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.

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.