Immobilizing otherwise unstable chiral selectors has great advantages, including the ability to separate enantiomers that
cannot be resolved on existing CSPs. The unique structure of Chiralpak IC often allows separations of different selectivity
from other polysaccharide-based CSPs. A combination of Chiralpak IC with Chiralpak IA and IB is replacing the older generation
coated phases. The three immobilized columns are complementary, and a separation can sometimes be found on only one column.
Figure 2 shows the example of prilocaine where the enantiomers are poorly separated with Chiralpak IA and IB but are well
resolved with Chiralpak IC (8).
Figure 2: Separation of the enantiomers of prilocaine. Green is Chiralpak IA, blue is Chiralpak IB, and red is Chiralpak IC.
Mobile phase is hexane–2-propanol 8:2; flow rate = 1 mL/min; column dimensions = 250 × 4.6 mm.
Immobilization of the chiral polymer increases solvent stability, which allows modification of the selectivity of chiral separations
by the modulation of the solvent composition and its components. Figure 3 shows the exploitation of the mobile-phase constituents.
Neither of the conventional mobile phases (hexane–2-propanol or hexane–ethanol) gave good resolution of the enantiomers of
indoprofen using Chiralpak IC (8). Dichloromethane-based mobile phase improved separation in a significantly shorter time.
Figure 3: Separation of indoprofen enantiomers. Column was Chiralpak IC, 250 × 4.6 mm. All mobile phases contained 0.1% trifluoroacetic
acid. Flow rate = 1 mL/min. Green is 30% ethanol in hexane; blue is 35% 2-propanol in hexane; and red is 2% methanol in dichloromethane.
Separation method development
The restrictions on solvents that applied to the early generation of coated polysaccharide CSPs made the development of separation
methods using them relatively straightforward. One screened with four mobile phase mixtures—hexane:ethanol, hexane:2-propanol,
acetonitrile, and 1:1 ethanol:methanol. Following this, the optimization also was simple because there were either "hits"
or not, and the opportunity for modifying the separations achieved were limited. This limitation is an important advantage
of these CSPs because the vast majority of separations can be found using these four solvents and only four CSPs—the tris-(3,5-dimethylphenylcarbamate)
of cellulose and of amylose, cellulose tris(4-methylbenzoate), and amylose tris(S)-α-methylbenzylcarbamate.
Using immobilized CSPs removes the restrictions of solvents to the extent where any organic solvent with a reasonable viscosity
is now accessible. This is a tremendous advantage in that it allows optimization of separations using solvent selectivity
effects not available for methods using the coated CSPs. Nonetheless, this also may complicate the development of separation
methods simply because of the bewildering array of solvents from which to choose.
To simplify the task, statistical studies have been conducted to assist in solvent selection and reduce the number that should
be tried to a reasonable few while retaining maximum resolution and selectivity. Of course, such statistical guidelines must
be based upon a wide range of applications to ensure their relevance, and there will always be some exceptions.
Two main sets of data, each based on the separation of a different set of 70 compounds, have been developed. Each set was
screened using a wide range of solvents, and the success rates arising from each were determined.