Agilent ZORBAX Rapid Resolution High Definition (RRHD) Columns

Importance of the topic

High-definition, high-throughput separations are fundamental to modern analytical laboratories across pharmaceuticals, biotechnology, and environmental monitoring. Advancing column technology to deliver faster analyses without sacrificing resolution or stability enables more efficient method development, greater sample throughput, and improved compound identification, particularly in complex mixtures of small molecules, peptides, and intact proteins.

Objectives and Overview

This application note introduces Agilent’s ZORBAX Rapid Resolution High Definition (RRHD) column family, detailing how these sub-2 µm UHPLC columns achieve unparalleled resolution, flexibility, and robustness. The goal is to illustrate their performance advantages across a range of chemistries—from standard C18 separations to specialized phenyl, HILIC, and wide-pore phases optimized for proteins and peptides—and to demonstrate method transfer and scalability with Agilent UHPLC systems.

Methodology and Instrumentation

• Column phases: Over twelve bonded phases including Eclipse Plus C18, Extend-C18, phenyl-hexyl, PAH, cyano, Bonus-RP, StableBond SB-C18/C8/C3, SB-Phenyl, SB-Aq, and HILIC.
• Particle size and design: 1.8 µm particles with 80–300 Å pores for maximum resolution of small to large biomolecules; stable to 1200 bar pressure.
• Chromatographic conditions: Demonstrated isocratic and gradient methods, flow rates from 0.3–1.7 mL/min, temperatures 24–80 °C, mobile phases containing acids (formic, trifluoroacetic) or organic modifiers (ACN, IPA).
• Detection: LC/UV and LC/MS in positive ESI mode, MS2 scans for endocannabinoids and anthocyanins, UV detection at 225–280 nm for proteins and biocides.

Main Results and Discussion

• Small-molecule separations: Sub-2 µm C18 phases resolved eleven analgesics and anti-inflammatories in under one minute with peak widths (PW½) reduced by >60% compared to 3.5 µm columns, improving throughput on the 1290 Infinity LC.
• Endocannabinoid profiling: Four C18 chemistries (Eclipse Plus, XDB-C18, Extend, SB-C18) demonstrated subtle selectivity differences for anandamide, PEA, 2-AG, OEA, and an impurity, highlighting the importance of phase selection for lipidomics.
• Phenolic compound analysis: Phenyl-hexyl versus SB-Phenyl phases showed enhanced π–π interactions for anthocyanin glycosides, resolving additional peaks from blueberry extracts under a rapid methanol/formic acid gradient.
• Protein and peptide separations: Wide-pore (300 Å) RRHD columns achieved sub-two-minute UPLC separations of insulin chains, high-resolution mapping of monoclonal antibody digests, and maintained consistent performance over 150–200 injections without pressure increases or peak degradation.
• Scalability and transfer: Gradient slope and column volume scaling between 1.8 µm RRHD and legacy 3.5–5 µm columns preserved selectivity while reducing run times by up to 50%, demonstrating seamless method migration.

Benefits and Practical Applications

• Maximum resolution with minimal analysis time, enabling higher laboratory productivity.
• Robust column stability at pH 1–11.5 and temperatures up to 80 °C for versatile method design.
• Wide phase portfolio for optimized selectivity in small-molecule, lipid, phenolic, peptide, and protein analyses.
• Enhanced MS compatibility and sensitivity through narrow peaks and reduced dispersion.
• Simplified method transfer and scalability across UHPLC and HPLC formats for legacy support.

Future Trends and Opportunities

Advances in particle technology and bonding chemistries will further expand phase selectivity, enabling even more targeted separations of isomeric and labile biomolecules. Integration with high-resolution mass spectrometry, microfluidic UHPLC, and automated method optimization will drive ultrafast, high-throughput screening in proteomics, metabolomics, and quality control. Novel hybrid-material particles and core-shell architectures promise to combine sensitivity enhancements with lower backpressures, opening new frontiers in bioanalysis.

Conclusion

Agilent ZORBAX RRHD columns offer a comprehensive solution for modern UHPLC demands, delivering sub-2 µm performance across a wide range of stationary phases. Their high-pressure stability, broad pH and temperature tolerance, and scalability ensure rapid, high-resolution separations for both small molecules and large biomolecules. When paired with the Agilent 1290 Infinity LC, laboratories can achieve unparalleled throughput and reproducibility, accelerating method development and routine analyses.

Used Instrumentation

• Agilent 1290 Infinity LC system with Intelligent System Emulation Technology (ISET).
• Agilent 6410 Triple Quadrupole LC/MS for MS/MS detection.
• ZORBAX RRHD columns in 2.1×50–150 mm and 3.0×50–150 mm formats, 1.8 µm particle size.