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 bandcompression 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.

Recent applications of retention modelling in liquid chromatography

“…Another objective of method optimization may be to maximize the sensitivity. One way to achieve this is by on‐column focusing, retaining all analytes at the column inlet by decreasing the solvent strength or by changing the temperature [56,57]. Rerick et al.…”

“…Rerick et al. [57] used an extended form of the NK model that included the effects of temperature. The model was extrapolated to predict retention in case cooling was employed.…”

Advances in ultra‐high‐pressure and multi‐dimensional liquid chromatography instrumentation and workflows

“…Another way of implementing thermal modulation interfaces was demonstrated by Verstraeten et al ., who studied the use of a low thermal mass heating sleeve as a preconcentration device and injector for the second dimension column of an LC×LC set‐up 131 . This concept was revisited by the Weber group, who demonstrated the use of a multiplicative thermal focusing device for on‐column focusing 132 …”

“… 131 This concept was revisited by the Weber group, who demonstrated the use of a multiplicative thermal focusing device for on‐column focusing. 132 …”