This article is part of a special issue on API Development, Formulation, Synthesis and Manufacturing.
Enantiomers of chiral compounds can have dramatically different pharmacological activities, thereby making the ability to
assess and isolate pure enantiomers vitally important to pharmaceutical developers. Chiral chromatography is traditionally
used as the stereoselective separation technique, with polysaccharide-based chiral-stationary phases (CSP) as the media of
choice for more than 20 years.
Although these CSPs have unparalleled application range and versatility, their main limitation has been the inability to tolerate
certain solvents. Traditionally, chromatographers have needed to exercise extreme caution to avoid even small quantities of
incompatible solvents that can rapidly degrade or destroy a column.
Novel CSPs, based on proprietary immobilization technologies, were recently developed to safely accommodate virtually any
organic solvent as a mobile phase or mobile-phase component. The CSPs derived using these technologies exhibit stability,
separation reproducibility, and durability when used in normal-phase, reversed-phase, and supercritical fluid chromatography
The ability to use an expanded range of solvents for mobile-phase components and solvent dissolutions, as well as elevated
temperatures, offers new possibilities for investigating conditions for obtaining separations that could not be achieved on
solvent-restricted columns. These new capabilities demand a new approach to method development.
Immobilized solid-phase selection.
Immobilization technology was used to manufacture three types of columns, referenced here as Column 1, Column 2, and Column
- Column 1 has an immobilized CSP with a tris-3,5-dimethylphenylcarbamate derivative of amylose.
- Column 2 has an immobilized CSP with a tris-3,5-dimethylphenylcarbamate derivative of cellulose.
- Column 3 has an immobilized CSP with a tris-3,5-dichlorophenylcarbamate derivative of cellulose that includes a unique chiral
A statistical evaluation of a large number of chiral compounds indicated that, when used for screening, the three columns
will separate 95% of chiral components. Using only a few mobile phases in the screening process delivers separations with
exceptionally high success rates.
Mobile phase solvent selection.
The immobilized columns were thoroughly tested for stability to most common organic solvents, particularly those in which
the chiral selector is soluble. These tests indicate complete stability to these solvents. For convenience, a limited range
of solvents is recommended for initial screening, but ultimately, there are no restrictions on the solvents that can be used
as mobile-phase components.
Using immobilized technology effectively requires a new approach for method development. Table I lists a number of the primary
solvents that may be used in a screening process to provide successful separations. Conventionally, the process is begun by
using one of the mobile phases listed in Table I.
Table I: First set of solvents for mobile-phase selection.
Following analysis of the results, a weaker or stronger solvent composition is used to adjust retention of chiral compounds
to achieve reasonable analysis time. For example, if the compounds are eluted too quickly, a weaker mobile phase should be
used. Note that dichloromethane (DCM) and methyl-tertiary-butyl ether (MTBE) will destroy conventional, coated polysaccharide-based chiral columns and should only be used with the
new immobilized columns.
The solvents in Table II can be used in those cases where resolution is not obtained using the primary screening solvents
in Table I. Note, again, that the extended-range solvents will destroy conventional, coated chiral columns and should only
be used with immobilized columns.
Table II: Second set of solvents for method development.
In the reversed-phase mode, the columns should not be operated below pH = 2 or above pH = 7. The upper range of Column 1 and
Column 3 operations can only be extended to pH = 9, provided that a borate buffer and ammonium bicarbonate buffer are used,
and that the guard column is changed at least once every 200 injections at this pH.