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

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.

(CHIRAL TECHNOLOGIES)

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.

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.

Sample dissolution solvent

Sample solubility is a key consideration in enantioselective high-performance liquid chromatography (HPLC) separations when scaling up from analytical, through semipreparative, to preparative columns. A wider variety of sample dissolution solvents can be used with a greater likelihood that sufficient sample solubility can be realized, thereby enabling higher sample loads. Chlorinated solvents, which are often preferred because they dissolve most organic compounds, are safe to use with the immobilized CSPs.

Studies have confirmed that chloroform, tetrahydrofuran, ethyl acetate, DCM, MTBE and acetone can be safely and effectively used as mobile phases and sample diluents with the immobilized CSPs. Dimethylsulfoxide can be used as sample solvent with a slight loss of column efficiency. Column 1 can be readily regenerated by flushing with dimethylformamide. Columns 2 and 3 can be regenerated using any solvent from the extended range listed in Table II.

Temperature range

The narrow temperature range tolerated by coated polysaccharide CSPs does not typically allow temperature to be used as a variable to control a separation. The immobilized CSPs are stable to at least 80 °C, giving an expanded temperature range, which makes temperature a variable worth investigating.

The effect of temperature on chromatographic separations is fairly well established. In general, increasing temperature increases column efficiency but decreases both retention and enantioselectivity. The decrease in selectivity will vary between compounds, and the rate of decrease depends on the difference in binding enthalpies of the enantiomers.

The effect of temperature on column efficiency depends on changes in mobile-phase viscosity, diffusion rates in the stationary phase, and kinetics of desorption. Some separations are improved using subambient temperature where the increased selectivity is sufficient to offset the loss in column efficiency.

Additives

Traditionally, diethylamine (DEA) is recommended as an amine additive for the analysis of basic compounds on polysaccharide phases. Studies conducted on Column 2 have shown that ethylenediamine, ethanolamine, and butylamine are likely to enhance the resolution and peak shape of basic compounds separated on this column when compared with the resolution obtained with the DEA additive. These additives provide enhanced compound resolution on Columns 1 and 3 only in specific cases where the compounds are relatively strong bases.

Conclusion

The robust immobilization technology for chiral-compound resolution provides, for the first time, the ability to use virtually any organic solvent as a mobile phase or mobile-phase component.

The ability to use a much wider variety of mobile-phase components, temperatures, and solvents for sample dissolution opens up new possibilities for investigating conditions to accomplish separations that cannot be obtained with coated CSPs. The indestructible qualities of the immobilized CSPs eliminate the need to take extreme precautions to avoid solvents that damage or destroy conventional columns.

Geoffrey B. Cox, PhD, is vice-president of technology and David W. Ellis* is senior manager of sales operations, both at Chiral Technologies, 800 North Five Points Road, West Chester, PA 19380, tel. 610.594.2100, fax 610.594.2325, dellis@chiraltech.com

*To whom all correspondence should be addressed.