Increasing Peak Capacity Using the Agilent 1290 Infinity 2D-LC Solution

Importance of the Topic

Comprehensive two-dimensional liquid chromatography (LC×LC) addresses the challenge of resolving complex mixtures by greatly increasing peak capacity. Higher peak capacity reduces coelution, enhances detection confidence, and supports applications in environmental analysis, pharmaceutical impurity profiling, metabolomics, and food safety.

Study Goals and Overview

This overview evaluates the Agilent 1290 Infinity 2D-LC solution under a 60-minute gradient. A 69-component test mixture was analyzed by classical HPLC (3.5 µm particles), UHPLC (1.8 µm), and comprehensive LC×LC using identical system dead volumes. The aim was to quantify improvements in effective peak capacity and resolving power.

Methodology and Used Instrumentation

First-dimension separations were performed on an Agilent ZORBAX RRHD Bonus-RP column (2.1×150 mm) with either 3.5 µm or 1.8 µm particles at 0.1 mL/min and a 60-min organic gradient. Fractions (50 µL) were transferred every 0.5 min to the second dimension, using an Agilent ZORBAX RRHD Eclipse Plus C18 column (3.0×50 mm, 1.8 µm) at 2 mL/min with fast modulated gradients.

• Agilent 1290 Infinity Binary Pumps (G4220A) for both dimensions
• Agilent 1290 Infinity Autosampler (G4226A)
• Thermostatted Column Compartment and Thermostat (G1316C, G1330A)
• Agilent 1290 Infinity Diode Array Detector (G4212A)
• 2 position/4-port duo-valve for 2D-LC (G4236A)
• OpenLAB CDS ChemStation C.01.07 with 2D-LC software A.01.02
• GC Image LC×LC Edition for contour plotting

Main Results and Discussion

One-dimensional analysis on the 3.5 µm column yielded an effective peak capacity of ~210; replacing it with a 1.8 µm UHPLC column increased capacity to ~300 under identical gradient time and system volumes. Comprehensive LC×LC delivered an effective peak capacity of ~800, representing a 3–4× improvement. Orthogonality between reversed-phase dimensions provided ~75% surface coverage; however, undersampling (β≈4 for UHPLC conditions) limited the practical gain below the ideal theoretical product (~4 330).

Benefits and Practical Applications

By tripling to quadrupling peak capacity without extending run time, the Agilent 1290 Infinity 2D-LC solution dramatically lowers peak overlap in complex matrices. This capability is critical for trace-level compound screening, detailed impurity profiling in pharmaceuticals, and comprehensive environmental and food safety analyses.

Future Trends and Applications

Ongoing developments in modulation speed, novel orthogonal stationary phases, and tighter integration with high-resolution mass spectrometry will further boost practical peak capacity and identification power in 2D-LC workflows. Automated method optimization and real-time data processing will expand routine adoption.

Conclusion

The Agilent 1290 Infinity 2D-LC solution achieves a 3–4× increase in effective peak capacity compared to 1D HPLC and UHPLC methods within the same 60-min gradient. This enhancement offers a robust strategy for high-resolution separations of complex samples.

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