Principles and Practice of Chromatography - The Control of Chromatographically Available Stationary Phase (V<sub>s</sub>) > Page 36

The Control of Chromatographically Available Stationary Phase (Vs)

The volume of stationary phase that is made available to the solutes can be controlled in a number of ways. Firstly, the stationary phase loading on the column can be varied to adjust the retention as required. A specific stationary phase loading may be selected, to either improve the resolution, or to reduce the analysis time, or in some instances, to increase the sample load. Sometimes, the stationary phase loading is reduced so the column is more amenable to specific compounds (e.g. to prevent proteins from being denatured).

Secondly, the stationary phase can contain molecules of a special shape that can only make close contact with molecules having a complementary shape. Other molecules can not interact so closely with the stationary phase and consequently, the stationary phase available to them will be restricted. This approach is exploited in chiral chromatography where the stationary phase is made to consist largely of a specific enantiomer that confers chiral selectivity to the distribution system

Thirdly, the stationary phase can be attached to the surface of a porous support, and the pore size chosen to be commensurate with the size of the solute molecules to be separated. Under such circumstances the molecules that are smaller than the pores will enter the matrix of the material and have more stationary phase available to them. Conversely, the larger molecules will be excluded from the pores and, consequently, come in contact with much less of the stationary phase. Size selectivity, achieved by the use of porous solids, is utilized in size exclusion chromatography (SEC) where solutes are separated almost exclusively on the basis of molecular size. The separation of chiral compounds can be successfully utilized in both GC and LC; size exclusion chromatography, however, is not greatly used in GC and is almost exclusively confined to LC.