Thin layer chromatography

A common, simple, inexpensive and relatively fast method for the separation of phenolic compounds from a mixture is thin layer chromatography (TLC). A small amount of the extract (40-100 ^l) is applied approximately 2 cm from the bottom of a thin layer chromatography plate, which is a matrix (typically cellulose or silica gel) attached to an inert carrier material, such as glass or plastic. The solvent is allowed to dry, either at room temperature, under a gentle stream of nitrogen, or with the use of warm air (a hair dryer can be convenient for this purpose). Application of multiple small amounts with drying in between applications will result in tighter spots and better resolution later on. A sharp pencil can be used to scratch off the matrix in order to clearly delineate individual lanes running along the length of the TLC plate. This will avoid samples from mixing later on.

The TLC plate is then placed in a glass container with a solvent filled to approximately 1 cm from the bottom. The solvent will move to the top of the TLC plate as a result of capillary action. Since each compound in the mixture will have a unique way of interacting with the matrix and the solvent, some compounds will move faster towards the top of the TLC plate than others. The Rf -value is the ratio of the distance of the compound has migrated divided by the distance the solvent has migrated, and has by definition a maximum value of 1. The Rf -value tends to be constant for a given combination of compound, solvent, and matrix so that comparisons can be made between separations performed at different times. If a given compound is colored, it is easy to determine the Rf -value. For non-colored compounds staining methods are available (see section 1.3).

The identification of phenolic compounds separated by TLC is somewhat challenging. The most common strategy is to include a set of reference compounds on the TLC plate. These compounds are applied individually, and if the mixture contains any of the reference compounds, they can be identified based on the Rf -value. Note that this approach always leaves some room for uncertainty, because two different compounds can have the same Rf -value. Further characterization is necessary to establish compound identity with more confidence. This can be achieved by scraping off the area on the TLC plate where the compound of interest has migrated to, followed by solvent extraction of the matrix, and more detailed chemical analyses, such as, for example gas chromatography-mass spectrometry or mass spectrometry (see Section 1.5 and Chapter 5).

Below is a step-by-step protocol for TLC. In this example the goal is to separate anthocyanins isolated from flower petals.

1. Pick the petals and place them in 1 ml methanol acidified with 0.1 or 1% (v/v) HCl.

2. Let the petals remain in the methanol overnight in the refrigerator. Alternatively, the tissue can be gently crushed with a glass rod or pipette.

3. Remove the supernatant with a Pasteur pipette and put it into a clean glass vial or a microfuge tube. Keep the material in the dark and in the cold as the compounds may break down easily.

4. Cellulose TLC plates are recommended for the separation of anthocyanidins, but silica gel will also work. If you use silica gel plates, it is helpful to wash the plates first with a mixture of chloroform and methanol (1:1 v/v). Simply run the solvent up the plates and let them air dry. This helps to remove some of the debris that is often associated with the plates. The plates will work even if you do not take the time to do this, but using pre-washed plates often provides a cleaner separation of compounds.

5. Spot a thin, fine band of pigment on the plate. To see as many spots as possible you need to put quite a lot of material onto the plate. It is best to apply the pigments in a band, while removing the solvent with a gentle flow of nitrogen.

6. There are many possible carriers, most of them mixtures of organic solvents. Table 4.1 lists some options that can be used for the separation of anthocyanins from petals.

Table 4-1. Solvent mixtures for thin layer chromatograpy of phenolic compounds

Solvent mixture

Ratio

Layer

«-butanol-acetic acid-water

4:1:5

upper layer

acetic acid-HCl-water (Forestal solvent)

30:3:10

miscible

ethyl acetate-formic acid-water

85:6:10

upper layer

ethyl acetate-water-formic acid-HCl

85:8:6:1

upper layer

«-butanol-2N hydrochloric acid

1:1

upper layer

«-butanol-acetic acid water

4:1:1

upper layer

The oxidation reaction of phenolic compounds can be used for the purpose of detection. While the phenolic compound is oxidized, a reagent is reduced, and the reduction can be monitored by a change in color. Two common reagents are ammoniacal silver nitrate and the Folin-Denis reagent.

Reactions of phenolic compounds with ammoniacal silver nitrate result in the formation of metallic silver. A simple procedure is to mix equal volumes of 0.1 N NH4OH and 0.1 N AgNO3 and to apply this as a spray to a thin layer chromatogram at room temperature. The oxidized phenols appear as brown spots because of the silver. The reaction is, however, not specific to phenolic compounds. The Folin-Denis reagent, discussed in Section 1.1.1, and produces a blue color upon reaction with phenolic compounds.

An alternative way to visualize certain phenolic compounds is with the use of a hand-held UV lamp in the dark. The presence of the phenolic compounds can be observed by the fluorescence.

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