From Proteins to Polymers-GPC/SEC - Understanding column selection and method considerations for your sample

Importance of the Topic

Size exclusion chromatography (GPC/SEC) is a fundamental technique for characterizing polymers and biomolecules by hydrodynamic volume rather than just molecular weight. It supports quality control in pharmaceutical formulations, polymer manufacturing, and biopharmaceutical development by providing reliable information on molecular size distribution, aggregation, and conformation.

Objectives and Overview

The primary goal of this presentation is to guide analysts in selecting appropriate columns, solvents, and instrumentation for GPC/SEC applications. It reviews terminology, explains the separation mechanism, and outlines method development considerations for diverse sample types including organic polymers, water-soluble polymers, proteins, and monoclonal antibodies.

Methodology and Instrumentation

• Terminology: Definitions of GPC, SEC, and GFC and their typical mobile phases.
• Separation mechanism: Porous bead columns separate molecules by hydrodynamic volume; large molecules elute first.
• Sample considerations: Differentiation between organic polymers and biomolecules; impact of solubility, aggregation, and sample cleanup.
• Solvent and mobile phase: Criteria include full solubility, avoidance of non-size interactions, column compatibility, detection requirements, and safety.
• Column selection: Comparison of individual, mixed, and multi-pore chemistries; choosing pore size and particle size; combining columns for extended range or higher resolution.
• Detection and instrumentation: Use of refractive index (RID), UV, ELSD, viscometry, and light scattering (static and dynamic) detectors. Importance of low-dispersion plumbing, proper fittings, and optimal data collection rates.

Used Instrumentation

• Agilent AdvanceBio SEC columns (1.9 μm, 2.7 μm, 3 μm, 5 μm) with pore sizes from 100 Å to 2000 Å for organic and aqueous applications.
• LC systems: Agilent 1260 Infinity II Bioinert LC, equipped for multi-detector SEC including MALS and DLS.
• Detectors: RID, UV/DAD, ELSD, viscometer, static and dynamic light scattering.

Main Results and Discussion

• Solvent optimization: Starting with 150 mM phosphate buffer (pH 7) for proteins; organic solvents such as THF, chloroform, DMF for polymers.
• Column performance: Demonstrated increased resolution by reducing particle size, increasing column length, or connecting columns in series.
• Rapid methods: High flow rates on sub-2 μm columns maintain baseline resolution while boosting sample throughput by up to fourfold.
• Detector enhancements: ELSD offers improved sensitivity for additives; multi-detector SEC provides absolute molar mass and size measurements.

Benefits and Practical Applications

• Accurate determination of molecular weight distribution and detection of aggregates or fragments in therapeutic proteins.
• Control of polymer physical properties (strength, viscosity, dissolution) by precise molar mass analysis.
• Streamlined method transfer and high sample throughput for routine QC laboratories.

Future Trends and Applications

• Wider adoption of multi-detector SEC (viscometry, MALS, DLS) for comprehensive characterization of branching and conformation.
• Development of novel column chemistries (multi-pore particles, bioinert surfaces) for challenging biomolecules.
• Integration with mass spectrometry for direct molecular identification and structural analysis.
• Automation and miniaturized systems for high-throughput and process analytical technology (PAT) environments.

Conclusion

Effective GPC/SEC method development hinges on matching solvent systems, column chemistries, and detectors to sample properties and analytical goals. Advances in column design and multi-detector platforms continue to expand the technique’s capability for polymer and biopharmaceutical analysis.

References

No formal literature references were provided in the source document.