Applications and Use of High Pressure and Ultra High Pressure LC ColumnsLC Column

Significance of the Topic

The use of high-pressure and ultra-high-pressure liquid chromatography (HPLC/UHPLC) with sub-2 µm particles has revolutionized analytical separations by delivering higher efficiency, faster run times and improved resolution. These advances are crucial for pharmaceutical quality control, environmental monitoring, metabolomics and proteomics, where speed and data quality directly impact decision-making and throughput.

Objectives and Study Overview

This study aims to evaluate the robustness, efficiency and lifetime performance of Agilent ZORBAX RRHD columns operated at pressures up to 1200 bar. Key goals include:

• Comparing column lengths (50 mm, 100 mm, 150 mm) in terms of resolution and analysis time
• Assessing column lifetime through extended low- and high-pressure injection lifetests (650 bar and 950 bar)
• Demonstrating the benefit of coupling columns for enhanced resolution
• Showcasing sub-one-minute separations and fast analysis of derivatized compounds

Methodology and Instrumentation

Columns evaluated:

• ZORBAX RRHD SB-C18, 2.1 × 50 mm, 1.8 µm
• ZORBAX RRHD Eclipse Plus C18, 2.1 × 100 mm and 2.1 × 150 mm, 1.8 µm

Chromatographic conditions:

• Mobile phases: water/acetonitrile mixtures (40:60 or gradient 60–70 % ACN)
• Flow rates: 0.3–2.3 mL/min depending on column and application
• Temperatures: ambient or 60 °C for fast separations
• Pressure range: up to 1200 bar on Agilent 1290 Infinity LC system

Main Results and Discussion

• Low-pressure lifetest at 650 bar and high-pressure at 950 bar over 5000 injections showed negligible loss in plate count (from ~21 722 to ~20 653) and consistent retention behavior.
• Column length selection balances resolution (higher for 150 mm) and analysis time (shortest for 50 mm). Effective Rs values improved from 5.6 (50 mm) to 11.98 (150 mm) under UHPLC conditions.
• Coupling 100 mm and 150 mm RRHD columns delivered N6 ≈ 49 200 and Rs3,2 of ~4.9, demonstrating scalable resolution gains.
• Sub-one-minute separations of small molecules and six-compound biocide mixes were achieved using 2.1 × 50 mm columns at 2 mL/min and 975 bar.
• Analysis of DNPH-derivatized aldehydes on two coupled 100 mm columns reached initial pressures >1080 bar and provided clear separation of up to 13 compounds within 9 min.

Benefits and Practical Applications

• Higher sample throughput due to faster gradients and sub-minute separations.
• Improved resolution enables detection of closely eluting or low-abundance analytes.
• Extended column lifetime under ultra-high-pressure conditions supports sustainable lab operations.
• Flexible column coupling strategies allow method tailoring for targeted applications in pharmaceuticals, food safety and environmental testing.

Future Trends and Applications

Emerging directions include:

• Further expansion of pressure limits beyond 1200 bar for even higher efficiency with sub-1 µm particles.
• Integration of UHPLC with high-resolution mass spectrometry for proteomics and metabolomics.
• Development of novel stationary phases optimized for ultra-high-pressure stability and selectivity.
• Green chromatography approaches leveraging reduced solvent usage with narrower bore columns.
• High-throughput two-dimensional LC systems utilizing coupled columns under ultra-high pressure.

Conclusion

Agilent ZORBAX RRHD columns, when operated on a 1290 Infinity LC system at pressures up to 1200 bar, deliver robust performance, exceptional efficiency and long lifetimes. Selection of column length and coupling strategies enables precise control over resolution and analysis speed, meeting diverse analytical demands from rapid screening to in-depth separations.

Reference

1. Link J, Brooks A, Henderson JW Jr, Long W, Martone N, Martosella J, Permar B. Applications and Use of High Pressure and Ultra High Pressure LC Columns. HPLC 2009, Agilent Technologies.