Solvent Systems, Developing Chambers and Development

Next to the choice of the stationary phase (precoated layer), the choice of solvent system is the factor with the greatest influence on a thin-layer chromatogram. In only a few cases does the solvent system consist of one component only. Normally, mixtures of up to six components are used, and these must have the appearance of single-phase systems with no sign of cloudiness. 

The solvent system performs the following main tasks  

They should also fulfill the following requirements  

In the following three Sections, matters of practical relevance are at the forefront of our description of solvent systems. Theoretical treatments and discussions can be foimd in the literature [2, 4, 5]. 

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A further but also more time-consuming advance is to employ the AMD system (Camag). Here the mobile phase and mobile phase vapor is sucked out of the chamber and from the TLC plate after every development. This reduces to a minimum the contamination of the place of work with possibly injurious solvent vapors. 

FIGURE 6.10 The cover of the prepared chromatoplate for gradient development in a fully online horizontal chamber 1 — solvent system inlet, 2 — Silcoflon cover sheet, 3 — chromatoplate, 4 — solvent system outlet, 5 — channel for solvent system. (From Nyiredy, Sz. and Benko, A., Proceedings of the International Symposium on Planar Separations, Planar Chromatography 2004, Nyiredy, Sz., Ed., Research Institute for Medicinal Plants, Budakalasz, 2004, pp. 55-60. With permission.)... 

The selection of a mobile diase for the separation of simple aixtures may not be a particuleurly difficult problem and can be arrived at quite quickly by trial and error. Solvent systems can be screened in parallel using either several development chambers or a device like the Camag Vario KS chamber, which allows the simultaneous evaluation of a number of solvents by allowing each of these to migrate along parallel channels scored on a single TLC plate [8]. However, whenever the number of components in a mixture exceeds all but a small fraction of the spot capacity for the TLC system, a more systematic method of solvent optimization is required. 

A wide selection of solvent systems is available in the biochemical literature. If a new solvent system must be developed, a preliminary analysis must be done on the sample with a series of solvents. Solvents can be rapidly screened by developing several small chromatograms (2X6 cm) in small sealed bottles containing the solvents. For the actual analysis, the sample should be run on a larger plate with appropriate standards in a development chamber (Figure 3.3). The chamber must be airtight and saturated with solvent vapors. Filter paper on two sides of the chamber, as shown in Figure 3.3, enhances vaporization of the solvent. 

Develop the plate in hexane-diethyl ether-acetic acid (80 20 1) by plac mg the TLC plate in a chamber containing the solvent system, making sun. the edge with the applied samples is down. The solvent level in the cham ber must not be above the application spots on the plate. (Why ) Leave the chromatogram in the chromatography jar until the solvent front rises to about 1 cm from the top of the plate (45 to 60 min). Remove the plate and make a small scratch at the solvent level. Allow the chromatogram to dry (Hood ) and then place it in an iodine chamber for several minutes. Remove the plate and lightly trace, with a pencil or other sharp object, around each red-brown spot. This should be done promptly, as the colors will fade with time. Calculate the mobility of each standard and unknown lipid relative to the solvent front (R() ... 

Method 2 (silica gel layers). The silica gel chromatoplates are activated at 105 °C for 30 min before use. After cooling, the samples are applied at a distance of 2 cm from the bottom of the plate and then developed with an appropriate solvent system (for example, hexane-acetone (5 1) for organothiophosphorus compounds [165]). The plate is dried for 5 min at 105 °C and is placed while still hot in a chromatography chamber containing a small beaker of a 10% solution of bromine in carbon tetrachloride. After 10 sec the chromatoplate is removed, cooled and sprayed with a 0.05% solution of flsetin, robinetin or flavonol in ethanol. The plate is heated for 5 min at 105 °C in order to produce the fluorescence. The plates are then observed at a wavelength of 370 nm. 

Development. All the plates investigated here were each kept overnight at a relative humidity of 20. The plates were then developed to heights of 5 cm (HPTLC) and 7 cm (TLC) in normal chambers, without chamber saturation, using the appropriate solvent systems. 

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