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

In comprehensive two-dimensional liquid chromatography (LC x 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 x 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 x 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 similar to 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 mu L/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 x RPLC-UV-ESI-TOF-MS.

Automated summary

In this paper, a solution to the peak broadening problem in comprehensive two-dimensional liquid chromatography (LC x LC) using temperature-responsive liquid chromatography (TRLC) in the first dimension (1D) to reconcentrate the analytes is introduced. The combination of TRLC with reversed-phase liquid chromatography (RPLC) allows for full solute refocusing in the second dimension (2D) column. Experimental results show that using narrower columns in the 2D enhances sensitivity and enables split-free coupling of the 2D effluent with electrospray ionization mass spectrometry (ESI-MS).