Nathan Cheung Chem 12A

Laboratory 16: Isolation of Chlorophyll and Carotenoid

Objective: In this experiment, the pigments responsible for photosynthesis were extracted from spinach leaves, separated into various fractions using column chromatography, and then analyzed using thin layer chromatography. Performing these procedures allowed for isolation of the components into various fractions on the basis of polarity and analysis and identification of the organic components via thin layer chromatography. Data:

Distance solvent traveled: 9.7 cm Starting distance: 1 cm Chemical Carotene Pheophytin a Pheophytin b Chlorophyll a Chlorophyll b Travel distance (cm) 6.5 4 3 2.6 2.3 1.5 Xantophylls 2.0 1.2 0.6 Rf .74 .4597 .3448 .2988 .26436 .1724 .2298 .1379 .6896

Discussion: The extraction procedure first required that the spinach leaves be blended with a mixture of water, salt, and a 75/25 hexanes/acetone solution. Grinding the spinach leaves

into small particles maximized the surface area, allowing more solvent contact and thus a more efficient extraction. Separation of the aqueous, organic, and solid layers was achieved via centrifuging the sample, and anhydrous sodium sulfate was then added to remove any excess water (present because water is somewhat soluble in organic solvents, even if only by a little bit). The crystalline lattice structure of anhydrous sodium sulfate allowed it to absorb water to form a hydrate, thus removing any remaining water from the solution. Vacuum filtration was then used to remove the drying agent from the mixture, causing evaporation of the solvent, leaving behind only the purified extract (which was then redissolved in hexanes). The active components found in the purified extract included xanthophyll, -carotene and -carotene, pheophytin A and pheophytin B, and chlorophyll. The carotenes are linear carbon chains with phenyl groups on each end and methyl groups attached throughout, and are therefore simply nonpolar hydrocarbons. Xanthophyll is simply -carotene with a hydroxyl group attached, giving it a single dipole and minimal polarity. The pheophytins possess multiple ketone and amine functional groups, the structures of which create electrical dipoles. Chlorophyll is nearly identical in structure, but the presence of a magnesium ion in the chlorophylls makes it more polar than the pheophytins. The relative polarities of these molecules ultimately determined their behavior during the chromatography analyses performed. Hexane, the least polar eluent, was used first. Next, a 75/25 hexanes/acetone mixture. Lastly, the most polar eluent, pure hexane, was used to separate the relatively polar chlorophyll. During this lab we were unable to see the yellow band separate from the green band. This could be due to several errors. One possible error was that the cotton wasnt packed tightly enough and the solution was running through the column too quickly. The second error could be due to the column had been allowed to run dry and had cracked allowing the solution to run through the cracks and flow out.

Questions: 1. 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.

2. 3. 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. 4. We were not able to separate into two bands but supposedly, each band would contain more than one pigment.

Conclusion: Through the use of extraction and TLC, we were able to separate the pigments of a spinach leaf and calculate its Rf values (and thusly, its polarity).