The Agilent InfinityLab 2D-LC Solution with Active Solvent Modulation

Significance of the Topic

Two-dimensional liquid chromatography (2D-LC) addresses the limitations of one-dimensional separations by improving peak capacity and resolving power for complex samples and coeluting analytes.

Aims and Study Overview

This overview presents active solvent modulation (ASM), a valve-based technique that balances solvent strengths between the first and second dimensions, and demonstrates its effectiveness in reversed-phase–reversed-phase (RPLC–RPLC) and hydrophilic interaction–RPLC (HILIC–RPLC) workflows.

Methodology and Used Instrumentation

Instrumentation:

• Agilent 1290 Infinity II 2D-LC System including two High Speed Pumps, Multisampler with cooler, Multicolumn Thermostats, Diode Array Detectors
• Agilent 1290 Infinity Valve Drive with ASM valve (G4243A) and Multiple Heart‐Cutting valves (G4242-64000)
• ASM capillaries of varying lengths and internal diameters to control dilution factors
• Agilent 6150 Single Quadrupole LC/MS with Jet Stream ESI source

Main Results and Discussion

RPLC–RPLC of polycyclic aromatic hydrocarbons:

• Without modulation, high organic content from the first dimension caused volume overload, broadening, and distortion of second-dimension peaks.
• Applying ASM with dilution factors of 1.5 and 2.0 restored focusing at the head of the second column, producing narrower, symmetrical peaks and enhanced sensitivity.

HILIC–RPLC of peptides:

• Direct transfer led to partial breakthrough of peptides due to solvent mismatch, as confirmed by matching mass spectra of retained and unretained fractions.
• ASM with factors of 3.0 and 5.1 eliminated breakthrough, yielding complete retention and well-focused chromatographic peaks across multiple heart cuts.

Benefits and Practical Applications

• Enhanced resolution, peak shape, and sensitivity for challenging 2D-LC separations.
• Seamless coupling of diverse solvent systems, expanding compatibility for reversed-phase and HILIC modes.
• Quantitative transfer and analyte focusing without extensive gradient redesign.
• Improved dynamic range and reproducibility for trace-level analysis in pharmaceutical, environmental, and proteomic applications.

Future Trends and Potential Applications

ASM is expected to drive broader adoption of comprehensive 2D-LC by simplifying method development and enhancing robustness.
Anticipated advancements include automated ASM factor optimization, real-time modulation control, integration with ultrahigh-resolution mass spectrometry, and miniaturized 2D-LC platforms for high-throughput screening.

Conclusion

Active solvent modulation effectively resolves solvent incompatibility in 2D-LC, enabling focusing at the second-dimension column head and delivering superior resolution and sensitivity across varied separation schemes.

References

1. Stoll, D. R.; Carr, P. W. Two-Dimensional Liquid Chromatography: A State of the Art Tutorial. Analytical Chemistry 2017, 89, 519–531.
2. Stoll, D. R.; et al. Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography. Analytical Chemistry 2017, 89, 9260–9267.
3. Li, D.; Jakob, C.; Schmitz, O. Practical Considerations in Comprehensive Two-Dimensional Liquid Chromatography Systems (LC×LC) with Reversed-Phases in Both Dimensions. Analytical and Bioanalytical Chemistry 2015, 407, 153–167.