Pigment Separation with Chromatography Lab report
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Pigment Separation with Chromatography Conclusion
The purpose of this laboratory experiment was to separate pigments found in spinach using a microscale column. Those isolated pigments were then placed on a Thin-Layer Chromatography sheet and analyzed using TLC. The spinach pigment extract was a very dark, almost black color. When isolated using a microscale column eluted with 90:10 hexane:acetone solution, the β-carotene moved through the column easily and purely. However, once the 20:80 hexane:acetone solution was used to elute, the other, more polar pigments (xanthophyll, chl b, and chl a) moved much slower than the b-carotene did, and required 25-30 elution cycles until the pigments reached the bottom of the column (…)
Once all three pigments were separated, there were still colors left in the column, mainly a faint green color (which was assumed to be chl b. After that, TLC was used to separate the pigments further. This specific experiment’s TLC plate only had two visible colors from all of the pigments after the mobile phase moved through the stationary phase, and no colors under UV light. This is most likely because the pigments extracted with the microscale column were very dilute, except for b-carotene, which did show a yellow color on the TLC plate. The Rf values for the marks were 0.94 for the original extract and 1.00 for the β-carotene. Unfortunately, no other marks were found on the TLC plate. The initial microscale column was very successful, because the xanthophyll was differentiated from the chl a, but the TLC could have been better done if the pigment solutions were not as dilute.