Thin layer chromatography (TLC) is a form of qualitative analysis called adsorption
chromatography used almost daily in synthetic organic chemistry labs. The composition of a
mixture can be assessed relative to known standards and separation of the mixture is roughly
based on polarity. In brief, a small portion of the sample is placed on the stationary phase
(polar silica) and is carried up the TLC plate by the mobile phase (solvent). A polar compound
will interact more with the polar stationary phase and will not travel as far up the plate.
Conversely, a less polar compound will move farther up the plate. Notice that this technique is
not terribly different from GC. Separation is based on polarity in both cases – relative boiling
points can be estimated from polarity. Mobile and stationary phases are still employed, except
that the states of matter are different. The principles of TLC will later be applied to column
chromatography, where larger sample volumes will be physically separated and isolated based
on polarity of the components. In this lab, students will use solvents of different polarity (mobile
phases) to determine the optimal separation of the pigments found in spinach extracts.
The most prominent types of plant pigments are chlorophylls, carotenoids, flavanoids,
and tannins. Chlorophylls contain a ring system formed by four pyrroles linked by four methine
bridges, a Mg2+ ion in its center, and a long nonpolar hydrocarbon chain (C20H39). Chlorophylls are
cousins of other biologically important molecules such as vitamin B12 and the heme found in
hemoglobin; they all have a tetrapyrrole ring system. There are two main types of chlorophylls
present in higher plants, a and b. Chlorophyll a is more abundant than chlorophyll b by a ratio of 3
to 1. The only structural difference between them is that a methyl group in a has been replaced by
a formyl group, CHO, in b.