Possibilities and Limitations of Aqueous Temperature Responsive Liquid Chromatography in Comprehensive 2D–LC

Abstract

In theory, comprehensive two-dimensional liquid chromatography (2D-LC) allows for significant enhanced peak capacity compared to one-dimensional high performance LC (1D-HPLC). However, reaching such a separation performance while also obtaining robust, easily implementable, and sensitive methods proves challenging. Because it can hinder the broader use of 2D-LC, there is a need for developing easier, more trouble-free approaches that feature the benefits of LC×LC while not compromising with what can be done with 1D-HPLC. Commercial 2D-LC interfaces are based on two-position multiport valves composed of two loops, which are alternatingly used either to collect the effluent from the first dimension (1D), or to inject their content to the second dimension (2D). This design implies that if large sampling volumes transferred to the second column need to be avoided, a comparatively (much) higher flow rate and broader column i.d. is required for the second column. However, doing so can lead to a loss in sensitivity because of dilution and impractical analytical chromatography as a result of the high flow rates involved. In most LC×LC column combinations, this problem is exacerbated thanks to the high eluotropic strength of transferred loop volumes. However, when the elution strength of the transferred solvent is very small, refocusing the analytes can be obtained, which allows the user to overcome such issues. For example, this is the case when a purely aqueous separation mode is combined with reversed-phase LC (RPLC) in the 1D and 2D, respectively. Temperature responsive LC (TRLC), which is an emerging LC mode requiring only water as the mobile phase and whereby retention is controlled via temperature only, is promising in this context. In this second installment about TRLC, we illustrate the unique benefits of the combination of this separation mode with RPLC in comprehensive 2D-LC. The potential of the approach is shown through the analysis of representative standard mixtures, active pharmaceutical ingredients, synthetic impurities and phenolics in natural products.