Temperature-assisted on-column solute focusing: A general method to reduce pre-column dispersion in capillary high performance liquid chromatography

Abstract

Solvent-based on-column focusing is a powerful and well known approach for reducing the impact of pre-column dispersion in liquid chromatography. Here we describe an orthogonal temperature-based approach to focusing called temperature-assisted on-column solute focusing (TASF). TASF is founded on the same principles as the more commonly used solvent-based method wherein transient conditions are created that lead to high solute retention at the column inlet. Combining the low thermal mass of capillary columns and the temperature dependence of solute retention TASF is used effectively to compress injection bands at the head of the column through the transient reduction in column temperature to 5°C for a defined 7mm segment of a 6cm long 150μm I.D. column. Following the 30s focusing time, the column temperature is increased rapidly to the separation temperature of 60°C releasing the focused band of analytes. We developed a model to simulate TASF separations based on solute retention enthalpies, focusing temperature, focusing time, and column parameters. This model guides the systematic study of the influence of sample injection volume on column performance. All samples have solvent compositions matching the mobile phase. Over the 45–1050nL injection volume range evaluated, TASF reduces the peak width for all solutes with k′ greater than or equal to 2.5, relative to controls. Peak widths resulting from injection volumes up to 1.3 times the column fluid volume with TASF are less than 5% larger than peak widths from a 45nL injection without TASF (0.07 times the column liquid volume). The TASF approach reduced concentration detection limits by a factor of 12.5 relative to a small volume injection for low concentration samples. TASF is orthogonal to the solvent focusing method. Thus, it can be used where on-column focusing is required, but where implementation of solvent-based focusing is difficult.