Agilent AdvanceBio SEC 1.9 μm Columns - User guide

Importance of the Topic

Size-exclusion chromatography (SEC) is a fundamental tool in analytical chemistry and biopharmaceutical research for characterizing macromolecules, detecting aggregates, and verifying sample purity. High-resolution, bio-inert SEC columns enable accurate separation of proteins, peptides, antibodies, and drug-protein conjugates under both denaturing and native conditions. Maintaining column performance through proper care, cleaning, and storage is critical to reproducible results and method robustness.

Objectives and Overview

This application note presents recommended operating conditions, maintenance protocols, and storage guidelines for Agilent AdvanceBio SEC columns (120 Å and 200 Å pore sizes, 1.9 μm particle size) in various dimensions. It also provides ordering details, quality control benchmarks, and best practices for LC-SEC and SEC-MS applications to maximize column lifetime and chromatographic performance.

Used Instrumentation

• Agilent 1260 Infinity II Bio-inert LC system
• AdvanceBio SEC columns:
   • 120 Å, 1.9 μm, 4.6×300 mm (PL1580-5250)
   • 120 Å, 1.9 μm, 4.6×150 mm (PL1580-3250)
   • 120 Å guard, 1.9 μm, 4.6×30 mm (PL1580-1250)
   • 200 Å, 1.9 μm, 4.6×300 mm (PL1580-5201)
   • PEEK-lined variants for 2.1 mm id columns (e.g., PL1980 series)
• Detectors: UV (220 nm or 280 nm), DAD, light scattering, SEC-MS
• Recommended fittings: Agilent InfinityLab Quick Connect (p/n 5067-5966) or Bio-inert UHP-FF (p/n 5067-5695)

Methodology and Instrumentation

Operating parameters were optimized to minimize dead volume and maintain low backpressure. Key conditions include:

• Mobile phase: 150 mM sodium phosphate buffer, pH 7.0 (≤0.5 M salt; ≤20% organic for denaturing mode; 20% MeOH for switching solvents)
• Flow rate: 0.35 mL/min (4.6 mm id); 0.05–0.10 mL/min (2.1 mm id)
• Temperature: ambient to 40 °C (max 80 °C)
• Injection volume: 1–5 µL
• QC standards: Thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), angiotensin II (1 kDa)

Main Results and Discussion

Quality control chromatograms demonstrate sharp, baseline-resolved peaks for monomer, aggregates, and fragments with consistent retention times. Column pressure remains stable under recommended conditions; any increase in backpressure is first attributed to guard column fouling or system filters. Cleaning protocols using high-salt, organic solvents, or acidic reagents can restore performance without sequential buffer overload, provided flow direction and pressure (<400 bar) are controlled.

Benefits and Practical Applications

AdvanceBio SEC columns deliver:

• High efficiency separations of peptides, proteins, mAbs, and ADCs
• Metal-free, bio-inert flow path for sensitive SEC-MS analysis
• Flexible mobile phase compatibility (denaturing/native)
• Rapid equilibration and minimal carryover with inline guard columns

Future Trends and Potential Applications

Advancements in SEC include further miniaturization (narrow-bore and capillary formats), integration with high-resolution MS for detailed mass profiling, and development of novel stationary phases for ultra-high throughput. Emerging bio-inert coatings and pore chemistries will broaden analyte scope, particularly in biosimilar characterization and real-time process monitoring.

Conclusion

Following the recommended conditions for AdvanceBio SEC columns ensures reliable, high-resolution separations and extends column lifetime. Proper cleaning, storage, and system configuration are essential for maintaining performance in diverse biopharmaceutical applications.

References

• Agilent Technologies. AdvanceBio SEC User Guide. Publication 5994-0739EN; July 9, 2021.