Separation Improvements with 2D LC

Significance of the Topic

Two-dimensional liquid chromatography (2D-LC) has become a critical tool in analytical chemistry for resolving complex mixtures, particularly in pharmaceutical impurity profiling and environmental analyses. By coupling orthogonal separation mechanisms in two sequential columns, 2D-LC significantly enhances peak capacity and resolution compared to conventional one-dimensional methods. The ability to isolate closely related compounds, including early-eluting polar species, supports regulatory compliance and quality control in drug development and environmental monitoring.

Objectives and Study Overview

This work evaluates the performance gains achievable with 2D-LC using a range of superficially porous Agilent Poroshell columns under both comprehensive and heart-cutting modes. A mixture of sulfa drugs serves as the test sample to demonstrate how different second-dimension column chemistries affect the separation of intentionally co-eluting analytes. The study compares reversed phase, phenyl-hexyl, cyano, polar-embedded and Bonus RP stationary phases, quantifying orthogonality and peak capacity improvements.

Methodology and Applied Instrumentation

The 2D-LC platform is built on the Agilent 1290 Infinity series and includes:

• G1329A 1200 Auto-sampler
• G1311A 1200 Quaternary Pump (400 bar)
• G4220A 1290 Infinity Pump
• G1316C 1290 Thermostatted Column Compartment with a 2-position/4-port valve (G4236A)
• G1315C 1200 DAD (1st dimension detector)
• G4212A Infinity DAD (2nd dimension detector)
• OpenLab C 1.04 with 2D control software

The columns screened (2.7 µm superficially porous) include:

• Poroshell 120 EC-C18 (100×2.1 mm)
• Poroshell 120 SB-C18 (50×3 mm)
• Poroshell 120 Bonus RP (100×3 mm)
• Poroshell 120 Phenyl Hexyl (50×3 mm)
• Poroshell 120 EC-CN (50×3 mm)
• Poroshell 120 SB-AQ (50×3 mm)

Two 2D-LC strategies were implemented:

1. Comprehensive LC×LC: the entire effluent of the 1st dimension is subjected to rapid cyclic gradients in the 2nd dimension and reconstructed to build a 2D map.
2. Heart-cutting 2D-LC: selected peaks from the first column are captured in a capillary loop via a counter-current fill/defill valve and eluted on the second column.

Main Results and Discussion

In comprehensive mode, up to a three- to four-fold increase in peak capacity was demonstrated by matching 2nd dimension runtime to the 1st dimension fractionation interval. The novel 2-position/4-port duo valve provided symmetric fill and flush, minimizing band broadening and eliminating the need for multiple valves or synchronization.

In heart-cutting experiments with co-eluted sulfa drug peaks, reversed-phase C18 vs. C18 yielded no additional resolution. However, switching to cyano and polar-embedded phases introduced π-π and polar interactions, resulting in clear separation. The Bonus RP phase exhibited the greatest hydrophobic subtraction model F-value (≈255 relative to Eclipse Plus C18) and delivered the most distinct selectivity, even under shallow gradients.

Benefits and Practical Applications

• Enhanced resolution of closely related impurities, improving analytical specificity in pharmaceutical QC.
• Increased peak capacity for complex environmental matrices, facilitating trace-level detection.
• Rapid cycle times in the 2nd dimension maintain throughput while amplifying separation power.
• Modular instrumentation and software enable flexible method development and automation.

Future Trends and Applications

Ongoing efforts will explore alternative mobile phases, including methanol and different buffer systems, as well as extending the approach to HILIC-based second dimensions for greater orthogonality. Integration with high-resolution mass spectrometry and advances in column chemistries promise to further expand 2D-LC capabilities in biologics, metabolomics and impurity profiling.

Conclusion

This study underscores the importance of selecting a chemically dissimilar second-dimension column to maximize orthogonality. The combination of superficially porous columns, a counter-current duo valve and optimized gradients delivers substantial gains in peak capacity and resolution, enabling more reliable separation of challenging analyte mixtures.

References

• Agilent Technologies. Agilent 1290 Infinity 2D-LC System; Publication 5990-5572EN.