Chromatography Photosynthesis requires the presence of special pigments that can absorb the energy of light. A pigment is a substance that absorbs light of a…

Photosynthesis requires the presence of special pigments that can absorb the energy of light. A pigment is a substance that absorbs light of a particular wavelength. Its colour depends upon the colour of light that it reflects. For example, a green substance appears green because it reflects green light and absorbs all other colours of light, especially red and blue.
The most important plant pigments in photosynthesis are the “chlorophylls”. Green plants contain both chlorophyll a and chlorophyll b. In addition to chlorophyll, the leaves of many green plants also contain one or more other pigments, including “carotenes”, which are orange, and “xanthophylls”, which are yellow. The presence of these other pigments is masked by the abundance of chlorophyll during most of the year.

The pigments in plant cells can be separated from one another by a technique known as chromatography. Chromatography is a technique for separating and identifying substances in a mixture, based upon their solubility in a solvent. It is one of the most valuable techniques chemists and biochemists use to determine ingredients that give flavour or scent, analyze environmental pollutants, identify drugs in urine, and even separate proteins that can identify evolutionary relationships. When a dye mixture is placed on a strip of chromatography paper and placed into a solvent solution, the individual substances in the mixture will migrate up the chromatography paper at different rates. The rate of migration is based upon the adsorption capacity of the chromatography paper and the solubility of the sample in the solvent. As the solvent moves up the chromatography paper strip substances in the mixture that are soluble in the solvent are carried along up the strip. On the other hand, the substances that are more attracted to the chromatography paper than to the solvent stop moving and form bands or spots along the paper strip. After chromatography, each band or spot on the chromatogram can be assigned a Relative Mobility Factor (Rf) which is characteristic of the specific substance that is associated with it. The Rf is a ratio of the distance a substance travels to the distance the solvent travels. It is calculated as follows:
Figure 1. Example of a chromatogram

The Rf value for a particular substance is constant with a consistent set of chromatographic conditions. Therefore, substances can be identified according to their Rf value when compared to a known standard. In this investigation, you will separate the individual pigments involved in photosynthesis.
Pre-Lab Questions (Lab adapted from WLHS)
1) What are the requirements for photosynthesis to occur?
2) Why do some trees appear green during the spring and summer, but then red / orange / yellow in the fall?
Most trees have green leaves in the summer containing the pigment chlorophyll. This pigment is also utilised to transform sunlight into energy for the tree. In summer, chlorophyll is frequently substituted in the leaves. When it gets cold, the plants stop producing chlorophyll, breaking it down into shorter pieces.
3) What are two factors that cause pigments to move at different rates during paper chromatography?
The solvent maintains the dissolved pigments as it proceeds up the paper. The pigments are maintained at different rates because they aren’t equally soluble. A pigment that is the most soluble will traverse the longest distance, and a pigment that is less soluble will move a smaller distance.
Goal: To determine using chromatography which pigments are present in fresh spinach leaves.
Materials and Methods:
· 2 pieces of chromatography paper
· coin
· pencil
· fresh spinach leaf
· 2 – large test tubes
· ruler
· marker
· 2 test tube stoppers
· scissors
· 70% isopropyl alcohol (rubbing alcohol) in acetone
1) Cut two pieces of chromatography paper that are each slightly longer than the test tube. Cut a point at the bottom. Using a pencil, draw a baseline 1.5 cm from the bottom of the long side of each rectangle.
2) Place a spinach leaf over the pencil line on one of the rectangles. Roll the coin over the leaf so that a horizontal green line is transferred to the pencil line.
**NOTE: you can’t have too much pigment! Make sure your line of pigment is dark!!
3) On the other rectangle, draw a marker line over the pencil line.
4) Add just enough isopropyl alcohol mixture to the test tubes to cover the bottom. Do NOT add more than 1 cm to ensure that the pigment line will not be submerged when the paper is lowered into the beaker. CAUTION: avoid inhaling the alcohol mixture
5) Lower each paper rectangle into the test tubes containing alcohol. The solvent will begin to move up the paper and cause the pigments to move as well.
6) Cover the test tubes lightly with stoppers. Do not disturb the test tubes for approximately 15 minutes, or until the solvent is about 1 cm from the top of the paper.
7) When the solvent is about 1 cm from the top of the paper, remove the paper and mark the farthest point of the solvent’s progress (front line) with your pencil before this line evaporates.
8) Allow the filter-paper strips to dry, and then make a sketch of each chromatograms. Some possible colors and the pigments they represent are:
faint yellow / orange – carotenes
olive green – chlorophyll b
yellow – xanthophyll
red – anthocyanin
blue green – chlorophyll a
(see sample chromatogram diagram on the last page for possible sequence results)
Table 1. Results of spinach leaf chromatography

Table 2. Results of ____________________ marker chromatography

Sketch your two chromatograms in the image here. Label the colored lines with their distance traveled from the origin, and the likely identity of that pigment.
Use the chromatogram shown here to complete the table below. 

Fig. 3 Sample Chromatogram
Table 3. Rf values for common photosynthetic pigments

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