Principles and Practice of Chromatography - The Control of Chromatographically Available Stationary Phase (V<sub>s</sub>) > Stationary Phase Limitation by Chiral Selectivity. > Page 38

The stationary phase loading of a an LC column is not modified the same way as a GC column. This is because, irrespective of the type of support material, the amount of stationary phase in an LC column is primarily determined by its surface area. In addition, the amount of available stationary phase on a bonded phase can be modified by adjusting the molecular size (chain length) of the bonded material.

The chain length of the bonded material can be critical when separating proteins as dispersive interactions between the alkane chains and the dispersive (hydrophobic) groups of the protein can be strong enough to cause structural deconformation; (i.e., the protein becomes denatured). Reducing the chain length of the bonded material, the dispersive forces can be reduced significantly and the deconformation diminished. In practice, carbon chains only two or four carbon atoms long are among those most commonly used for separating labile proteins.

Stationary Phase Limitation by Chiral Selectivity.

The extent to which an enantiomer can interact with the stationary phase depends on how close it can approach the molecules of the stationary phase. If the stationary phase is also chiral in nature, it is likely that one enantiomer in the sample will fit closely to the stationary phase surface whereas the other will be stearically excluded and thus have less stationary phase with which to interact. The first chiral separations in GC were reported by Gil-Av et al. as in 1966 (7), but, surprisingly, the use of GC for the separation of enantiomers has only recently been investigated and developed into a practical system. The use of chiral stationary phases in GC has been dogged by entantiomeric instability arising from the racemization of both the chiral stationary phase and the chiral solutes at elevated temperatures. In addition, at the elevated temperatures necessary to elute the solutes in a reasonable time, the chiral selectivity of the stationary phase can also be impaired.