because green is not a vital component of photosynthesis while, red light will have a high absorbency due to its significant role in photosynthesis (Campbell, 1996). This will be done using the proven method of paper chromatography to separate the pigments for analysis (Nishio, 2000). The analysis of the pigments will be done using the spectrophotometer to give an absorbency spectrum that clearly indicates the absorption rates of green versus red light.
Materials and Methods
The light absorbency in spinach leaves was tested on 30 September 2003 using a Principles of Biology I (BIOG 161) class from Lorain County Community College. In order to complete this experiment the protocol from Karohl was followed (2003). Due to the size of the class the individuals were divided into six groups for the chromatographic separation of the spinach pigments. The groups where then redrawn into five groups for the pigment analysis. Because of the cuvettes being to long for the particular brand of spectrophotometer being used an individual from each group needed to hold down the cover while reading the absorbance scale.
Once the chromatographic separation of the spinach pigment was concluded the chromatography paper was analyzed for the separation of the different photosynthetic pigments. Four distinct pigments where found. In order from the origin to the solvent front they are chlorophyll b (olive green), chlorophyll a (blue-green), xanthophylls (yellow) and carotene (yellow-orange). Figure 1 shows how the bands where arranged and their colors.
Figure 1: A drawing of the chromatography paper clearly showing the separation and colors of the photosynthetic pigments found in spinach leaves.

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Once all the groups where done with the spectrophotometric analysis of the their pigment the absorbencies where charted and then graphed out. The data (Table 1) was placed into a graph creating an absorbance spectrum (Figure 2). The graph shows that the pigments peak in absorbency at the red and blue wavelengths, while dipping to a low absorbency rate during the green wavelengths.

An absorption spectrum of the photosynthetic pigments show the different wavelengths of light an organism can use for photosynthesis (Campbell, 1996). On the absorbency spectrum (see Figure 2) for this experiment one can see that red light had a better absorption rate than green light. The absorption rate indicates that red light is absorbed for photosynthesis while green light is transmitted giving the spinach leaves their green color. This is consistent with research on how chlorophyll a absorbs red and blue light the best, making these two colors vital to photosynthesis (Campbell, 1996).
Chlorophyll a and b have a significantly higher absorption rate indicating their primary role in photosynthesis (see Figure 2). This is in line with the fact that chlorophyll a is the only pigment that can directly participate in light reactions (Campbell, 1996). The carotene and xanthophylls absorbed a slightly different set of wavelengths allowing the plant to have a wider spectrum of colors it can use for photosynthesis (Campbell, 1996). The data and earlier research concludes that spinach has less ability to absorb green light than red light.

Table 1: This data shows specifically the range of absorbency rates for the individual photosynthetic pigments at different wavelengths.
Absorbance Spectrum of Spinach Pigment Extract
WavelengthChlorophyll aChlorophyll bXanthophyllCaroteneTotal Pigment
Figure 2: The absorabancy spectrum shows how absorbent the photosynthetic pigments are at different wavelengths of light. Note: Green light is between 500 to 570 nm and red light is between 630 to 720 nm.

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