Agilent Biomolecule Reversed-Phase LC Columns User Guide

Importance of the topic

Biomolecule separations by reversed-phase liquid chromatography are critical for the analysis of peptides, proteins, and oligonucleotides in biopharmaceutical and life science research. Ultra inert column hardware and optimized stationary phases minimize metal interactions, improving sensitivity, reproducibility, and column lifetime.

Objectives and overview

This guide summarizes best practices for installation, conditioning, operation, cleaning, and storage of Agilent Altura, AdvanceBio, and BioHPLC reversed-phase columns. It provides recommended parameters to achieve consistent high-performance separations of biomolecules.

Instrumentation used

• Agilent Altura HPH-C18 (1.9 µm), Altura Oligo HPH-C18 (2.7 µm), ZORBAX Eclipse Plus C18, Poroshell 300, AdvanceBio and PLRP-S stationary phases
• InfinityLab Quick Turn and Quick Connect fittings for zero-dead-volume connections
• Standard HPLC systems configured for QC testing, capable of up to 1300 bar

Methodology and column conditioning

Columns arrive tested in shipping solvents and must be flushed with the user’s mobile phase for at least 10–20 column volumes. For formic acid-modified phases, use the specific flow rates, temperatures, and volumes listed in the conditioning table. Observe flow-direction labels, start at reduced flow rates, and avoid backflushing sub-2 µm packings. Follow pressure and pH limits during operation.

Main results and discussion

Operating pressures span from 200 bar for 10–30 µm packings to 1200 bar for sub-2 µm materials. Recommended pH windows range from 1 to 14 depending on phase. Mobile phases typically employ water/acetonitrile or methanol gradients with 0.1% TFA or formic acid. Column cleaning of small-molecule fouling uses reversed solvent strength and backflush (where permitted). Protein contaminant removal employs acidic, organic, or chaotropic solutions as needed. Storage in pure organic solvents after buffer removal prevents salt corrosion.

Benefits and practical applications

• Ultra Inert technology blocks active metal sites for enhanced peak shape and sensitivity
• Wide pH and temperature compatibility supports diverse biomolecule classes
• High-pressure capability enables fast, high-resolution separations

Future trends and possibilities for use

Continued development of column hardware coatings will further reduce metal-sensitive analyte interactions. Advances in high-pressure and high-temperature stationary phases, along with seamless integration with mass spectrometry and automated workflows, will expand applications in proteomics, oligonucleotide therapeutics, and high-throughput screenings.

Conclusion

Adhering to the outlined preparation, operating, and maintenance procedures ensures optimal chromatographic performance and column longevity. Agilent’s Ultra Inert reversed-phase columns deliver reliable, high-quality separations for challenging biomolecule analyses.