Continuous step gradient elution for preparative separations

Abstract

A continuous step gradient elution process is suggested using several columns fixed in parallel to a multifunctional valve. The positions of the columns are changed in a periodic manner by shifting the valve at discrete times. Thus, the inlet streams into a certain column can be modulated in each time interval. Through one port, the feed is supplied whereas through the next port, eluent with increasing modifier concentration is dosed.

In this work, the principle was studied for a normal-phase high-performance liquid chromatography system using the separation of a binary mixture of two cycloketones as an example. In these experiments, four columns were used. The feed stream was introduced in a relatively weak solvent and then three gradient steps using stronger solvents followed. In order to study the concept quantitatively, a mathematical model describing the column dynamics was developed. In preliminary experiments, the most relevant thermodynamic and kinetic parameters of the chromatographic system have been determined. In particular, the dependence of the adsorption isotherms on the mobile phase composition was analyzed. The influence of extra column volumes on the process performance was also considered. Four gradient elution experiments were carried out varying the shape of the step gradients. Besides a modulation of the eluent composition, flow rate gradients have also been considered. In the experiments performed, the separation process turned out to be stable and reproducible. The experimental results were compared with the model predictions. Due to the relatively good agreement found, the model can be applied to design the step gradient process and to evaluate its potential compared to conventional isocratic elution.

Keywords:

• Preparative chromatography
• Continuous overloaded step gradient elution

Acknowledgments

The support of Schering AG (Berlin) and Fonds der Chemischen Industrie is acknowledged gratefully.