Agilent AdvanceBio SEC 2.7 μm Columns - User guide

Significance of the topic

Size exclusion chromatography (SEC) plays a vital role in the characterization and quality control of biotherapeutics and biomolecules. High-resolution separation of proteins and peptides allows researchers and quality control laboratories to monitor aggregate levels, detect degradation products, and ensure batch-to-batch reproducibility. The development of robust, low-nonspecific-interaction stationary phases enables more reliable analyses of sensitive biomolecules while extending column lifetime.

Objectives and study overview

This application note presents recommended operating parameters and maintenance guidelines for Agilent AdvanceBio SEC columns. Key goals include establishing starting conditions for mobile phase composition, flow rate, temperature, and pressure limits, as well as providing protocols for column cleaning, storage, and performance monitoring. The overview highlights how to achieve consistent separations of monoclonal antibodies, proteins, and peptides in routine and SEC-MS workflows.

Methodology

The technique utilizes 2.7 μm silica-based SEC columns with hydrophilic bonding chemistry to minimize analyte–stationary phase interactions. Typical conditions start with a 150 mM phosphate buffer at pH 7.0, a flow rate of 1 mL/min, ambient column temperature, and detection at 220 nm. Injection volumes of 5 μL (up to 1 % of column volume) are recommended. For MS-compatible separations, aqueous volatile buffers such as ammonium acetate are advised. Performance is assessed using a batch-specific test mixture of protein standards, including thyroglobulin, γ-globulin, ovalbumin, myoglobin, and angiotensin II.

Instrumentation used

• Agilent AdvanceBio SEC 300 Å, 2.7 μm, 7.8 × 300 mm (p/n PL1180-5301)
• Agilent AdvanceBio SEC 300 Å, 2.7 μm, 4.6 × 300 mm (p/n PL1580-5301)
• Agilent AdvanceBio SEC 300 Å guard column, 2.7 μm, 7.8 × 50 mm (p/n PL1180-1301)
• Agilent AdvanceBio SEC 130 Å, 2.7 μm, 4.6 × 300 mm (p/n PL1580-5350)
• Agilent InfinityLab Quick Connect fittings (p/n 5067-5966) and Bio-inert UHP-FF PEEK fittings (p/n 5067-5695)

Main results and discussion

Under the recommended conditions, AdvanceBio SEC columns deliver high-resolution separations of monoclonal antibodies and protein standards, clearly resolving monomers, aggregates, and smaller fragments. System backpressure remains below 200 bar at standard flow rates, ensuring extended column life. Method precision and reproducibility are confirmed by consistent retention times and peak shapes across multiple injections and column batches.

Benefits and practical applications

• Minimal nonspecific adsorption due to specialized hydrophilic bonding chemistry.
• Wide pH stability (2.0–8.5) and temperature tolerance (20–80 °C) for application flexibility.
• Compatibility with UV, DAD, light scattering, and MS detection.
• Standardized performance monitoring using Agilent molecular weight calibration standards.

Future trends and potential applications

Advances in SEC stationary phase design and coupling to mass spectrometry will expand the scope of biomolecule characterization, enabling more detailed analysis of glycoforms, post-translational modifications, and complex assemblies. Integration with automated sample preparation and high-throughput platforms will further streamline biopharmaceutical quality control and support accelerated product development.

Conclusion

Agilent AdvanceBio SEC columns provide a robust, reproducible platform for size exclusion analysis of proteins and peptides. By following the outlined operating conditions, cleaning protocols, and storage practices, laboratories can achieve consistent, high-resolution separations with minimal downtime and extended column lifespan. This approach supports critical workflows in biotherapeutic research, development, and quality assurance.