Stationary Phase Limitation by Exclusion

Size Exclusion Chromatography might imply that solute retention was determined solely by the size of the molecule. However, this can only be true if the magnitude of the interaction forces between the solute and each phase is the same. This situation can be closely approached by the appropriate choice of the mobile phase. Under such circumstances the larger molecules, being partially or wholly excluded, will elute first and the smaller molecules elute last.

It is important to understand that, even when the dominant retention mechanism is controlled by molecular forces, if the stationary phase or supporting material contains pores of size commensurate with those of the solute molecules, exclusion will still partly control retention. This is because the larger molecules will interact with less stationary phase and be eluted relatively faster than if they had interacted with the same amount of stationary phase as the smaller molecules. The two most common exclusion media used in LC are silica gel and macroporous polystyrene divinylbenzene resins. Figure 19 shows an exclusion chromatogram of a series of molecular weight standards obtained on silica gel.

The column length was 50 cm and the mobile phase tetrahydrofuran (THF). The THF would be strongly adsorbed on the silica surface and, thus, the solutes would be distributed between pure THF in the mobile phase and THF on the surface of the silica. As a consequence, the interactions are virtually identical in the two phases and the retention was determined almost exclusively by stationary phase availability. The molecular diameter of solute standards were (1) 11,000Å, (2) 240Å, (3) 49.5Å, (4) 27.1Å and (5) 7.4Å.

Courtesy of Supelco Inc.

Figure 19 The Separation of a Mixture by Exclusion Chromatography

Until relatively recently, silica has been the most commonly used exclusion media for the separation of high molecular weight hydrocarbons and polymers. However, it was not so successful in the separation of polymeric materials of biological origin. More recently the micro-reticular macroporous polystyrene gels have been introduced and found to be very useful for the separation of biopolymers by size exclusion. These materials have even replaced many of the traditional applications of silica gel. In summary, in all types of chromatography, solute retention is controlled by either the magnitude and probability of interaction and/or by the amount of stationary phase that is available. However, even if, by appropriate choice of the phase system, the solutes are separated, unless the peak dispersion is constrained to allow the individual solutes to be eluted discretely, the components of the mixture may still not be resolved.