Thin Layer Chromatography - Choice of Phase System in TLC

Choice of Phase System in TLC

The choice of the best phase system to employ in TLC, as with all chromatography techniques, is the most challenging and the most difficult. As discussed previously, in TLC, the complex elution processes that results from the frontal analysis of the eluting solvent complicates the separation. The choice is simplified, however, by the relative rapid separations that can be obtained by TLC and, thus, many separations can be rapidly carried out and a range of possible solvent mixtures can be quickly scanned. Furthermore as gradient development is not generally practiced in TLC (that is, gradient programming as used in LC) this is not a variable that normally needs to be optimized.

The approach to phase selection is more or less the same as that used in GC and LC. From the nature of the sample, the dominant interactive mechanism to control retention has to be identified and this will determine the type of stationary phase that is likely to be successful

Stationary Phase Selection

A selection procedure for a TLC stationary phase is represented diagramatically in table 2. If the solutes are ionic in nature (or can be made so by buffering the solution to a particular pH) then an ion exchange media is likely to be appropriate for the stationary phase. The table shows that there are three to choose from. Alternatively, the components of the mixture may have significantly different polarities in which case a polar stationary phase might be appropriate and the table indicates that in this case there are four different phases to choose from. However, this choice is much oversimplified. If the solutes are strongly polar (e.g. a group of peptides or perhaps proteins), despite being strongly polar, as a result of their complexity and the abundance of polar groups present in each molecule, they all might exhibit approximately the same polarity. As a result they would not be necessarily separated on a polar stationary phase. In fact, they may differ to a far greater extent in their dispersive characteristics and therefore might be better separated on a dispersive stationary phase. For simple polar molecules however, a polar stationary phase would be a logical choice. Finally, if the solutes differ largely in their dispersive interactive capability, then a dispersive stationary phase would be appropriate. The general type of stationary phase can thus be estimated but there remains a choice to be made within the groups of ionic, polar and dispersive stationary phases themselves.

Table 2. Stationary Phase Selection

Choice of a Specific Ion Exchange Stationary Phase

The different stationary phases shown in table 2 encompass both anionic and cationic type of phases together with strong and weak ion exchangers. However, they include ion exchange materials that possess interactive capabilities in addition to ionic. Considering first the type of ion exchange mechanism, obviously anions will be separated by an anion exchanger and cations by a cation exchanger. On the other hand, the choice of the strength of the ion exchanger will be determined by the strength of the ions to be separated together with the magnitude of their dissociation constants. Strong ions are nomally separated on weak ion exchange stationary phases and on the other hand weak ions are generally separated on a strong ion exchange stationary phases. The rationale for the choice of a specific ion exchange medium is shown in table 3. It is seen that the dominant interactive mechanism is not the only type of interaction that is to be considered in relation to the solutes that are to be separated. If the solutes are strongly dispersive in character, such as the fatty acids (e.g. the alkane sulfonates) then the separation could be augmented by dispersive interactions between the solutes and the stationary phase.

Table 3. Choice of Ion Exchange Stationary Phase

Ion Exchange Resin Ion Exchange Cellulose Ion Exchange Bonded Phase
Ion exchange resin has a matrix of a cross-linked styrene polymer which exhibits strong dispersive interactions. Ion exchange cellulose has a matrix of carbohydrate-type structures that exhibit strong polar interactions. Ion exchange bonded phases, although silica based, have closely associated alkane chains that can exhibit strong dispersive interactions.

In which case an ion exchange resin might be appropriate. Alternatively, if the ion exchange interactions could take place at intermediate pH values, then an ion exchange bonded phase would also provide dispersive interactions with the solute to augment the separation. (It should be recalled that bonded phases are unstable at extreme pH values). Conversely, if the solutes are strongly polar (hydroxy carboxylic acids) then the separation would need to be augmented by polar interactions in which case ion exchange cellulose might produce the necessary interactive polar groups and make the separation more effective.