Agilent HPlC Column Selection guide

Importance of the topic

Reversed-phase liquid chromatography (RP-LC) is the workhorse technique for small-molecule and biopolymer analysis across pharmaceutical, environmental, food-safety and industrial laboratories. Selecting the optimal column chemistry, hardware configuration and operating conditions is essential to maximize resolution, speed, robustness and lifetime of the separation method.

Objectives and overview

This guide presents a systematic approach for HPLC column selection and method development, covering both conventional HPLC and UHPLC platforms. It details: decision trees for column chemistry and phase selection; guidelines for particle size, pore size, column dimensions and pH; fast-LC options; and peripherals such as guard cartridges, injectors and detectors. Key product families from Agilent – Poroshell 120, ZORBAX RRHD/RRHT, Eclipse Plus, Eclipse XDB, StableBond, Extend-C18, Bonus-RP, Pursuit and Polaris – are described with their unique properties and recommended applications.

Methodology and instrumentation

Column and method development should begin at low pH (2.0–3.0) using endcapped C18 phases (Eclipse Plus or Poroshell EC-C18) and volatile buffers for LC/MS. Selectivity can be fine-tuned by switching organic modifiers (ACN, MeOH, THF) and bonded phases (C8, CN, phenyl, polar-embedded). For basic compounds, low pH suppresses silanol interactions; for neutral acids, elevated pH may be used on Extend-C18. UHPLC readiness requires minimization of extra-column volume with narrow-bore tubing, low-volume flow cells and high-frequency data sampling. Instrument platforms include the Agilent 1290 (1200 bar), 1260 (600 bar) and 1220 (600 bar) Infinity LC systems, complemented by Agilent SPE and filtration products for sample cleanup.

Main results and discussion

• Poroshell 120 columns achieve sub-2 μm column performance with 40–50% lower backpressure than conventional sub-2 μm phases, enabling faster separations of cephalosporin impurities, phenols and alkylphenones on standard 400–600 bar LC systems.
• ZORBAX RRHD (1.8 μm) phases—stable to 1200 bar—and RRHT (1.8 μm) phases—stable to 600 bar—deliver maximum efficiency and resolution for rapid screening (sub-minute runs) and method transfer across HPLC and UHPLC.
• Eclipse Plus (C18, C8, phenyl-hexyl), Eclipse XDB (C18, C8, phenyl, CN) and StableBond (C18, C8, CN, phenyl, C3, Aq) phases extend pH range to 2–9 (or 1–8 for StableBond), providing excellent peak shape for acids, neutrals and bases.
• Extend-C18 enables high-pH (up to 11.5) separations with bidentate bonding, enhancing retention of basic analytes in free base form and supporting high temperature operation to modify selectivity.
• Bonus-RP and Polaris phases embed polar functionality into the alkyl ligand to improve aqueous compatibility and peak shape for polar analytes without phase collapse.
• Pursuit, Pursuit XRs and Pursuit XRs Ultra columns expand selectivity options with diphenyl and pentafluorophenyl phases, high loadability and compatibility with high-throughput LC/MS.

Benefits and practical applications of the method

• Accelerated analysis (80–95% time savings) for pharmaceutical impurities, environmental pollutants and food contaminants using fast-LC columns.
• Enhanced method flexibility through one-family scalability from 5 to 1.8 μm particles and from 4.6 to sub-2.1 mm to 1.0 mm ID columns.
• Improved robustness and lifetime under harsh conditions (low pH, high pH, high temperature) with StableBond, Extend-C18 and polymeric PLRP-S phases.
• Reduced solvent consumption and waste via narrow-bore, Solvent Saver and Poroshell 120 columns.
• Optimized LC/MS sensitivity and throughput for bioanalysis and small-molecule quantitation.

Future trends and possibilities of use

Advancements in superficially porous particle technology and high-pressure UHPLC are driving even higher throughput and resolution. Integration of AI-driven method development tools, green solvents and microfluidic systems will further reduce analysis times and solvent use. Emerging polar-embedded phases and novel surface chemistries will extend reversed-phase LC to more polar and ionizable analytes. Continuous improvements in column lifetime under extreme pH and temperature are expected to broaden application ranges in proteomics, metabolomics and high-throughput screening.

Conclusion

Successful HPLC column selection relies on a clear understanding of sample properties, desired selectivity, instrument pressure limits and operational pH. Starting method development at low pH on a robust endcapped C18 phase, then optimizing organic modifier and bonded phase, offers a reliable path to high performance. Fast-LC options such as Poroshell 120, RRHD and RRHT enable dramatic speed gains without sacrificing resolution. Specialized phases—StableBond, Extend-C18, Bonus-RP and Polaris—address challenging separations under extreme pH and polar conditions. Guard columns, optimized connections and proper instrumentation settings complete a workflow that delivers reproducible, high-quality results.

References

• Agilent Technologies, LC Columns & Supplies Catalog, publication 5990-6674EN
• Agilent Poroshell 120 Columns Selection Guide, publication 5991-0165EN
• Agilent LC Handbook: Guide to LC Columns and Method Development, publication 5990-7595EN