Extraction Of Chlorophyll From Spinach Lab Report

E5 & E6 Extraction of Chlorophyll from Fresh Spinach and Investigation of the Photochemistry of Chlorophyll

Chlorophyll a

Chlorophyll b

E5 - Extraction of Chlorophyll from Fresh Spinach
E6 - Investigation of the Photochemistry of Chlorophyll

The aim of this experiment is to investigate the photochemistry of chlorophyll. This experiment will be performed in two lab periods. In the first lab period you will extract chlorophyll, the green pigment in leaves, from spinach. In the second lab period you will investigate the interaction of light with chlorophyll. Read in Appendix B the reference section titled "Electronic Structure of Matter" copied from reference 1 before performing experiment investigating the photochemistry
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
Add sterilized sand to the column to form a 0.5-1.0 cm layer above the cotton. Using a pipette and bulb, rinse the sides of the column with a little petroleum ether to wash down any sand sticking to the sides of the column.
4. Add about 30 mL of petroleum ether to a second 150-mL beaker. Then add approximately 5 g of alumina (aluminum oxide) and swirl to make a slurry. To transfer the slurry to the pipette column, use a pipette with its narrow end broken off. Break the pipette carefully in a towel. The column will run quickly at first, so in order to prevent the alumina from drying out, hold a finger over the bottom of the column during the addition of the slurry. Pack 6 cm of alumina evenly. You may wish to mark off the 6 cm beforehand. Avoid any air bubbles in the packing of the alumina, and maintain the petroleum ether level above the level of the alumina.
5. In order to protect the surface of the alumina, add another 1.5-2 cm of sand. At this point, the packing should be just below the neck of the pipette.
6. Pre-weigh two clean, dry, 250 mL round bottom flasks. To weigh them, place the flasks on top of a beaker, and then subtract the weight of the beaker. You will rotary evaporate your carotene and chlorophyll extracts in these flasks.

Part III – Chromatographic Separation of