Flexible HPLC instrument setups for double usage as one heart-cut-2D-LC system or two independent 1D-LC systems

Importance of the Topic

Advanced two-dimensional liquid chromatography (2D-LC) significantly expands separation space by applying orthogonal separation mechanisms. Heart-cut 2D-LC offers targeted analysis of single or multiple fractions from a conventional one-dimensional (1D) LC run. In quality control and research laboratories, many LC-UV methods use nonvolatile buffers that prevent direct mass spectrometry (MS) coupling. Implementing elution strength reduction and trapping during heart-cut fraction transfer enables direct MS confirmation of peak identity and purity without modifying established 1D methods.

Goals and Study Overview

• Demonstrate flexible heart-cut 2D-LC configurations using the Thermo Scientific Vanquish UHPLC platform
• Highlight the Vanquish Dual Split Sampler for seamless switching between 2D-LC and two independent 1D-LC systems
• Compare chromatographic performance and operational flexibility of three fluidic setups

Methodology and Instrumentation

The proof-of-principle mixture comprised isomeric agrochemicals desmedipham and phenmedipham, which coelute under common 1D-LC-UV conditions with phosphoric acid in the mobile phase. Sample preparation involved 10 µg/mL solutions in acetonitrile/water (30/70 v/v).

Chromatographic conditions:

• 1D Stream: Hypersil GOLD C18, 2.1 × 100 mm, 1.9 µm; gradient from 90% aqueous (0.1% H₃PO₄) to 90% acetonitrile
• 2D Stream: Accucore PFP, 2.1 × 50 mm, 2.6 µm; gradient from 70% aqueous (0.1% formic acid) to 90% methanol
• Flow rates: 0.6 mL/min (1D), 0.3 mL/min (2D)
• MS Detection: Q Exactive Plus Hybrid Quadrupole-Orbitrap, positive full-scan 100–1000 m/z, 70 000 resolution

Instrument modules:

• Vanquish System Base with Quaternary and Binary Pump F
• Vanquish Dual Split Sampler FT
• Two Vanquish Column Compartments
• Vanquish DAD FG (1D) and VWD F (2D)
• Biocompatible 2-position/6-port valves and capillaries for fluidic routing
• Q Exactive Plus MS

Main Results and Discussion

Under non-MS-compatible 1D conditions, the two isomers produced a single sharp UV peak. Switching to volatile MS-compatible eluents enabled baseline separation in a conventional 1D test. All three heart-cut setups achieved complete transfer, trapping, and backflush elution with no significant changes in peak area or width relative to direct 1D injection. Setup comparisons:

• Setup 1 (trap column interface) offered simple implementation but limited parallel 1D usage and required low flow rates to protect detectors
• Setup 2 (trapping at 2D column head) enabled dual 1D-LC use but retained flow-rate limitations during trapping
• Setup 3 (trap column plus dual sampler and two column compartments) combined full elution strength reduction, direct 2D injection, parallel dual 1D-LC operation, independent temperature control, and robust valve logic

Setup 3 was identified as the most versatile configuration, supporting both heart-cut 2D-LC and two simultaneous 1D-LC workflows without manual replumbing.

Benefits and Practical Applications

• Direct MS confirmation of UV peaks from methods using nonvolatile buffers
• Flexible alternation between 2D-LC and two independent 1D-LC workflows increases instrument utilization and ROI
• Ability to develop 2D-LC methods via direct injection under MS-compatible conditions
• Automated dilution-assisted trapping reduces peak distortion and removes nonvolatile salts prior to MS
• Enhanced operational efficiency in QC, pharmaceutical, and industrial laboratories

Future Trends and Applications

• Integration of comprehensive and multi-heart-cut 2D-LC for highly complex samples
• Automated workflows with dynamic cut selection and intelligent run control
• On-column sample spiking or derivatization at trap interfaces for enhanced quantitation
• Expansion of column chemistries and dimensions for improved orthogonality
• High-throughput screening by combining dual samplers with rapid second-dimension cycles

Conclusion

Thermo Scientific Vanquish UHPLC systems, particularly with the Dual Split Sampler, deliver highly flexible heart-cut 2D-LC solutions. The optimal setup combines trap columns, dual samplers, and dual column compartments to enable both targeted 2D separations and parallel 1D-LC applications without reconfiguration. Chromeleon CDS offers advanced method scripting and intelligent run control, though basic functionality is supported in other control software. These capabilities streamline peak confirmation workflows and maximize laboratory productivity.

References

• 1 Guiochon G The limits of the separation power of unidimensional column liquid chromatography J Chrom A 2006 1126 6–49
• 2 Guiochon G et al Implementations of two-dimensional liquid chromatography J Chrom A 2008 119 109–168
• 3 Grübner M Steiner F Complementary Dual LC as an alternative to multiple heart-cut 2D-LC TN73184 2019
• 4 Stoll DR Carr PW Two-dimensional liquid chromatography A state-of-the-art tutorial Anal Chem 2017 89 519–531
• 5 Petersson P Haselmann K Buckenmaier S Multiple heart-cutting 2D-LC-MS J Chrom A 2016 1468 95–101
• 6 Stoll DR et al Evaluation of detection sensitivity in comprehensive 2D-LC Anal Bioanal Chem 2015 407 265–277