Thermo Scientific Vanquish UHPLC Systems for Online 2D LC

Importance of Two-Dimensional Liquid Chromatography (2D-LC)

Two-dimensional liquid chromatography addresses the limitations of one-dimensional separations when analyzing highly complex samples or closely related isomers. By combining orthogonal separation modes in sequence, 2D-LC enhances peak capacity, resolution, and confidence in compound identification, making it essential for applications in pharmaceuticals, metabolomics, food analysis, and quality control.

Objectives and Overview

This presentation introduces the Thermo Scientific Vanquish UHPLC platform configured for online 2D-LC. It outlines user requirements, describes three versatile 2D-LC solutions—multi heart-cut with parking loops, single heart-cut with trap column (basic), and single heart-cut with trap column (advanced)—and demonstrates their performance in real-world applications.

Methodology and Instrumentation

The Vanquish UHPLC system integrates:

• Dual pumps and switching valves for precise heart-cutting
• Parking loops or trap columns for fraction collection and solvent modulation
• Thermo Scientific Viper fingertight fittings for leak-free connections
• Dual Split Sampler for advanced routing in dual-channel operation
• Chromeleon Chromatography Data System with Smart Instrument Integration for method control

Three configurations:

• Solution 1: Multi heart-cut using loops to collect multiple fractions in a single run
• Solution 2 (Basic): Single heart-cut with trap column, enabling eluent strength reduction before 2D separation
• Solution 3 (Advanced): Single heart-cut with trap column and Dual Split Sampler for simultaneous or independent 1D/2D operation

Main Results and Discussion

Demonstrations include:

• Antioxidant analysis in tea and olive oil: Multi heart-cut separated up to five coeluting compounds, illustrating enhanced resolution without modifying the primary 1D method.
• Herbicide isomer separation: Single heart-cut (basic setup) improved peak shape and resolved desmedipham and phenmedipham isomers via solvent modulation and trap column backflush.
• Simultaneous dual-channel operation (advanced setup) enabled independent LC-UV and LC-MS streams or full 2D workflows without manual replumbing, maximizing lab throughput.

Method flexibility was achieved through Chromeleon CDS workflows supporting hard-coded and smart-coded heart-cut logic, custom variables, and Intelligent Run Control.

Benefits and Practical Applications

Key advantages include:

• Comprehensive profiling of complex mixtures in pharmaceutical impurity testing, metabolomics, and environmental samples
• Seamless transfer of existing 1D methods to MS-compatible analysis without revalidation
• Space-saving single-system setup for 1D and 2D analyses
• Automated fraction collection and washing improve reproducibility and reduce manual intervention
• Configurable workflow supports both targeted heart-cut and exhaustive comprehensive 2D analyses

Future Trends and Potential Applications

Emerging directions include:

• Integration with advanced detectors (e.g., ion mobility, high-resolution MS) for multidimensional characterization
• Expansion of smart-coded method libraries via online repositories (AppsLab) for rapid adoption
• Miniaturized and microfluidic 2D-LC formats to reduce solvent consumption
• AI-driven workflow optimization to select optimal heart-cut points and gradient conditions in real time

Conclusion

The Thermo Scientific Vanquish UHPLC platform delivers flexible, high-performance online 2D-LC solutions tailored to diverse analytical challenges. By offering multi heart-cut and single heart-cut strategies in a unified system, it streamlines method transfer, enhances resolution, and maximizes instrument utilization, supporting the evolving demands of modern analytical laboratories.

Reference

• Grübner M., Steiner F. Complementary Dual LC as a convenient alternative to multiple heart-cut 2D-LC for samples of medium complexity. Thermo Fisher Scientific Technical Note 73184, 2019.
• Technical note TN73298: Flexible HPLC instrument setups for double usage as one heart-cut-2D-LC system or two independent 1D-LC systems (to be published).