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10 Cards in this Set
- Front
- Back
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Purpose
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To extract the chlorophyll and carotenoid pigments from spinach leaves using acetone as the solvent
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Pigments in spinach (decreasing in Rf order)
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Chracteristics of:
1. Chlorophyll a 2. Chlorophyll b 3. B Carotene |
1. Chlorophyll a
- Blue-green, polar 2. Chlorophyll b - Green, polar 3. B Carotene - Yello, nonpolar |
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Procedure
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1. Grind the spinach while adding cold acetone
*Cold acetone will dissolve pigments and water *Cellulose and other plant compounds are insoluble *Cold acetone because we only want to dissolve polar components including pigments *Acetone will dissolve pigments because theyre both consisted with nonpolar/polar parts 2. Centrifuge and transfer supernatant. Then add hexane to the supernatant *Aqueous layer: water, acetone and other polar cmpds *Organic layer: Hexane, pigments, trace acetone&H2O *Pigments will dissolve in hexane because it contains a lot of aromatic rings therefore has nonpolar parts 3. Dry water with Na2SO4 4. Evaporate to get rid of hexane and acetone |
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Picture of scheme
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Column Chromatography
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Solvents used:
1. Hexane 2. 95% Hexane / 5% Acetone 3. 70% Hexane / 30% Acetone 4. Acetone 5. 80% Acetone / 20% Methanol |
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How does column work?
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1. Nonpolar components, Carotene pigments eluted first
*Because polar alumina doesn't absorb it as much 2. Polar components, Chlorophyll pigments eluted after |
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Postlab Question #1
Why are the chlorophylls less mobile on column chromatography and why do they have lower Rf values than the carotenes. |
If one were to examine the structure of chlorophylls (both a and b) they are consisted with nonpolar cyclopentenes and polar ester, ketone and aldehydes. Column is prepared with polar alumina and when chlorophyll is run through the column it will interact with polar functional groups of chlorophyll molecules.
Similarly in TLC, polar parts of chlorophyll interacts with polar layer of absorbent and partially polar solvent in chamber; therefore, it will ascend slowly. |
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Postlab Question #2
Propose structural formulas for pheophytin a and pheophytin b |
Overall, the structure of pheophytins will contain less polar parts than chlorophylls or xanthophylls but more polar parts than carotene.
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Postlab Question #3
What would happen to the Rf values of the pigments if you were to increase the relative concentration of acetone in the developing solvent? |
If you increase the concentration of acetone in the chamber, then solvent would be too polar. All bands will ascend much faster because polar components won't interact with the solvent as much. It is impoartant that the solvent used in chamber is intermediately polar.
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