Lab+4+Plant+Pigments+and+Photosynthesis


 * Lab 4 Plant Pigments and Photosynthesis **


 * Overview **

In this laboratory you will separate plant pigments using chromatography. You will also measure the rate of photosynthesis in isolated chloroplasts. The measurement technique involves the reduction of the dye, DPIP. The transfer of electrons during the light-dependent reactions of photosynthesis reduces DPIP and changes its color from blue to colorless. Any color changes will monitored using a Spectrometer.


 * Objectives **

At the completion of this laboratory you should be able to
 * Understand the principles of chromatography.
 * Calculate R f values.
 * Design an experiment in which chromatography is used as a separation technique.
 * Describe how light intensity, light wavelength, and temperature can affect photosynthesis.
 * Design an experiment to measure how light intensity, light wavelength, and temperature can affect photosynthetic rates.


 * Part A: Plant Pigments **

Paper chromatography is a useful technique for separating and identifying pigments and other molecules from cell extracts that contain a complex mixture of molecules. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of solvent molecules to the paper and the attraction of solvent molecules to each other. As the solvent moves up the paper, it carries along any substances dissolved in it, in this case plant pigments. The pigments are carried along at different rates because they are not equally soluble in the solvent and because they are attracted to different degrees to the cellulose in the paper through the formation of hydrogen bonds. Examination of the molecular structure of the pigments may reveal chemical functional groups that help determine the pigment's solubility in the solvent. Four pigments are typically found in leaf extracts from plants. They are: Chlorophyll //a// (bluish green), Chlorophyll //b//, (yellow to olive green), carotene (orange-yellow), and xanthophyll (lemon yellow). The molecular structures of each pigment are shown on the next page. Note that the difference between chlorophylls is a slight difference in the functional group on the ring. Carotene and xanthophyll are also similar chemically, differing only slightly in their functional groups. __ Procedure __
 * Chromatography of Plant Pigments **

Students will work in groups of two in this section. Each group should do the following:

1. Each student should obtain a 6-10 cm strip of chromatography paper. Using a pencil (not pen), draw a baseline approximately 1.5 cm from the bottom of the paper. Try to touch the paper as little as possible because skin oils can interfere with the chromatogram development.

2. Using a coin, smash the leaf juice onto the pencil line. This will leave a green line of pigment.

3. Repeat the application of the pigment several times until a dark streak is present. Allow the paper to dry between applications and try to keep the streak as narrow as possible.

4. Each group will be given a chromatography development jar. Keep the jar sealed until ready to use. (Caution - avoid inhaling the solvent fumes from the jar). The solvent used for this experiment is a mixture of petroleum ether, a highly lipophilic solvent and acetone. (9:1) 5. Place the __completely dried__ chromatography paper into the jar and use the cork to hold the paper in place against the jar mouth. The chromatograph paper should hang so that the bottom touches the solvent in the bottom of the jar, but the pigment strip must be __above__ the solvent. Hold the strip next to the jar and determine were the length is satisfactory __before__ opening the jar. Each jar can hold both partner’s papers. 6. When the solvent is about 1 cm from the top margin of the paper (several minutes), remove the paper and immediately mark the location of the solvent front in pencil before it evaporates. Your instructor will help you identify the spots. Label your pigment spots (1-4) from top to bottom. Reseal the jar after removing the paper.

//Note –// Some individuals may find more than 4 spots. If more are present, adjust your data recording as needed.

__ Analysis of Results __ Calculate the R f (Rate of flow) value for each of the pigments using the formula:

R f = __distance pigment migrated__ distance solvent front migrated

//Note// - because R f is a relative value, there are no units.

__ For the Lab report __ Report your data by completing Table 1. Use the colors, R f values and molecular structure to match each spot with a pigment name.

Table 1

Minoriteam || Color || Rf Value || Pigment Name ||
 * Spot Number
 * APALA Interns** || Color || R f Value || Pigment Name ||
 * 1 || yellow-green || .25 || chlorophyll b ||
 * 2 || blue-green || .428571 || chlorophyll a ||
 * 3 || yellow || .571429 || xanthophyll ||
 * 4 || bright yellow || .96 || carotene ||
 * Spot Number
 * The Wolf Pack** || Color || Rf Value || Pigment Name ||
 * 1 || yellow green || .167 || Chlorophyll b ||
 * 2 || blue green || .25 || Chlorophyll a ||
 * 3 || light yellow || .5 || Xanthophyll ||
 * 4 || Bright yellow || .967 || Carotene ||
 * Spot Number
 * Alpha and Omega** || Color || Rf Value || Pigment Name ||
 * 1 || Pale-Green || .34285 || Chlorophyll b ||
 * 2 || Green || .52857 || Chlorophyll a ||
 * 3 || Yellow || .67142 || Xanthophyll ||
 * 4 || Dark Yellow || .94280 || Alpha-Carotene ||
 * Spot Number
 * Combo Team** || Color || Rf Value || Pigment Name ||
 * 1 || light green || .2428 || chlorophyll b ||
 * 2 || sea green || .3857 || chrolophyll a ||
 * 3 || misty yellow || .5714 || xanthophyll ||
 * 4 || pale yellow || .95 || carotene ||
 * Spot Number
 * 1 || olive green || .2027 || chlorophyll b ||
 * 2 || blue-green || .3378 || chlorophyll a ||
 * 3 || light yellow || .6081 || xanthophyll ||
 * 4 || orange-yellow || .9834 || carotene ||
 * Spot Number || Color || Rf Value || Pigment Name ||
 * 1 ||  ||   ||   ||
 * 2 ||  ||   ||   ||
 * 3 ||  ||   ||   ||
 * 4 ||  ||   ||   ||
 * Spot Number || Color || Rf Value || Pigment Name ||
 * 1 ||  ||   ||   ||
 * 2 ||  ||   ||   ||
 * 3 ||  ||   ||   ||
 * 4 ||  ||   ||   ||
 * Spot Number || Color || Rf Value || Pigment Name ||
 * 1 ||  ||   ||   ||
 * 2 ||  ||   ||   ||
 * 3 ||  ||   ||   ||
 * 4 ||  ||   ||   ||
 * Spot Number || Color || Rf Value || Pigment Name ||
 * 1 ||  ||   ||   ||
 * 2 ||  ||   ||   ||
 * 3 ||  ||   ||   ||
 * 4 ||  ||   ||   ||

