Reversed-Phase for Biomolecules: From Column Selection to Troubleshooting

Importance of Reversed-Phase Chromatography for Biomolecules

Reversed-phase liquid chromatography (RP-LC) is a cornerstone technique for analyzing peptides, proteins and oligonucleotides. Its ability to separate biomolecules based on hydrophobic interactions with a nonpolar stationary phase makes it essential for characterizing critical quality attributes (CQAs) of biotherapeutics, ensuring safety, efficacy and process consistency.

Objectives and Overview

This application note explores best practices for column selection, method development and troubleshooting in RP-LC of biomolecules. It covers stationary phase chemistries, particle and pore size considerations, mobile phase composition, gradient design and ion-pairing strategies for intact proteins, peptide mapping and oligonucleotide separations.

Methodology and Instrumentation

Columns evaluated include silica-based (Zorbax SB300, Poroshell 300 SB) and polymeric phases (PLRP-S), with pore sizes from 100 Å to 4000 Å and particle diameters from sub-2 µm to 5 µm. Both totally porous (TPP) and superficially porous (SPP) particles are compared. Ion-pairing reagents such as trifluoroacetic acid (TFA), formic acid, triethylammonium acetate (TEAA) and hexafluoroisopropanol (HFIP) enable controlled retention and compatibility with UV or mass spectrometry detection. Temperature, gradient steepness and flow rate are optimized using HPLC and UHPLC platforms capable of handling backpressures up to 600 bar.

Main Results and Discussion

• Silica modifications (end-capping, steric protection) extend column lifetime and reduce secondary interactions.
• Superficially porous particles deliver comparable efficiency to sub-2 µm totally porous materials at lower backpressure.
• Shorter alkyl chains (C3–C8) improve intact protein separations, while C18/diphenyl phases excel for peptides.
• Higher temperatures (60–80 °C) and optimized gradient slopes sharpen peaks and shorten run times by up to 60 %.
• Ion-pair reagent concentration and chain length directly influence oligonucleotide retention and resolution.

Benefits and Practical Applications

RP-LC methods described provide high-resolution profiles for intact mAbs, peptide mapping workflows achieving >99% sequence coverage, and oligonucleotide purity assessments critical for therapeutic development and quality control.

Future Trends and Potential Use

Emerging trends include integration with high-resolution mass spectrometry, development of ultra-high-pH stable phases for improved selectivity, and application of larger-pore stationary phases for mRNA and complex oligonucleotide constructs.

Conclusion

A systematic approach to column chemistry, particle architecture and mobile phase design enables robust, high-throughput RP-LC separations of biomolecules. Tailored method parameters ensure reproducible, high-resolution analyses across a range of molecular weights and modalities.

Reference

1. Kirkland J.J.; Glajch J.L.; Farlee R.D. Analytical Chemistry 1989, 61, 2.