The photosynthetic pigments are responsible for absorbing and trapping light energy in the early steps of photosynthesis. Before coming to lab develop a hypothesis relating to pigments and light (i.e. why are there several pigments in green leaves?). What benefits do the plants get by having several pigments? Also, consider what happens to leaves on deciduous trees in the Northern Hemisphere in the Fall and pose a hypothesis to explain this phenomenon. Which of the hypotheses developed can be tested using this experiment? Why? Be sure to include these hypotheses in the introduction of your lab report.

The major pigments of photosynthesis are the chlorophylls. The two chlorophylls found in green plants are chlorophyll a (chl a) and chlorophyll b (chl b). Certain other chlorophylls (chlorophyll c and bacteriochlorophylls) are found in non-green algae, protistans, and photosynthetic bacteria. Other pigments include carotenoids and phycobilins, sometimes referred to as the accessory pigments. Carotenoids occur in all photosynthetic organisms, while phycobilins occur in the red algae and cyanobacteria.

In this experiment, the photosynthetic pigments from spinach leaves will be extracted and separated using the technique of paper chromatography. After separating the pigments, their absorption spectra will be obtained using a spectrophotometer.

Chlorophylls

Chlorophylls are greenish pigments which contain a porphyrin ring. This is a stable ring-shaped molecule around which electrons are free to migrate. Because the electrons move freely, the ring has the potential to gain or lose electrons easily, and thus the potential to provide energized electrons to other molecules. This is the fundamental process by which chlorophyll captures the energy of sunlight. See Plant Pigments for an image of chlorophyll. As is the case for other compounds, the specific absorption maxima of any chlorophyll depends on the solvent in which it is dissolved.

Carotenoids

There are two classes of carotenoids, the carotenes and the carotenols. All carotenoids have long isoprenoid chains, with alternating double and single bonds. Structurally, the carotenes are composed entirely of carbon and hydrogen, whereas the carotenols also contain oxygen in the form of hydroxyl or keto groups. See Plant Pigments for an image of b-carotene.

Paper Chromatography

Chromatography is a technique used to separate the components of a mixture. There are various types of chromatography (column, paper, thin-layer, gas), but in all cases the separation is achieved by distribution of components between a fixed or stationary phase and a moving or mobile phase. In paper chromatography, the components of a mixture are separable into discrete zones on a sheet of filter paper.

The mixture is initially spotted or streaked near one end of the paper. If the separated substances are to be extracted later for further analyses, the procedure is called preparative paper chromatography. With a capillary tube, the mixture is streaked on the chromatography paper: enough sample is applied so that there will be an adequate amount for subsequent extraction and spectrophotometric analysis.

For ascending paper chromatography, the appropriate solvent is added to the bottom of a chromatography jar. The atmosphere in the jar should be saturated with solvent vapor prior to adding the paper. The paper is placed in the jar so that the streak is above the level of the solvent. Then, the solvent moves up the paper by capillary action, past the sample, toward the end of the paper.

During this process, termed development, the solutes separate and form a trail of discrete bands on the chromatogram. Separation of the components is usually measured by the R_f value. The R_f value is given by the equation:

For the numerator, the distance is measured from the origin either to the center or to the leading edge of each spot or band. The denominator is the distance from the origin to the solvent front. The Rf values can be used to identify the various solutes when the experimental conditions are very carefully controlled.

Analysis of Spinach Pigments

The photosynthetic pigments are extracted from spinach by grinding the leaves in acetone. The paper is then streaked with the spinach extract and suspended in a chromatography jar previously equilibrated with vapors of the solvent, a 9:1 mixture of petroleum ether and acetone.

When separation is completed, identify the pigment bands by their colors and relative positions on the chromatogram. The major pigments appear in 5 bands: in order, from the origin to the solvent front, they are chl b (olive-green), chl a (blue-green), violaxanthin (yellow), lutein (yellow), and b-carotene (yellow-orange). Click here to view images of the major photosynthetic pigments found in spinach.

Each pigment or pigment group will be eluted from the chromatogram by cutting out each band and soaking the strips of paper in acetone. Violaxanthin and lutein, the carotenols, will be combined and treated as a single group. The absorption spectrum of each pigment or pigment group will then be determined.

You will also perform a quantitative analysis for the two major pigments, chl a and chl b. The absorption coefficients (a) for chl a and chl b in 80% acetone are: for chl a, a₆₆₃ = 82.04, and for chl b, a₆₄₅ = 45.60. Using the Beer-Lambert equation you will determine the concentration (mg/ml) of each chlorophyll in a dilution of the eluted sample. Finally, you will calculate the ratio (chl a)/ (chl b), a value that is characteristic for each plant species.

Beer-Lambert equation:

• Experimental Procedures
• Sec 001 2004 data
• Sec 002 2004 data
• Previously Collected Data