4th pd E. Perkins
In this lab, we were to separate pigments and calculate Rf values using plant pigment chromatography, describe a technique to determine the photosynthetic rate, compare photosynthetic rates at different light intensities using controlled experiments and explain why rate of photosynthesis varies under different environmental conditions. In the second part of the lab, we used chloroplasts extracted from spinach leaves and incubated then with DPIP and used the dye-reduction technique. When the DPIP is reduced and becomes colorless, the resultant increase in light transmittance is measured over a period of time using a spectrophotometer. If pigments are separated, then Rf values can be determined.
Paper chromatography is a useful technique for separating and identifying pigments and other molecules from cell extracts that contain a complex mixture of molecules. As solvent moves up the paper, it carries along any substances dissolved in it. The more soluble, the further it travels and vice-versa. Beta carotene is the most abundant carotene in plants and is carried along near the solvent front since it is very soluble and forms no hydrogen bonds with cellulose. Xanthophyll contains oxygen and is found further from the solvent front since it is less soluble in the solvent and is slowed down by hydrogen bonding to cellulose. Chlorophyll a is primary photosynthetic pigment in plants. Chlorophyll a, chlorophyll b, and carotenoids capture light energy and transfer it to chlorophyll a at the reaction center.
Light is part of a continuum of radiation or energy waves. Shorter wavelengths of energy have greater amounts of energy. Wavelengths of light within the visible spectrum of light power photosynthesis. Light is absorbed by leaf pigments while electrons within each photosystem are boosted to a higher energy level. This energy level is used to produce ATP and reduce NADP to NADPH. ATP and NADPH are then used to incorporate CO2 into organic molecules. In place of the electron accepter, NADP, the compound DPIP will be substituted. It changes chloroplasts from blue to colorless.
Obtain a 50 ml graduated cylinder which has about 1 cm of solvent at the bottom. Cut a piece of filter paper which will be long enough to reach the solvent. Draw a line about 1.5 cm from the bottom of the paper. Use a quarter to extract the pigments from spinach leaf cells and place a small section of leaf on top of the pencil line. Use the ribbed edge of the coin to crush the leaf cells and be sure the pigment line is on top of the pencil line. Place the chromatography paper in the cylinder and cover the cylinder. When the solvent is about 1 cm from the top of the paper, remove the paper and immediately mark the location of the solvent front before it evaporates. Mark the bottom of each pigment band and measure the distance each pigment migrated from the bottom of the pigment origin to the bottom of the separated pigment band and record the distances. Then, turn on the spectrophotometer to warm up the instrument and set the wavelength to 605 nm. Set up an incubation area that includes a light, water flask, and test tube rack. Label the cuvettes 1, 2, 3, 4, and 5, respectively. Using lens tissue, wipe the outside walls of each cuvette. Using foil paper, cover the walls and bottom of cuvette 2. Light should not be permitted inside cuvette 2 because it is a control for this experiment. Add 4 mL of distilled water to cuvette 1. To 2, 3, and 4, add 3 mL of distilled water and 1 mL of DPIP. To 5, add 3 mL plus 3 drops of distilled water and 1mL of DPIP. Bring the spectrophotometer to zero by adjusting the amplifier control knob until the meter reads 0% transmittance. Add 3 drops of unboiled chloroplasts and cover the top of cuvette 1 with Parafilm and invert to mix. Insert cuvette 1 into the sample holder and adjust...