Method Development in Comprehensive 2D-LC

Significance of the Topic

Comprehensive two-dimensional liquid chromatography (2D-LC) using reversed-phase separations in both dimensions offers a major advance in resolving complex mixtures that exceed the capabilities of one-dimensional LC. By combining two orthogonal reversed-phase (RPLC) systems and leveraging peak capacity multiplication, 2D-LC enables analysis of diverse analytes in environmental, pharmaceutical, or biochemical samples with improved resolution and quantitation.

Study Objectives and Overview

This work presents a systematic method development strategy for RPLC×RPLC to maximize the fractional coverage of the two-dimensional separation space. The approach begins with one-dimensional column and solvent screening guided by the Hydrophobic Subtraction Model (HSM) to identify pairs of columns with complementary selectivity. Correlation of retention factors is used to preselect low-correlation combinations. Selected column/solvent pairs are then evaluated in a comprehensive 2D-LC setup, and the fractional coverage—defined by a binning algorithm based on peak widths—is calculated. Finally, a shifted second-dimension gradient is introduced to enhance coverage and mitigate bandwidth suppression effects.

Experimental Methodology

A complex test mixture containing 70 compounds (polycyclic aromatic hydrocarbons, phthalate esters, phenones, and pesticides) was used to benchmark performance. Six reversed-phase stationary phases were screened with two organic solvents (acetonitrile and methanol) and variable pH mobile phases. Retention correlations and coverage metrics guided the selection of optimal column pairs. A modulation scheme with 80 μL loops and a 0.5-minute modulation time was applied to construct 2D chromatograms.

Used Instrumentation

• Agilent 1290 Infinity II 2D-LC system with dual high-speed pumps and multicolumn thermostats
• Agilent 6530 Accurate-Mass Q-TOF LC/MS for peak identification
• Agilent 1290 diode array detectors for UV detection
• OpenLAB CDS, MassHunter, and GC Image software for data acquisition and 2D analysis

Main Results and Discussion

One-dimensional screening identified the Agilent ZORBAX Eclipse Plus C8 with ammonium acetate/acetonitrile and the Eclipse PAH with ammonium acetate/methanol as the most orthogonal pair (R²≈0.96). Initial 2D-LC evaluation yielded ~18% fractional coverage. Implementation of a shifted second-dimension gradient tailored to first-dimension retention times increased coverage to over 50%, substantially improving peak distribution across the 2D space and resolving previously coeluting species.

Benefits and Practical Applications of the Method

This strategy delivers enhanced resolution of complex samples, supports high-throughput analyses, and is adaptable to various matrices. It enables comprehensive profiling in environmental monitoring, pharmaceutical impurity testing, and metabolomics.

Future Trends and Applications

Advances may include integration of machine learning for column selection, novel stationary phase chemistries, automated method optimization, and coupling with ion-mobility MS and multidimensional separations for deeper sample characterization.

Conclusion

A robust workflow combining HSM-guided screening, retention correlation analysis, fractional coverage metrics, and shifted gradients achieves high orthogonality and separation power in RPLC×RPLC. This methodology can be generalized to diverse complex-sample analyses.

Reference

• Rutan et al. J. Chromatogr. A, 2012
• Li et al. Anal. Bioanal. Chem., 2015
• Vanhoenacker et al. Agilent Technologies Technical Overview, 2015
• Giddings. Anal. Chem., 1984
• Carr & Stoll. Agilent Technologies Primer, 2015
• Graesboll et al. J. Chromatogr. A, 2014
• Allen et al. J. Chromatogr. A, 2014
• Bassanese et al. Talanta, 2015
• Gilar et al. Anal. Chem., 2005
• Davis et al. Anal. Chem., 2008
• Li & Schmitz. Anal. Bioanal. Chem., 2013