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

Abstract

In comprehensive two-dimensional liquid chromatography (LC × LC), solvents of high eluotropic strength are frequently used in the first dimension ( 1 D), which lead to peak broadening in the second dimension ( 2 D). 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 1 D paired with a 3 mm (or broader) column in the 2 D 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 2 D. 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 1 D, which is a purely aqueous separation mode. Full solute refocusing at the 2 D 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 ∼1.5and 3-fold sensitivity enhancement when using a 1 mm I.D. column compared to 2.1 or 3 mm I.D. columns in the 2 D, 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 2 D is that it allows split-free coupling of 2 D 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.

Gain deeper insights into traditional Chinese medicines using multidimensional chromatography combined with chemometric approaches

“…For example, the use of parallel column arrays in the 2D dimension achieves complementarity between rapid sampling rates and 2D separation, maximizing peak capacity ( Foster et al, 2022 ). In another approach, detection limits were enhanced and the dilution problem was addressed in 2D-LC by combining a broader 1 D column with a narrower 2 D column ( Wicht et al, 2022 ). To overcome the issue of mobile phase incompatibility, researchers have proposed various strategies, including double column modulation, vacuum-assisted solvent evaporation modulation, solvent switch modulation, and on-line dilution modulation with bypass ( Chapel & Heinisch, 2022 ).…”

Formation of Micelles by Nonionic Detergent Molecules Leads to the Breakthrough Peak in Reversed-Phase Ultraperformance Liquid Chromatography (UPLC)

“…Typically, solvents of high eluotropic strength are used in the first separation (1D), which in turn impacts peak shapes in the second dimension (2D) and eventually affects detectability. 4 This breakthrough phenomenon in liquid chromatography has been noted for decades on both 1Dand 2D-LC systems. 1,5 Breakthrough phenomena are not limited to reversed-phase liquid chromatography (RPLC) columns; they can also occur in hydrophilic interaction liquid chromatography (HILIC) columns when the injection volume is relatively large.…”

“…Beyond the conventional one-dimensional (1D) approach utilizing a single column, two-dimensional liquid chromatography (2D-LC; LC × LC) is employed when a greater separation capability is required for complex samples. Typically, solvents of high eluotropic strength are used in the first separation (1D), which in turn impacts peak shapes in the second dimension (2D) and eventually affects detectability . This breakthrough phenomenon in liquid chromatography has been noted for decades on both 1D- and 2D-LC systems. , …”

Physicochemical modelling of the retention mechanism of temperature-responsive polymeric columns for HPLC through machine learning algorithms

“…The potential of TRLC has been exploited in many applications and used to solve numerous issues that would be present with other modes. It proved useful to use TRLC as a first dimension in comprehensive 2D-LC to overcome refocusing problems and increase sensitivity [6,7]. It was used with a refractive index detector (RID) to perform temperature gradient elution as an alternative to solvent gradient which is not possible with RID [8].…”