Multiplicative On-Column Solute Focusing Using Spatially Dependent Temperature Programming for Capillary HPLC.

Abstract

The benefits of capillary liquid chromatography columns are truly realized when small, limited sample volumes require signal enhancement, but the available sample volume does not permit on-column focusing during injection onto a larger column. This dilemma is common when samples are naturally small or precious (such as in biological, forensic, art, and archeological investigations) and analyte concentrations are low. Signal enhancement by solvent-based focusing is effective with capillary columns, but it is limited to a single band-compression step and can only be achieved at the inlet. Here we evaluate multiplicative temperature-assisted solute focusing using a linear array of ten independently controlled 1.0 × 1.0 cm thermoelectric cooling elements (TECs) to generate dynamic temperature changes along the length of the column. The evaluation has two prongs: simulation and experimental. Simulation is required to understand the effect of a particular temperature change at a particular place and time on the column to determine optimal timing of temperature changes. Because the accuracy of the simulations is good, as long as the effect of temperature on retention factor is known, experimental conditions required to achieve a particular focusing objective can be estimated. We evaluated the capability of the technique to selectively focus only one of two solutes. This was achieved using three adjacent zones with temperature controlled by (upstream first) four, two, and one TECs. The three focusing steps occurring on column gave a 20-fold increase in peak height without solvent-based focusing for a solute with modest retention enthalpy.