Agilent InfinityLab 2D-LC Solutions

Significance of the Topic

Two-dimensional liquid chromatography (2D-LC) offers enhanced separation power for complex mixtures that challenge conventional one-dimensional methods. It enables the resolution of coeluting isomers, accurate determination of trace impurities, and analysis of difficult matrices. Implementing 2D-LC improves productivity and data quality across pharmaceutical quality control, biopharmaceutical characterization, forensic toxicology, food analysis, and research applications.

Objectives and Study Overview

The Agilent InfinityLab 2D-LC Solutions aim to streamline complex separations through an integrated hardware and software platform. Key goals include:

• Providing flexible 2D modes (heart-cutting, multiple heart-cutting, comprehensive 2D-LC, high-resolution sampling).
• Maximizing peak capacity and orthogonality to resolve challenging compounds in a single automated run.
• Simplifying workflows by eliminating offline steps such as manual sample preparation, desalting, and eluent exchange.
• Allowing fast method setup and intuitive data analysis through dedicated OpenLab software.

Methodology

The approach combines two chromatographic dimensions connected by innovative switching valves. The first dimension separates the bulk sample under ultrahigh-performance liquid chromatography (UHPLC) conditions. Selected fractions are transferred to the second dimension for high-resolution analysis or desalting. Modes include:

• Heart-cutting: targeted transfer of specific peaks.
• Multiple heart-cutting: sequential sampling for impurity profiling.
• Comprehensive LC×LC: full chromatogram transfer for maximum peak capacity.
• High-resolution sampling: narrow fraction zones to separate broad peaks such as biopharmaceutical variants.

Used Instrumentation

• Agilent 1290 Infinity II High Speed Pumps: precise, low-delay volumes for fast gradient delivery.
• Agilent 1290 Infinity II Multisampler: automated sample introduction.
• Agilent 1290 Infinity II Diode Array Detectors: UV detection in both dimensions.
• Agilent 1290 Infinity II Multicolumn Thermostat: temperature control for reproducible separations.
• Dedicated 2D-LC switching valves: single and multiple heart-cutting valves with symmetrical flowpaths and Active Solvent Modulation (ASM) valve for solvent compatibility.
• OpenLab CDS ChemStation Edition: easy 2D-LC method setup, shifted gradients, and 2D-LC Viewer for chromatogram browsing and quantitation.

Main Results and Discussion

The InfinityLab 2D-LC platform demonstrated:

• Orthogonal separation power to resolve coeluting isomers and hidden impurities in pharmaceuticals.
• Automated desalting for MS compatibility, enabling direct analysis of peptides and synthetic oligonucleotides with high sensitivity.
• Reduction of ion suppression in forensic cannabinoid analysis, improving quantitation without extensive sample cleanup.
• Fingerprint profiling of complex beverages using comprehensive 2D-LC, distinguishing subtle compositional differences in beer varieties.
• Simultaneous aggregation and charge variant profiling of monoclonal antibodies via high-resolution sampling.
• Streamlining of pharmacokinetic drug–metabolite workflows, reducing analysis time by up to 75% and minimizing sample consumption.

Benefits and Practical Applications

• Enhanced resolution and peak capacity in a single automated run reduces method development time and potential errors from manual handling.
• Versatile application across regulated and research laboratories, including QA/QC, biopharma, toxicology, and food quality analysis.
• Seamless integration of hardware, software, and services ensures reproducible performance and rapid adoption.
• Automated sample desalting and focused solvent modulation enable direct coupling to mass spectrometry without off-line manipulation.

Future Trends and Opportunities

Advances may include:

• Tighter integration with high-resolution mass spectrometry and real-time data analytics for deeper sample profiling.
• Miniaturized 2D-LC formats to reduce solvent consumption and enhance sensitivity for trace analysis.
• AI-driven method optimization to predict optimal 2D conditions based on sample characteristics.
• Expanded applications in metabolomics, lipidomics, and natural product discovery, leveraging comprehensive peak mapping.

Conclusion

Agilent InfinityLab 2D-LC Solutions deliver robust, user-friendly two-dimensional separations that address the most challenging analytical problems. By combining advanced hardware, intuitive software, and flexible valve technologies, this platform maximizes peak capacity, simplifies workflows, and extends the capabilities of modern laboratories across diverse application areas.