Page 320 - Chiral Separation Techniques

312 12 Sub- and Supercritical Fluid Chromatography for Enantiomer Separations

Modifier additives also play a role in method optimization and are typically added
to the modifier at concentrations less than 1 % (v/v). Additives can provide increased
efficiency by minimizing undesirable interactions between the analyte and the CSP,
and may be necessary to elute certain types of compounds. The type of additive
(acidic or basic) that will produce the best results depends upon the functionality of
the analyte [72]. Certain additives are strongly retained on the stationary phase, and
their effect may persist even after they are removed from the eluent [22]. The impact
of both modifiers and additives can also be affected by the proximity of the operat-
ing conditions to the critical point of the eluent [73].

12.5.3 Temperature

Temperature can also be used to optimize enantioselectivity in SFC. The selectivity
of most CSPs increases as temperature decreases. For this reason, most chiral sepa-
rations in SFC are performed at ambient or subambient temperatures [50, 74]. Sub-
ambient temperatures are particularly useful for compounds having low conforma-
tional stability [75]. Stringham and Blackwell explored the concept of entropically
driven separations [76]. As temperature increased, enantioselectivity decreased until
the enantiomers co-eluted at the isoelution temperature. Further increases in tem-
perature resulted in reversal of elution order of the enantiomers. The temperature
limitations of the CSP should be considered before working at elevated tempera-
tures.

12.5.4 Pressure

Changes in pressure typically have a greater impact on retention than on selectivity.
Most studies of CSPs have indicated little effect of pressure on stereoselectivity [28,
31]. However, Bargmann-Leyder et al. reported pressure-related changes in selectiv-
ity for an amylose-based CSP, though the magnitude of the pressure effect was not
the same for all the compounds studied [58]. Pressures in the range of 15–20 MPa
are common for chiral SFC.

12.5.5 Flowrate

Optimum flowrates are higher in packed column SFC than in LC. Flowrates as high
as 5.0 mL min –1 generally do not dramatically reduce efficiency in SFC [12]. Bier-
manns and co-workers reported the separation of β-blockers at a flowrate of 4.0 mL
–1
min , a rate eight times higher than the flowrate recommended for LC [56]. No
deterioration of column performance was observed.