Agilent ZORBAX Rapid Resolution High Definition 300SB-C18 Threaded Column

Significance of the topic

High-resolution, high-throughput reversed-phase columns are critical for modern analytical workflows, especially in proteomics, pharmaceutical QC, and bioanalysis. The ability to withstand elevated pressures, aggressive solvents, and a wide pH range ensures reliable separations of small molecules, peptides, and intact proteins with minimal background interference.

Objectives and overview of the article

This data sheet presents the design, performance characteristics, and operational guidelines for the Agilent ZORBAX RRHD 300SB-C18 threaded column. It aims to demonstrate the column’s stability, efficiency, and suitability for ultra-high-performance liquid chromatography (UHPLC) applications, including peptide mapping and protein analysis.

Methodology and instrumentation

The column uses a sterically protected C18 bonded phase on 300 Å porous silica particles, yielding high efficiency at particle sizes of 1.8 µm. Key operating conditions for a QC test include:

• System: Agilent 1290 Infinity UHPLC
• Column dimensions: 2.1 mm × 50 mm, threaded
• Mobile phase: 45 % acetonitrile / 55 % water
• Flow rate: 0.50 mL/min
• Temperature: ambient (23 °C)
• Detector: UV at 254 nm
• Sample: model compounds (uracil, phenol, 4-chloro-nitrobenzene, naphthalene)

Main results and discussion

The QC chromatogram shows sharp, well-resolved peaks with high theoretical plate counts, reflecting uniform packing and consistent bed density. The column tolerates pressures up to 1200 bar and temperatures up to 90 °C (low pH), with optimal lifetimes under 1000 bar. Its StableBond chemistry resists degradation in acidic or ion-pair mobile phases, while the high-purity silica minimizes basic compound adsorption.

Operational guidelines emphasize correct end-fitting torque, solvent compatibility, pH limits (0.8–8.0), and storage in pure acetonitrile to maintain performance and longevity.

Benefits and practical applications

• High sensitivity and low background for mass spectrometry coupling
• Robustness under aggressive mobile phases and wide pH range
• Rapid analysis times with high resolution for small molecules and biomolecules
• Extended column life through steric protection of bonded phase

Future trends and potential applications

Advancements in UHPLC instrumentation and microflow systems will further reduce analysis times and solvent consumption. Emerging applications include intact protein separations, high-throughput peptide mapping in biopharma, and coupling with high-resolution mass spectrometers for deeper proteome coverage.

Conclusion

The Agilent ZORBAX RRHD 300SB-C18 threaded column combines high pressure tolerance, wide pH compatibility, and steric protection of the bonded phase to deliver reliable, high-resolution separations in demanding analytical workflows. Its design supports rapid method development and routine use in pharmaceutical, environmental, and proteomic laboratories.

References

1. Claessens HA, van Straten MA, Kirkland JJ. J Chromatogr A. 728 (1996) 259.
2. Introduction to Modern Liquid Chromatography, 2nd ed., Snyder LR, Kirkland JJ. John Wiley & Sons, 1979.
3. Practical HPLC Method Development, 2nd ed., Snyder LR, Kirkland JJ, Glajch JL. John Wiley & Sons, 1997.