Lifetime Stability and Comparative Performance Analyses of Reversed-Phase Ultra High Performance Liquid Chromatography (UHPLC) Columns

Importance of the Topic

Recent advances in ultra high performance liquid chromatography (UHPLC) demand columns that deliver high resolution, fast separations and sustained stability under extreme pressures. Reliable column lifetime with reproducible performance is crucial for high-throughput analysis in pharmaceutical, environmental and biological laboratories to reduce downtime and operational costs.

Objectives and Study Overview

This work evaluates the mechanical and chemical stability of Agilent ZORBAX Rapid Resolution High Definition (RRHD) reversed-phase UHPLC columns under challenging conditions. Comparative tests were conducted against leading competitor columns to assess lifetime reproducibility, bed stability, pressure tolerance and resistance to sample particulates.

Methodology and Instrumentation

The study employed multiple lifetime tests, including low pH stress (1000 bar), repeated injections of protein-precipitated plasma and microparticulate suspensions until a 10% backpressure increase was observed. Column efficiency (theoretical plates), peak tailing and pressure profiles were monitored throughout extended injection sequences. Sample mixes comprised model compounds (uracil, phenol, chloronitrobenzene, naphthalene) and biological matrices.

Instrumentation Used

• Agilent 1290 Infinity UHPLC system configured for pressures up to 1200 bar
• Agilent RRHD Eclipse Plus C8 and ZORBAX SB-C18 columns (1.8 µm particles; 2.1 × 50 mm, 2.1 × 100 mm and 3.0 × 100 mm)
• Detector wavelengths: 254 nm (plasma tests) and 210 nm (low pH tests)
• Mobile phases: 55/45 ACN/H2O with 0.1% formic acid or 0.1% TFA
• Sample preparation: protein precipitation (ACN), particulate suspensions of microcrystalline cellulose and excipients

Main Results and Discussion

Agilent RRHD columns maintained consistent efficiency and peak shape over >5000 injections at 1000 bar in low pH conditions, showing less than 10% loss in theoretical plates. Microparticulate tests demonstrated >2800 injections with minimal backpressure increase, outperforming competitor columns which reached failure earlier. Repeated plasma injections confirmed stable pressure profiles, indicating robust sample tolerance.

Benefits and Practical Applications

• Extended column lifetimes reduce replacement frequency and cost.
• High-pressure tolerance allows faster flow rates and higher throughput.
• Resilience to complex biological and particulate-laden samples enhances method robustness.
• Broadens UHPLC applicability in pharmaceutical QC, proteomics and environmental analysis.

Future Trends and Opportunities

Continued particle engineering may extend operational pressures beyond 1200 bar and support nano-UHPLC formats. Integration with mass spectrometry for high-throughput proteomics and further development of chemically stable stationary phases will address emerging analytical demands.

Conclusion

Agilent RRHD UHPLC columns exhibit superior mechanical stability and lifetime performance under extreme conditions, providing reliable, high-throughput separations with reduced downtime. Their robust design makes them ideal for demanding analytical workflows.

References

• Martosella J., Broske A.D., Henderson J.W., Link J., Liu J., Martone N., Permar B., Wang H.L. Lifetime Stability and Comparative Performance Analyses of Reversed-Phase Ultra High Performance Liquid Chromatography (UHPLC) Columns. HPLC 2010, Boston, MA; Application Note P-2916-M; Agilent Technologies.