Thin Layer Chromatography - Reversed Phases

Reversed Phases

Bonded phases, and in particular reversed phases, are now quite popular in TLC. Unfortunately, due to their strong dispersive (hydrophobic)character they exhibit poor wettability when used with solvent mixtures with high water content. The wetting characteristics of a number of commercially available dispersive phases have been reported (8) and the results are shown in table 1.

Table 1 Wetting Characteristics of Some Reversed Phases

Maximum Water Content to Permit Wetting

Reversed Phase Carbon Content (%w/w) Length of Alkane Chain Methanol % v/v Acetonitrile % v/v Isopropanol % v/v
ODS 5.0 18 100 100 100
ODS2 16.9 18 32 64 74
RP2 5.0 2 50 76 84
RP8 12.2 8 50 68 84
RP18 19.8 18 55 58 82

With the exception of ODS (which is a partially reacted silica carrying only 5% w/w of a C18 alkane chain) the reversed phases are not wetted by solvents carrying a high water content. RP2 that has only 2 carbon atoms per aliphatic chain bonded to the surface can tolerate 76% of water in the mobile phase when acetonitrile is the solvent and the stationary phase is still wetted. However, the other reversed phases can only tolerate about 65% of water if acetonitrile is the solvent and only about 50%, if methanol is the solvent. Poor wettability is not important in liquid chromatography, as the mobile phase is forced through the packed bed under pressure. In thin layer chromatography, however, the flow of mobile phase depends on the capillary attraction of the solvent between the particles and thus, if the particles are not wetted, there can be little or no flow of mobile phase.

Unfortunately, some of the most useful applications of reversed phases (particularly in the biotechnology field) requires the use of mobile phases carrying relatively high concentrations of water. Thus, the poor wettability of the reversed phases precludes much of their use in the more (theoretically) advantageous conditions for the separation of substances of biological origin. The results shown for ODS might suggest that a reversed phase produced with just enough silica remaining unreacted to render it wettable with pure water, might make a more useful material for thin layer chromatography. As the matrix is wetted, the mobile phase would flow across the surface driven by surface tension forces between the particles in the normal way. The silica (as opposed to the reversed phase) would be virtually completely deactivated by the water and, thus, offer minimal interaction with the solutes. Consequently, under such circumstances, the retentive mechanism between the stationary phase and the solutes would be due almost exclusively to dispersive interactions with the reversed phase.