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Extraction of Spinach

Samir Mohandes CHEM 243A-034 Instructor: Sean Campbell September 21, 2011

Abstract 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. Analysis of Rf values led to the conclusion that the hexane fraction contained carotenes and xanthophyll, the 75/25 hexane/acetone fraction contained xanthophyll, pheophytins, and chlorophylls, and the acetone fraction contained chlorophylls.

Results Table 1. Spot travel distances and corresponding Rf values. Sample data is grouped by test tube. Colorless spots refer to spots that were invisible under the UV lamp but left distinct marks after exposure to iodine vapors. Sample Solvent Fraction 2 Spot color N/A Yellow Colorless Colorless Gray Fraction 3 Gray Gray Dark green Light green Fraction 4 Dark green Yellow Colorless Gray Spinach extract Gray Gray Dark green Light green Travel distance (cm) 7.39 6.68 6.12 6.19 2.94 2.46 2.05 1.69 1.35 1.2 6.71 6.19 3.05 2.15 2.12 1.9 1.41 Rf N/A 0.904 0.828 0.838 0.398 0.333 0.277 0.229 0.183 0.162 0.908 0.838 0.413 0.291 0.287 0.257 0.191

Table 2. Rf ranges and speculated component arranged by test tube number. Speculated components are based on both sample color and polarity sequence, which corresponds to the Rf ranges. A more detailed analysis of each components relative polarity can be found in the discussion section of this report. Fraction 2 3 4 Spot colors Yellow Green Green Rf range 0.828 0.904 0.183 0.838 0.162 Speculated components Carotenes, xanthophyll xanthophyll, pheophytin A, pheophytin B, chlorophyll A, chlorophyll B Chlorophyll A, chlorophyll B


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. The salt served to create abrasion against the spinach cell walls and chloroplast membranes, causing them to decompose more thoroughly. Additionally, it decreased the solubility of the mixture, causing the active ingredients to favor the organic layer upon separation. 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 nominally so). 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 utilized to remove the drying agent from the mixture, causing evaporation of the solvent, leaving behind only the purified extract (which was then re-dissolved 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 respective beta versions of these molecules contain aldehyde functional groups, making them slightly more polar than their alpha counterparts. The relative polarities of these molecules ultimately determined their behavior during the chromatography analyses performed. On the alumina column, eluting solvents of increasing

polarities were used to separate the components in order of increasing polarities. Hexane, the least polar eluent, was used first to separate the carotenes and xanthophyll, the least polar components from the extract. TLC analysis confirmed these results; the fraction drawn from the alumina column using hexanes as an eluent traveled the farthest away from the polar stationary phase of the TLC plate, indicating that it was the most nonpolar of the samples tested. Two distinct spots were developed, presumably one each for carotenes (with a higher Rf, being nonpolar) and xanthophyll (with a slightly lower Rf due to the dipole created by the hydroxyl group). Next, a 75/25 hexanes/acetone mixturemore polar than pure hexanes but less polar than pure acetonewas used as an eluting solvent; as expected, this separated the pheophytins (the next most polar component) from the column, which gave lower Rf values than the carotenes and xanthophyll (Table 1). Lastly, the most polar eluent, pure hexane, was used to separate the relatively polar chlorophyll; correspondingly, the consequent Rf values on the TLC plate were the lowest of all samples. With both the pheophytins, the B variants developed separately lower on the plate than the A variants due to the polarity of their aldehyde groups. Comparison against the pure extract on the TLC plate seemed to indicate that fraction 3 contained all components except for the carotenes (Table 2). If separated ideally, fraction 2 would have contained only the carotenes and xanthophyll, fraction 3 would have contained only the pheophytins, and fraction 4 would have contained only the chlorophylls. However, the number of spots per fraction (Table 1) shows that this is not the case. In all probability, this was due to a series of errors in timing; the fraction 2 test tube was replaced with the fraction 3 test tube before the orange band had fully reached the bottom of the column, causing excess xanthophyll to appear in fraction 3, and the fraction 4 test tube was introduced too late, causing the chlorophylls to appear in fraction 3 as well, explaining the spot disparity in Table 1.