3A Extraction of Spinach Author: Holly Polk Instructor: Oleksandr Zhurakovskyi Organic Chemistry Lab 243A, Section 010 Date Work Performed: February 12 , 2009 Date Submitted: February 19, 2009 Abstract The objective of the lab is to isolate different fractions of spinach components using column chromatography and to analyze the different fractions by TLC. Column chromatography and different eluents will be used to separate compounds based on differences in partitioning between mobile and stationary phases. When the extracts were used for TLC, the most distinctive spots were of carotene and chlorophyll. The spinach extract had a spot at 0.86, corresponding with carotene which had spots with Rf factors of 0.79 and 0.86. The spinach extract also had a spot at 0.23, corresponding with chlorophyll which had a spot at 0.16.
Introduction The objective of the lab is to isolate different fractions of spinach components using column chromatography and to analyze the different fractions by TLC. Column chromatography will be used to separate the different components in different fractions based on the polarity of the different compounds. Another objective of this experiment is to isolate and separate the spinach pigments using differences in polarity to effect the separation. A TLC plate will be used for analysis of the different pigments in spinach. In the crude extract, the following components may be seen (in order of decreasing Rf values): Carotenes (1 spot) (yellow-orange), Pheophytin a (gray, may be nearly as intense as chlorophyll b), Pheophytin b (gray, may not be visible), Chlorophyll a (blue-green, more intense than chlorophyll b), Chlorophyll b (green), Xanthophylls (possibly 3 spots: yellow). The different compounds should rise to different heights on the TLC plates. The basis of the experiment focuses on pigments. The leaves of plants contain a number of colored pigments generally falling into two categories, chlorophylls and carotenoids. Chlorophylls a and b are the pigments that make plants look green. There are also carotenoids which are part of a larger collection of plant derived compounds called terpenes. Spinach leaves contain chlorophyll a and b and b -carotene as major pigments as well as smaller amounts of other pigments such as xanthophylls which are oxidized versions of carotenes and pheophytins which look like chlorophyll except that the magnesium ion Mg 2+ has been replaced by two hydrogen ions 2H + . Since the different components are colored differently, the separation can be observed visually.
Physical Constants Chlorophyll a Blue-green, polar C 55 H 72 MgN 4 O 5 M. W. 893.5026
Chlorophyll b Green, polar C 55 H 70 MgN 4 O 6 M. W. 907.4862
beta-Carotene--yellow, nonpolar C 40 H 56 M.W. 536.8824 Chlorophyll a C 55 H 72 O 5 N 4 Mg a-carotene xanthophyll
pheophytin Other Physical Constants: Compound name Structure Mole cular weig ht (g/m ol) Melting Point Celsius Boiling Point Celsius Safety considerations (if any) Iodine I–I 254
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