In this laboratory you will separate plant pigments using paper 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, changing it from blue to colorless. (DPIP = 2,6-dichlorophenol-indophenol; Sigma Chem Co. ,www.sigma-aldrich.com/order, Cat. No. D1878, approx $20 for 5 gm).
Before doing this laboratory you should understand:
- how chromatography separates two or more compounds that are initially present in a mixture (see
- the relationship between light intensity and photosynthetic rate.
After doing this laboratory you should be able to:
- separate pigments and calculate their Rf values;
- describe a technique to determine photosynthetic rates;
- describe a technique for comparing photosynthetic rates at different temperatures, or different light intensities, or different wavelengths of light using controlled experiments; and
- explain why the rate of photosynthesis varies under different environmental conditions.
The first part of this lab time will be used to demonstrate paper chromatography. The second period of time will be used for the measurement of photosynthesis using the DPIP-reduction technique.
CHROMATOGRAPHY OF PLANT PlGMENTS
It was in the early 1940's that paper chromatography was invented by an observant person who saw application in how inks made rainbox hues when they "ran" on rain spattered paper. If you do not understand the principles behind "affinity" chromatography, you might find it helpful to look at an analogy.
Equipment
- Various leaves
- a coin (quarters and halves work well)
- chromatography paper
Remember to handle paper strips by the edges only. - solvent: 9 parts petroleum ether/l part acetone (5 mls/group). (Ordinary lighter fluid is petroleum ether!)
Place a leaf over a piece of chromatography paper and roll the edge of a coin over the leaf (using a ruler as a guide) so that the pigments of the leaf are driven into the chromatography paper 1.5 cm from the bottom. This will produce a straight line of pigment that can be chromatographed in a system such as:
The solvent of petroleum ether and acetone should be used in a fume hood or outside.
ISOLATION OF CHLOROPLASTS AND STUDY OF THEIR PHOTOSYNTHETIC ACTIVITY
0.1M Phosphate buffer: 174 g K2HP04 (dibasic) brought to 1 liter with distilled H20 and 136 g KH2P04 (monobasic) brought to 1 liter with distilled H20 (monobasic has a lower pH). Mix some monobasic with dibasic until the pH is 6.5 (try 685 mL of monobasic into 315 mL of dibasic). Since this solution is 1 M, 0.1 liter of the solution must be diluted with O.9 liter distilled H20 to prepare a 0.1 M solution.
Chloroplast suspensions: To prepare and prime the chloroplasts, incubate fresh spinach leaves under a light for a few hours. Do not allow the leaves to become hot. Pour 0.5-M cold sucrose into a blender so that it just covers the blender blades. This is probably 100 mL or 200 mL of solution. Pack fresh spinach leaves into the blender to a level one inch above the blades. Set up a beaker in ice with 2 layers of cheesecloth folded over a funnel. Blend spinach (about three short bursts - errrrrrr; errrrrr; errrrrrr!). Squeeze through cheesecloth into a large funnel leading into a large beaker that is placed in an ice bucket. The results should resemble those shown the accompanying graph.
Legend
diamond = unboiled/dark
square = unboiled/light
triangle = boiled/light
X = no chloroplasts- ALTERNATIVE: For sake of shortening the time spent here, the following qualitative technique may be used:
- Obtain three test tubes, and place all three in a test tube rack. To each tube, add 6 mL of distilled H20, 2 mL of phosphate buffer, and 2 mL of DPIP. To test tubes 1 and 2, add 4 drops of chloroplast suspension (some groups might make tubes of differing number of drops). Immediately place tube 1 in a dark cabinet. To test tube 3, add 4 drops of boiled chloroplast suspension. Place tubes 3 and 4 outside in daylight, or in the upstairs greenhouse if it is raining.
- Observe each tube and record the time needed for the tube to lose its blue color.
- Obtain three test tubes, and place all three in a test tube rack. To each tube, add 6 mL of distilled H20, 2 mL of phosphate buffer, and 2 mL of DPIP. To test tubes 1 and 2, add 4 drops of chloroplast suspension (some groups might make tubes of differing number of drops). Immediately place tube 1 in a dark cabinet. To test tube 3, add 4 drops of boiled chloroplast suspension. Place tubes 3 and 4 outside in daylight, or in the upstairs greenhouse if it is raining.
- Now look at the chloroplasts under a microscope; draw what you see.
- Question: Would you expect chloroplasts to migrate through a semi-permeable (dialysis) membrane? Explain you answer.
- Would you expect chloroplasts to diffuse and migrate up a paper chromatogram? Explain your answer in light of the fact that your chromatography showed that the colored bands did move up the paper.
I want to go to the TOP OF PAGE or ESCAPE! or go back to the Intro Lab's Home Page! or W&M course info
- ALTERNATIVE: For sake of shortening the time spent here, the following qualitative technique may be used: