High Resolution Fast LC - Easier Than You Think

Importance of the Topic

High-resolution fast liquid chromatography (LC) has become a critical tool in pharmaceutical, environmental, food and chemical analysis, offering rapid separation of complex mixtures while preserving or even enhancing chromatographic resolution. By shortening run times and reducing solvent consumption, laboratories can improve throughput, lower operating costs and accelerate method development.

Objectives and Study Overview

This work explores how shortening column length, narrowing column internal diameter, reducing packing particle size and applying elevated temperature and higher flow rates can deliver 2–10× faster LC analyses on conventional HPLC systems. It presents practical examples using Agilent 1100/1200 series instruments, addresses the impact of extra-column and dwell volumes, and demonstrates method scaling between different column formats.

Methodology and Instrumentation

• Column strategies:

• Traditional 5 μm particles vs. sub-2 μm porous vs. 2.7 μm superficially porous (“Poroshell 120”) coreshell particles
• Column lengths from 150 mm down to 15 mm and diameters from 4.6 mm to 0.075 mm

• Operating conditions:

• Flow rates up to 4 mL/min on 4.6 × 30 mm, 1.8 μm columns
• Gradient time and composition adjustments to maintain constant gradient retention factor (k*)
• Elevated temperatures (25–60 °C) to reduce back pressure and analysis time

• Key instrumental considerations:

• Agilent 1100/1200 series modules (binary high-pressure mixing pump, low-volume autosampler, high-speed detector with up to 80 Hz acquisition)
• Minimized extra-column volume using 0.12 mm ID tubing and sub-2 μL flow cells
• Dwell volume measurement and reduction techniques

Main Results and Discussion

• Separation of 15 analgesics in 2 min on 3 × 100 mm columns (1.8 μm and 2.7 μm) with comparable plate counts and signal-to-noise ratios.
• Ultra-fast isocratic separation of seven analgesics in 21 s using a 4.6 × 30 mm, 1.8 μm column at 4 mL/min.
• Method scaling from 4.6 × 250 mm, 5 μm columns to 3.0 × 100 mm, 3.5 μm columns yielded a 57% reduction in run time and an 83% reduction in solvent use.
• Doubling flow rate reduced gradient time by 50% without loss of resolution; increasing temperature from ambient to 60 °C cut analysis time by over 40% and lowered system pressure by ~25%.
• Demonstrated the critical influence of extra-column and dwell volumes on peak broadening when using low-dispersion columns.

Benefits and Practical Applications

• Faster sample turnaround supports high-throughput quality control and research workflows.
• Lower solvent consumption reduces environmental footprint and operating costs.
• Adoption on existing HPLC systems avoids expensive UHPLC upgrades.
• Enhanced method development flexibility through rapid screening of column and condition parameters.

Future Trends and Opportunities

• Wider implementation of UHPLC with sub-2 μm and core-shell particles for routine high-speed separations.
• Integration with high-resolution mass spectrometry for rapid screening and quantitation.
• Development of automated method optimization software using machine learning.
• Advances in green analytical chemistry driving ultra-low solvent and energy consumption.
• Expansion of micro- and nano-LC formats for proteomics and biomarker discovery.

Conclusion

High-resolution fast LC is readily achievable on conventional HPLC platforms by combining shorter, narrower columns packed with sub-2 μm or core-shell particles, optimized gradients, elevated temperatures, high-speed detection and minimized extra-column volumes. These adjustments deliver substantial gains in throughput, sensitivity and sustainability without major hardware investments.

Reference

1. Majors, L., et al. “Are You Getting the Most out of Your HPLC Column?” LC/GC Magazine, December 2003, Vol. 21(12), pp. 1124–1133.
2. Joseph, T., et al. The Influence of Sub-Two Micron Particles on HPLC Performance. Agilent Technologies Publication 5988-9251EN.