Solvent used in the elution process would be the mobile phase and solvents of different polarity would have a significant impact on the separation due to the varying solubility of compounds in different solvents. Hexane, being the less polar solvent, interacts mainly with the less polar analytes but very slowly with polar analytes. Therefore using hexane at the start of the elution process allows the less polar compound to be eluted out first. After the complete collection of less polar analyte, the mobile phase was changed to the more polar hexane/ethyl acetate solvent, which has stronger interaction with the more polar component, allowing it to be eluted out faster. The change in solvents throughout the elution process would allow for an effective and efficient separation of the compounds β-carotene and chlorophyll in the crude extract of green leaves. The polar silica gel in the column is the stationary phase and acts as an adsorbent, depending on the affinity of the component towards the stationary phase. In general, the more polar component would have a stronger interaction with the stationary phase, and the less polar component would be eluted out first.
From the observations of the column chromatography, yellow S2 collected is β-carotene, indicating that
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This problem could have arisen from an overly dilute extract used when running the TLC. Prior to conducting TLC, the extracts S2 and S3 were used in the UV-vis spectroscopic analysis. Failing to consider the use of the samples for TLC afterwards, huge proportions of both extracts were disposed off after the spectroscopic analysis. Even though the remaining extract volumes were concentrated by evaporating off the solvent, the number of moles of compounds present were still not concentrated enough for clear visible spots to be obtained from the TLC