Thin Layer Chromatography - Selection of the Mobile Phase 2
To return to solute retention by polar interactions in the example employing silica gel as the stationary phase, to reduce retention, the polar interactions between the solutes and the mobile phase must be increased to compete with the polar interactions between the solutes and the silica gel. To do this a small quantity of a polar solvent such as ethyl acetate or isopropanol is usually added to the mobile phase causing the polar solutes to be eluted more rapidly. However, in this case, yet another factor will be included in the retention reduction. The increased isopropanol concentration will not only increase the polar interactions in the mobile phase but some will also be absorbed as a layer on the surface of the silica gel, thus, moderating (or deactivating) its surface. Consequently, in this example, the solutes are eluted more rapidly due, not only as a result of increased interactions with the mobile phase, but also due to reduced interactions with the stationary phase.
Correspondingly, counter ions must be employed in the mobile phase in ion exchange chromatography to interact with the solute and cause elution. As discussed previously, solute retention is reduced by increasing the concentration of the interacting moiety in the mobile phase (the counter ion). Changes in the pH of the mobile phase will also affect retention. Generally, a change in pH that increases the dissociation of the solute will increase retention whereas an opposite change in pH that will reduce the dissociation of the solute will decrease retention. The outcome, however, is complicated by the effect of pH on the dissociation of the ion exchange material itself. If a weak ion exchange material is being used as the stationary phase and the pH effects the dissociation of the ion exchanger, then the more dissociated it becomes the more charged ionic sites become available for interaction and the greater the solutes will be retained.
To identify a suitable solvent mixture as the mobile phase for a sample that is completely unknown, a good start would be to use a silica gel plate and try a range of n-hexane/methylene dichloride mixtures. If the spots do not move even at high methylene dichloride concentrations, start adding small quantities of ethyl acetate or isopropanol. Conversely, if the spots move too fast reduce the quantity of methylene dichloride. If sample solubility is a problem in dispersive solvents, use a reversed phase plate and start with an 80 : 20, methanol : water mixture. If the spots move too fast reduce the concentration of methanol. If they move too slowly, or do not move at all, increase the concentration of methanol. If the spots still do not move replace the methanol with acetonitrile.
These recommendations are basic and much oversimplified. However, if the solvent selection is developed in the manner suggested, if a rational approach is used based on the concept of molecular forces, as more results are obtained, a satisfactory separation will eventually be obtained. As the separations become more difficult, more and different solvents will be necessary, but again, the nature of these solvents can be educed from the type of interactions that are needed to produce the chromatographic selectivity or the differential migration rates that are required.
The successful selection of the optimum solvent mixture for TLC separations will become less time consuming with increased practical experience and as chromatography skills are developed and improve.