Page 319 - Chiral Separation Techniques

12.5 Method Development in Chiral SFC 311

12.5 Method Development in Chiral SFC

Rapid method development remains one of the principal advantages of SFC for
enantiomeric separations, particularly when the analyst is faced with a structurally
diverse array of analytes. However, the applicability of SFC for a given separation
should be assessed prior to attempting method development. Berger [12] has sug-
gested that solubility of the analyte in methanol or a less polar solvent can be used
to gauge the likelihood of success. Compounds requiring aqueous conditions are
generally not good candidates for SFC [66].

12.5.1 Stationary Phase Selection

Column selection remains the most important factor in successful enantiomeric sep-
arations. The CSPs most likely to be effective in SFC are those that have been
employed under normal phase conditions in LC. In fact, the tremendous body of
knowledge that has been accumulated for LC can also guide column selection in
SFC [66]. The likelihood of success with a particular CSP can generally be gauged
after one or two injections [67]. If no evidence of separation is observed, another
CSP should be investigated.

12.5.2 Modifiers

The nature of the modifier and the modifier concentration impact both retention and
selectivity in packed column SFC. SFC offers considerable flexibility in modifier se-
lection because nearly all commonly used organic modifiers, including methanol and
acetonitrile, are miscible with CO . In contrast, methanol and acetonitrile are rarely
2
used as modifiers in normal phase LC because they are immiscible with hexane [68].
In general, retention decreases as the modifier concentration increases because the
modifier competes with the analytes for sites on the stationary phase. The effect on
retention of changes in modifier concentration seems to be more pronounced for
CSPs than for achiral stationary phases in SFC, and peak shapes are apt to degrade
rapidly at low modifier concentrations [12]. Efficiency tends to decrease as the mod-
ifier concentration increases because analyte diffusion is slowed by the increased
viscosity of the eluent [39].
Methanol remains the most widely used modifier because it produces highly effi-
cient separations, but it does not always produce the highest selectivity [8]. Recent
studies have provided insight into the role of the modifier in enantioselectivity in
SFC [69]. Blackwell and Stringham examined a series of phenylalanine analogues
on a brush-type CSP and developed a model that allowed prediction of selectivity
based on the bulk solvation parameters of various modifiers [70]. Careful choice of
modifiers can be used to mask or enhance particular molecular interactions and ulti-
mately provide control of selectivity [71].