Agilent ZORBAX Rapid Resolution High Definition (RRHD) Columns

Importance of the Topic

The rapid and reliable separation of small molecules, peptides, proteins and complex natural products underpins modern analytical chemistry, impacting pharmaceutical development, proteomics, food analysis and environmental monitoring. Achieving high-definition chromatographic resolution without compromising throughput or stability is critical for confident compound identification and quantitation across diverse applications.

Objectives and Overview

This whitepaper introduces Agilent ZORBAX Rapid Resolution High Definition (RRHD) columns and demonstrates their performance across a wide range of bonded phases and pore sizes. It provides comparative data on selectivity, speed, pressure tolerance and long-term reproducibility in key application areas including endocannabinoid profiling, anthocyanin analysis, intact protein and peptide separations, monoclonal antibody chain resolution and fast small-molecule screening.

Methodology and Instrumentation

All studies employed sub-2 µm silica particles configured for ultra-high pressures up to 1200 bar. Key methodological elements included:

• Agilent 1290 Infinity LC system with Intelligent System Emulation Technology
• Column dimensions: 2.1×50–150 mm, 3.0×50–150 mm packed with 1.8 µm particles
• Range of bonded phases: C18 (Eclipse Plus, StableBond SB), C8, C3, phenyl-hexyl, cyano, Bonus-RP, HILIC and wide-pore 300 Å phases
• Mass spectrometric detection: Agilent 6410 Triple Quad MS/MS and LC/MS2 with ESI in positive mode
• MS parameters: typical scan ranges 200–1000 m/z, fragmentor voltages ~135 V, drying gas ~12 L/min

Main Results and Discussion

Selectivity comparisons for endocannabinoids demonstrated distinct retention profiles across four C18 variants, with SB-C18 offering enhanced stability at low pH and Eclipse Plus providing symmetrical peaks. Phenyl-hexyl and SB-Phenyl columns resolved anthocyanin glycosides more effectively via π–π interactions than standard alkyl phases. In protein work, 300 Å SB-C18 columns enabled sub-two-minute separations of insulin chains, while 100 mm columns delivered high-resolution peptide maps. Monoclonal antibody reduction products were reproducibly separated into light and heavy chains across C3, C8 and diphenyl phases with no loss in performance over 150 runs. Ultra-fast screening of pharmaceutical and biocide standards was achieved in under one minute on 50 mm RRHD columns.

Benefits and Practical Application

RRHD columns empower analysts to:

• Increase throughput by using shorter columns or higher flow rates without sacrificing peak capacity
• Adapt to challenging matrices through tailored phase selection (phenyl, cyano, Bonus-RP, HILIC)
• Ensure method transfer from legacy 3.5 µm and 5 µm chemistries to sub-2 µm formats with identical selectivity
• Maintain long-term robustness over hundreds of injections under UHPLC conditions
• Enhance MS sensitivity and confidence via narrow peak widths and defined separations

Future Trends and Possibilities

Advances will focus on integrating novel stationary phase materials (core-shell, hybrid organic-inorganic) with UHPLC for deeper molecular characterization. Coupling RRHD columns with high-resolution mass spectrometry and multidimensional LC techniques promises even greater insights in proteomics and metabolomics. Digital workflows and artificial intelligence–driven method optimization will further streamline analytical pipelines.

Conclusion

Agilent ZORBAX RRHD columns deliver a versatile, high-pressure UHPLC solution that balances speed, resolution and stability across small-molecule and biopolymer separations. Their broad phase portfolio and scalable designs facilitate method development, transfer and routine high-definition separations in pharmaceutical, biotech and industrial laboratories.

Reference

• Agilent publication number 5990-7166EN
• Agilent publication number 5990-8470EN
• Agilent publication number 5990-8124EN
• Agilent publication number 5990-4899EN
• Agilent publication number 5991-1435EN