Answer the following questions:

1. What factors are involved in the separation of the pigments? Does molecular weight appear to have a role in pigment separation?

2. What functional group differences separates the almost identical pigments of Chlorophyll //a// and Chlorophyll //b//?

3. Is it possible to have a R f value greater than 1.00? Explain.

4. Would the R f value of a pigment be the same if a different solvent were used? Explain.
 * Part B: Photosynthesis **

Light is a part of a continuum of radiation or energy waves. Shorter wavelengths have greater amounts of energy (e.g. high-energy ultraviolet rays can harm living cells). Wavelengths of light within the visible part of the radiation spectrum are used in photosynthesis. Visible light has enough energy to be useful but not enough energy to break chemical bonds. The photosystems found in the chloroplasts of leaf cells contain pigments that absorb light. These chloroplasts have two different kinds of pigment systems, **Photosystem I** (PS I) and **Photosystem II** (PS II). PS I contains a specialized type of chlorophyll //a// molecule called P700 (its absorption spectrum peaks at 700 nm), and PS II contains a specialized chlorophyll //a// molecule called P680 (its absorption spectrum peaks at 680 nm). When light is absorbed by leaf pigments, electrons within each photosystem are boosted to a higher energy level and the energy is captured in the chemical bonds of ATP and NADPH. These high-energy products are then used to incorporate CO 2 into organic molecules. In this experiment, a dye-reduction technique will be used. The dye-reduction experiment tests the hypothesis that light and chloroplasts are required for the light reactions to occur. In place of the electron-accepting compound NADP, a dye DPIP (2,6-dichlorophenol-indophenol), will be substituted. If the dye accepts electrons from the chloroplasts, it will become colorless. In this experiment, chloroplasts are extracted from spinach leaves and incubated with DPIP in the presence of light. As DPIP is reduced and becomes colorless, the resultant increase in light transmittance can be measured over time using a Spectrometer. Table 2 Boiled chloroplasts Light ||= 5 No Chloroplast || Cuvettes were set up with the contents listed in the table above. Cuvette 2 was covered with foil to prevent exposure to light. A spectrophotometer was used to measure the initial percentage of like transmitted through each cuvette. Cuvette 1 was used to calibrate and recalibrate the spectrophotometer. To see how the spectrophotometer in this experiment was set up and calibrated, see pictures A-C on the following pages.
 * = Amount of Solution ||= 1 Blank ||= 2 Unboiled chloroplasts Dark ||= 3 Unboiled chloroplasts light ||= 4
 * = Phosphate Buffer ||= 1 mL ||= 1 mL ||= 1 mL ||= 1 mL ||= 1 mL ||
 * = Distilled Water ||= 4 mL ||= 3 ml ||= 3 ml ||= 3 ml ||= 3 ml + 3 drops ||
 * = DPIP ||= 0 ||= 1 mL ||= 1 mL ||= 1 mL ||= 1 mL ||
 * = Unboilded Chloroplasts ||= 3 drops ||= 3 drops ||= 3 drops ||= 0 ||= 0 ||
 * = Boiled Chloroplasts ||= 0 ||= 0 ||= 0 ||= 3 drops ||= 0 ||

Go to Lab Bench to do the prodecure. **Lab Bench**

Results of the experiment are below:

Table 3


 * Cuvette || 0 minutes || 5 minutes || 10 minutes || 15 minutes ||  ||
 * 2 || 31.3 || 32.5 || 35.5 || 34.8 ||  ||
 * 3 || 32.7 || 54.5 || 63.7 || 65.1 ||  ||
 * 4 || 32.7 || 32.9 || 33.1 || 32.5 ||  ||
 * 5 || 31.3 || 31.3 || 31.3 || 31.3 ||  ||

1. What is the purpose of DPIP in the experiment? 2. What molecule, found in chloroplasts, does DPIP “replace” in this experiment? 3. What is the source of electrons that will reduce DPIP? 4. What was measured with the spectrophotometer in this experiment? 5. What was the hypothesis for this experiment? 6. What is the effect of darkness on the reduction of DPIP? 7. Why did this happen? 8. What is the effect of boiling the chloroplasts on the reduction of DPIP? 9. Why did this happen? 10. Why was there a difference in the percentage of transmittance between the live chloroplasts that were incubated in the light and those that were kept in the dark? 11. What conclusions can you make about this experiment?