Agilent 1290 Infinity 2D-LC Solution for Multiple Heart-Cutting

Significance of the topic

Two-dimensional liquid chromatography (2D-LC) with targeted heart-cutting addresses challenges in separating and quantifying trace analytes within highly complex matrices that exceed the resolving power of conventional one-dimensional LC. The Agilent 1290 Infinity 2D-LC solution for multiple heart-cutting introduces advanced interfacing technology that enables high-frequency sampling of 1D peaks, parking of multiple heart-cuts, and detailed secondary analysis for enhanced selectivity, sensitivity, and reliability.

Objectives and study overview

This technical overview presents the design, operation modes, and performance evaluation of the Agilent 1290 Infinity 2D-LC multiple heart-cutting system. Key aims include demonstrating improved chromatographic resolution, exploring software-assisted method development, and validating repeatable targeted quantification for trace compounds.

Methodology and instrumentation

The system configuration and experimental workflow comprised:

• First-dimension: Agilent 1290 Infinity Quaternary (G4204A) or Binary (G4220A) Pump, Infinity Autosampler (G4226A), Thermostatted Column Compartment (G1316C), optional Diode Array Detector (G4212A) with 10 mm flow cell;
• Interface: Agilent 2-position/4-port duo-valve connected to two 6-position/14-port valves (G4242A upgrade kit) with dual parking decks holding twelve 40 µL sampling loops;
• Columns: ZORBAX SB-C18, 100 × 2.1 mm, 1.8 µm for 1D at 40 °C; ZORBAX Bonus-RP, 50 × 2.1 mm, 1.8 µm for 2D at 40 °C;
• Solvents: 0.2 % formic acid in water (A) and methanol for 1D, acetonitrile for 2D (B); gradients and flow rates optimized for each dimension;
• Software: Agilent OpenLAB CDS ChemStation with 2D-LC Heart-Cut Viewer, gradient preview, peak- and time-based sampling modes.

Sampling modes included peak-triggered and time-based strategies to collect multiple heart-cuts, with automated flush cycles ensuring loop cleaning and result integrity.

Main results and discussion

High-frequency heart-cutting enabled parking of up to twelve aliquots with minimal valve switching, preserving chromatographic stability. The gradient preview tool simplified method setup by synchronizing 1D sampling times with 2D gradient shifts. Quantification studies using a 16-compound mixture demonstrated linear response from 0.5 to 1 000 µg/mL (R² > 0.999) and sensitivity gains in 2D achieved via a 60 mm UV flow cell. Repeatability tests (20 injections) yielded relative standard deviations below 10 % for individual components, improving to below 3 % when summing areas across cuts.

Benefits and practical applications

• Enhanced separation and detection of target analytes in complex samples;
• High-throughput sampling with simultaneous loop parking and 2D analysis;
• Improved sensitivity and extended dynamic range through optimized 2D gradients and detector configurations;
• User-friendly software tools for rapid method development and data evaluation;
• Versatile applications in pharmaceuticals, natural products, polymer additives, biopharmaceutical glycan profiling, and environmental analyses.

Future trends and opportunities

Integration with mass spectrometry and charged aerosol detection will further expand compound coverage. Advances in automated method optimization, machine learning–guided sampling strategies, and high-throughput workflows are expected to drive broader adoption. Emerging applications may include quality control in manufacturing, comprehensive omics profiling, and real-time process monitoring.

Conclusion

The Agilent 1290 Infinity 2D-LC multiple heart-cutting solution offers a robust platform for targeted analysis of complex samples, combining superior chromatographic resolution, high sensitivity, reliable quantification, and intuitive operation to enable routine 2D-LC workflows across diverse analytical fields.

Instrumentation used

• Agilent 1290 Infinity Quaternary Pump (G4204A) or Binary Pump (G4220A);
• Agilent 1290 Infinity Autosampler (G4226A);
• Agilent 1290 Infinity Thermostatted Column Compartment (G1316C);
• Agilent Diode Array Detector (G4212A) with 10 mm or 60 mm flow cells;
• 2-position/4-port duo-valve (5067-4170) and 6-position/14-port valves (G4242A);
• ZORBAX SB-C18 100 × 2.1 mm, 1.8 µm; ZORBAX Bonus-RP 50 × 2.1 mm, 1.8 µm;
• Agilent OpenLAB CDS ChemStation software with Heart-Cut Viewer.

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