Switching solvent and enhancing analyte concentrations in small effluent fractions using in-column focusing

A novel approach to achiveve solvent switching and focusing of sub-column-volume analyte fractions in liquid chromatography is presented. By altering the temperature between loading and elution in back-flush mode, solvent transfer of analytes and focusing occurs, provided that the analytes exhibit temperature dependent retention on a given trap column. When retention on the trap decreases with increasing temperature, which is almost always the case, the loading of the trap-column takes place at a higher temperature than the elution. This principle is demonstrated using three small aromatic molecules (toluene, p-xylene and benzophenone) on a capillary monolithic column. On this column, the analytes show a traditional van’t Hoff dependence on temperature with enthalpy effects of, −15, −16 and −18 kJ mol−1, respectively, for a mobile phase of 25% acetonitrile in water. The column was loaded at 110 °C, cooled in an ice bath and eluted in back-flush mode at 0 °C. When operated in this way, the analytes are transferred from the loading solvent to the elution solvent, achieving solvent switching. Substantial focusing can also be obtained if the desorption solvent is stronger than the loading solvent.