Pigment Extraction and Chromatographic Analysis of Spinach Leaves for Photosynthesis Components

In this experiment, the pigments responsible for photosynthesis were extracted from spinach leaves. These pigments were separated into different fractions using column chromatography and subsequently analyzed using thin layer chromatography. By performing these procedures, the components were isolated based on their polarity, and thin layer chromatography was employed for the identification of organic components. The analysis of Rf values revealed that the hexane fraction contained carotenes and xanthophyll, the 75/25 hexane/acetone fraction contained xanthophyll, pheophytins, and chlorophylls, while the acetone fraction contained chlorophylls.

Results from the experiment, including spot travel distances and corresponding Rf values, were organized in Table 1. The data was grouped by test tube, and colorless spots, indicating those invisible under the UV lamp but leaving distinct marks after exposure to iodine vapors, were also recorded.

Table 2 presents Rf ranges and speculated components organized by test tube number. The speculation about components is derived from both sample color and the sequence of polarity, corresponding to the Rf ranges. A more in-depth analysis of the relative polarity of each component is provided in the discussion section of this report.

The discussion begins with an overview of the extraction procedure, emphasizing the importance of blending spinach leaves with a water, salt, and hexanes/acetone mixture to maximize surface area for efficient extraction.

The addition of salt aids in breaking down cell walls and chloroplast membranes while promoting the organic layer's separation. Centrifugation separates the aqueous, organic, and solid layers, followed by the addition of anhydrous sodium sulfate to eliminate excess water. Vacuum filtration removes the drying agent, allowing solvent evaporation and leaving behind the purified extract, which is redissolved in hexanes.

The purified extract contains xanthophyll, α-carotene, β-carotene, pheophytin A, pheophytin B, and chlorophyll. The unique structures of these components dictate their polarities. Carotenes are nonpolar hydrocarbons, xanthophyll has minimal polarity due to a hydroxyl group, pheophytins possess multiple functional groups creating dipoles, and chlorophyll is more polar due to the presence of a magnesium ion. The beta versions of these molecules are slightly more polar due to aldehyde functional groups.

Chromatography analyses on the alumina column involve eluting solvents of increasing polarities. Hexane separates carotenes and xanthophyll, as confirmed by TLC analysis. The 75/25 hexanes/acetone mixture isolates pheophytins, and pure hexane separates chlorophyll, resulting in distinct Rf values on the TLC plate. The observed differences in Rf values for the pheophytins' A and B variants are attributed to the polarity of their aldehyde groups.

A comparison with the pure extract on the TLC plate suggests that fraction 3 contains all components except carotenes. Ideally, fraction 2 should contain carotenes and xanthophyll, fraction 3 only pheophytins, and fraction 4 solely chlorophylls. However, discrepancies in spot numbers per fraction indicate likely timing errors during fraction collection. The premature replacement of the fraction 2 test tube with fraction 3 and the late introduction of the fraction 4 test tube led to the appearance of excess xanthophyll and chlorophylls in fraction 3, explaining the disparities in Table 1.