Procedure
part 1: Preparation of a spinach extract
1. One-half bag of supermarket spinach leaves should be placed in a commercial blender with about 200 mL of water and ground thoroughly for 5-10 minutes, or until all of the leaves are blended.
2. Split the extract into two and filter each using a large Buchner funnel into a 500-mL filter flask. Use additional filter flasks and Buchner funnels as necessary and change the filter paper in the funnel when filtration slows due to clogging. You may need to add excess water to remove all of the spinach remnants from the blender.
3. Meanwhile, prepare 375 mL of petroleum ether/acetone solution in a ratio of 8:2. Pour about 300 mL of this solution into a 1000 mL separatory funnel along with an equal amount of the filtered aqueous extract. Cap and shake the funnel vigorously, remembering to occasionally vent the system by opening the stopcock. Add another 50 mL of the solution and shake again. An emulsion may form. Drain off the aqueous layer (bottom green layer) and then decant the organic layer (light green layer) into a 250-mL Erlenmeyer. Use about 75 g anhydrous sodium sulfate to dry the organic layer and to break up any emulsions. Allow to dry for at least forty-five minutes with regular swirling. You may need to add more sodium sulfate if the sodium sulfate layer looks caramelized due to wetness.
4. Filter a small amount of the combined organic extracts into a 250-mL round bottom flask using a funnel. Use a rotary evaporator to remove the solvent, then filter more organic extract into the round bottom flask and continue the evaporation process. Do not let the temperature of the bath exceed 45 C, although a temperature of 25-35 C should be sufficient to evaporate the petroleum ether/acetone (hot plate set between 2 and 3). The result is a dark green liquid (1-3 mL), which may be diluted with a small amount of 8:2 Petroleum ether/acetone solution and placed immediately on columns using a Pasteur pipette (One partner should finish the evaporation process while the other is setting up the column).
Part II – UV Visible Absorption Spectrum
You will record an absorption spectrum of chlorophyll using the stock solution. An absorption spectrum reveals the range of wavelengths at which the molecule absorbs light.
1. Turn on UV Visible Spectrophotometer (Lambda 19) and computer.
2. On the main menu, click on ‘UV Win Lab' icon.
3. When the methods page appears, select ‘chloro.msc' method. The parameters for recording the absorption spectrum of chlorophyll have been entered into this method. Note the start and end wavelengths that will be scanned.
4. Go to the ‘Sample' tab. Change the ‘sample info' to your email address.
5. Return to the ‘Scan' selection. Click on the ‘setup' key on the toolbar. Wait for the start button to turn green and click on it. Click on ‘ok' if the ‘methods info' box appears.
6. The program will ask you to insert the sample ‘blank'. Insert reference and sample cuvettes filled with heptane, into their respective slots (The reference cuvette goes in the back slot and the sample cuvette goes in the front one). If your cuvette is clear on two sides and frosted on the other two sides, make sure the clear sides of the cuvette are facing the open sides of the holder in the spectrophotometer. This means that the frosted side of both cuvettes should be facing you. Press ‘ok'.
7. After the background correction with the blank is complete, the program will ask you to insert the sample. Remove the sample cuvette and discard the solvent (heptane). Fill the cuvette to about 3/4 level with the stock solution. Return the cuvette to the sample slot, and click on ‘ok.' You should see the absorption spectrum of chlorophyll appear as the scan progresses.
8. After the sample has been read, click on ‘print' under the ‘file' selection. Make sure that the printer switch is set to ‘B' first.
9. Do not discard the stock solution in the cuvette. You will use this cuvette filled with the stock solution to record a fluorescence spectrum.
10. Record an absorption spectrum of the β-carotene stock solution, repeating the procedure described above
Part III: leaf structure:
Examine a cross section of a plant leaf.
What are the functions of different parts of the leaf?
What are some of the characteristics of plants living in different environments?
What are some adaptations of leaves to different water availability?
