Enhanced Sensitivity in Comprehensive Liquid Chromatography: Overcoming the Dilution Problem in LC × LC via Temperature-Responsive Liquid Chromatography

In comprehensive two-dimensional liquid chromatography (LC × LC), solvents of high eluotropic strength are frequently used in the first dimension (¹D), which lead to peak broadening in the second dimension (²D). In the majority of the current LC × LC column combinations, analytes are less than optimally refocused upon transfer to the second column, which negatively affects sensitivity. Furthermore, the typical combination of 1 or 2.1 mm columns in the ¹D paired with a 3 mm (or broader) column in the ²D leads to at least a 9- or 4-fold dilution and a corresponding loss of sensitivity when using concentration-sensitive detectors. This occurs due to the enhanced radial dilution of the analytes in a broader column, while the sensitivity problem is further exacerbated in LC × LC due to the high flow operated ²D. In this paper, we introduce a solution to neutralize and inverse this dilution problem through a reconcentrating solution using temperature-responsive liquid chromatography (TRLC) in the ¹D, which is a purely aqueous separation mode. Full solute refocusing at the ²D column head is thereby obtained when TRLC is combined with reversed-phase liquid chromatography (RPLC). This is shown for the combination of a 2.1 mm I.D. TRLC column with decreasing RPLC column diameters (3–2.1–1 mm) operated at the same linear velocities, hence a resulting decrease in dilution, respectively. Ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection were used to determine the experimental detection limits. Sensitivity improvements with UV detection were somewhat lower than expected, but represent ∼1.5- and 3-fold sensitivity enhancement when using a 1 mm I.D. column compared to 2.1 or 3 mm I.D. columns in the ²D, respectively. This is attributed to extra-column dispersion and the poorer performance of 1 mm I.D. columns. A major benefit of the use of 1 mm I.D. columns in the ²D is that it allows split-free coupling of ²D effluent with ESI-MS (at 450 μL/min), making the coupling robust and simple. When using ESI-MS even better, albeit more variable, sensitivity enhancements were obtained on the narrower columns. The benefits of the methodology are demonstrated for paraben test solutes and for phenolic compounds in a blueberry extract by TRLC × RPLC-UV-ESI-TOF-MS.