Rapid Method Screening for High Resolution Aqueous SEC Measurements

Significance of the Topic

The development of rapid, high resolution aqueous size exclusion chromatography (SEC) is critical for efficient polymer characterization in research, quality control and industrial analytics. Optimizing column chemistry and instrument performance enables faster method screening, reduced sample adsorption and improved reproducibility for a wide range of polymer types, from synthetic resins to biopolymers.

Objectives and Overview

This study introduces a new platform combining Waters ACQUITY Advanced Polymer Chromatography (APC) system with novel bridged ethylsiloxane/silica hybrid (BEH) columns. The main goals are to achieve high operating pressures, low system dispersion and flexible solvent compatibility while screening multiple column and solvent conditions within a four-hour workflow.

Methodology and Instrumentation

BEH hybrid particles feature robust mechanical strength, a low-energy surface to minimize adsorption, and tunable pore size distributions. Two column chemistries were evaluated:

• APC XT with small particle size and TMS end-cap for organic compatibility
• APC AQ diol-bonded columns with no residual bonding for aqueous applications

The ACQUITY APC system delivers precise mobile phase flow up to 1000 bar, low dispersion, and flexible detection including refractive index (RI) with 1.3 and 10.3 µL flow cells. An optional six-solvent select valve allows automated solvent switching for rapid condition screening.

Main Results and Discussion

Low molecular weight polystyrene standards (266–15 000 Da) were separated on two 45 Å APC XT columns, demonstrating high resolution and reduced analysis time. Pulse studies with pullulan and PEG standards highlighted the diol column’s superior peak shapes under aqueous conditions. Temperature experiments (30 °C vs 50 °C) showed minimal retention shifts (<3 %) across various polymers, confirming column stability.
Method development screening with two BEH Diol 125 Å columns under different water/organic mixtures was completed in four hours, mapping resolution versus backpressure trade-offs. Diol ligand coverage effectively reduced accessible pore size to ~80–90 Å. Comparison to XT 45 Å columns indicated complementary operating ranges (1–30 kDa vs 200–5000 Da).

Benefits and Practical Applications

• Rapid screening of mobile phase conditions and column chemistries within a single instrument setup
• Enhanced polymer peak resolution in aqueous and mixed solvents
• Low adsorption surfaces suitable for sensitive biopolymers and PEGylated compounds
• High pressure tolerance enabling high flow rates with small particles

Future Trends and Opportunities

Further work will focus on optimizing ligand density and alternative surface chemistries to fine-tune pore accessibility. Development of intermediate pore size columns (~90 Å) may provide a balance between low dispersion and resolution. Expanding the platform to protein SEC and other biopolymer separations will leverage the robust BEH particle architecture and low dispersion instrument design.

Conclusion

The integration of BEH hybrid diol columns with the ACQUITY APC system enables rapid, high resolution aqueous SEC method development. This platform delivers robust performance, minimal adsorption and flexible solvent handling, streamlining polymer characterization workflows.

References

No external literature cited in the source document.