Method Development for Analysis and Isolation of Chiral Compounds Using Immobilized Stationary-Phase Technology

The authors discuss the capabilities of immobilization technologies and the ability to use an expanded range of solvents for mobile-phase components and solvent dissolutions. This article is part of a special issue on APIs.
Sep 01, 2010
Volume 2010 Supplement, Issue 4

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 modes.

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.

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 3.

  • 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 selector.

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.

Table I: First set of solvents for mobile-phase selection.
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.

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.

Table II: Second set of solvents for method development.
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.

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.

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