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Abstract:
In preparative chromatography often the solubility of samples to be separated is low in mobile phases suitable for the separation. Thus, large injection volumes have to be applied, if the same solvent is used for the injection, which leads to reduced resolution. To overcome such limitations in practice, often solvents with higher solubility are used for the injection. However, this introduces the danger of causing undesired effects of peak splitting1,2 and, less frequently, crystallization and blocking phenomena.
In this work a methodology is presented to evaluate the use of such an injection strategy. As an example the separation of D and L-threonine on a Chirobiotic-T stationary phase (Astec, U.S.A.) with ethanol/water mixtures as the mobile phases is considered. This methodology consists of:
- experimental determination of the adsorption isotherms of the solutes from overloaded chromatograms using a fast peak fitting method3 as a function of the mobile phase composition
- determination of the excess isotherm of the components of the mobile phase by perturbations at various mobile phase compositions
- determination of a best mobile phase composition by numerical optimization of injections of the solutes:
o dissolved in the mobile phase (i.e., cinj=2-16g/l - close to solubility limit)
o dissolved in water (cinj=150g/l)
An optimal mobile phase composition (25/75 water/ethanol) and optimal injection amounts have been identified. The results of the parametric study have been experimentally verified. For the example studied the high concentration injections in water outperformed by roughly 20% injections in the mobile phase used for the elution. This effect is mainly due to the fact, that much smaller injection volumes can be used to dose the same amount of threonine compared to the injections in the mobile phase. Finally, a discussion on the limits of the application of injections in a different solvent than for the elution will be given.
1P. Janderea, G.Guiochon, J.Chromatogr. 588 (1991) 1-14.
2K. Gedicke, M. Tomusiak, D. Antos, A. Seidel-Morgenstern, J. Chromatogr. A 1092 (2005) 142-148.
3A. Felinger, A. Cavazzini, G. Guiochon, J. Chromatogr. A 986 (2003) 207-225.
