Capillary-flow LC-MS: combining high sensitivity, robustness, and throughput

Importance of Capillary-Flow LC-MS

Capillary-flow LC-MS (capLC-MS) bridges the sensitivity of nano-flow separations with the throughput and robustness of analytical-flow methods. By operating at 1–15 µL/min through 100–300 µm ID columns, capLC-MS reduces solvent use and source contamination while enhancing concentration-sensitive electrospray ionization (ESI) efficiency. This approach fills a critical gap for laboratories requiring both high sensitivity and rapid analysis — for example, in large‐scale proteomics cohorts, targeted bioanalysis, and small‐molecule screening.

Study Objectives and Overview

This technical note presents a turnkey capLC-MS workflow: it aims to demonstrate reliable hardware configurations, standardized fluidic designs for direct injection or trap‐and‐elute modes, and performance benchmarks against nano and analytical flow systems. The goal is to enable high-throughput targeted quantification or shotgun profiling of complex samples with minimal method development and sustained robustness.

Methodology and Instrumentation

Key components include:

• Thermo Scientific UltiMate 3000 RSLCnano: interchangeable nano, capillary, and micro flow‐meters, low‐flow HPG pump (<25 nL delay volume), micro LPG pump for preconcentration, and column compartment with precise temperature control
• Plug‐and‐spray EASY-Spray source coupled via 20 µm silica emitter or alternative Ion Max/HESI-II probe with 50 µm stainless steel needle
• Capillary columns (0.3 × 150 mm, 2 µm C18) and matching trap cartridges (0.3 × 5 mm, 5 µm C18) in direct‐injection or back‐flush trap‐and‐elute configurations
• High-resolution MS detectors (Q Exactive HF or Q Exactive Plus) operating in full MS, PRM, or DDA modes

The system uses nanoViper fingertight fittings to eliminate dead volumes and tool requirements, enabling fluidic reconfiguration in minutes.

Key Results and Discussion

Sensitivity and throughput comparisons showed:

• CapLC-MS at 5–10 µL/min yields 5–30× higher sensitivity versus 450 µL/min analytical flow, and approaches nanoLC-MS sensitivity with sample capacity 10–15× greater
• Direct injection and trap‐and‐elute methods achieve 10 min gradients for peptide separations with baseline resolution and minimal carryover
• Retention time repeatability ≤1% RSD over 500 injections using scheduled MRM/PRM, essential for targeted assays
• Peak area precision of ~5% RSD in complex (E. coli digest spiked with cytochrome c) over 100 injections without internal standards
• Targeted quantification of monoclonal antibody infliximab: PRM calibration down to 16 amol on-column, linear over five orders of magnitude
• Shotgun proteomics of HeLa digest: capLC-MS identifies ~70% of proteins and peptides versus nanoLC-MS at equal loads, rising to ~80% at 4× increased sample injection

Practical Benefits and Applications

CapLC-MS offers:

• High‐throughput targeted bioanalysis (e.g., peptide quantification, biotherapeutic monitoring) with short sample‐to‐sample cycle times
• Robust profiling of complex proteomes for biomarker discovery and quality control in pharmacology
• Reduced solvent consumption and maintenance intervals compared to nanoLC-MS
• Versatility for small‐molecule metabolomics or pesticide screening using alternative stationary phases and trap chemistries

Future Trends and Opportunities

As capLC-MS gains adoption, further innovations may include:

• Integration with microfluidic sample preparation for end‐to‐end automation of low‐volume workflows
• Advanced emitter designs (e.g., integrated sheathless interfaces) to extend sensitivity at higher flow rates
• AI‐driven method optimization to tailor gradients and source parameters dynamically for diverse sample types
• Expansion of capillary column chemistries (e.g., hydrophilic interaction, core–shell) to broaden small‐molecule and intact‐protein applications

Conclusion

The described capLC-MS workflow unites sensitivity, robustness, and throughput in a single platform. It provides an accessible route to high‐performance targeted quantification and proteome profiling, reducing complexity and downtime. By standardizing fluidic configurations and leveraging plug‐and‐spray ESI sources, capLC-MS fills the gap between nano and analytical flow, meeting the evolving demands of modern analytical laboratories.

References

1. Bruins AP. Mass spectrometry with ion sources operating at atmospheric pressure. Mass Spectrom Rev. 1991;10:53–77.
2. Hopfgartner G et al. Ion spray mass spectrometric detection for liquid chromatography: concentration‐ versus mass‐flow‐sensitive device? J Chrom A. 1993;647:51–61.
3. Page JS et al. Ionization and transmission efficiency in an electrospray ionization‐mass spectrometry interface. J Am Soc Mass Spectrom. 2007;18:1582–1590.
4. Boychenko A et al. Sensitive, fast and robust quantification of antibodies in complex matrices by capillary flow UHPLC and high‐resolution accurate‐mass MS. ASMS 2016;64787.
5. Meding S, Boychenko A. Capillary flow LC‐MS unites sensitivity and throughput. Chromatography Today. 2016;43–45.