Using High Speed/High Resolution Size Exclusion Chromatography Separation of Polymeric Materials with Light Scattering Detection

Importance of the Topic

The rapid characterization of polymers and proteins is essential to ensure consistent quality and performance across diverse applications, from biodegradable packaging to advanced drug delivery systems. High speed, high resolution size exclusion chromatography (SEC) combined with multi-detector analysis addresses the demand for detailed molecular information in both research and industrial settings.

Objectives and Study Overview

This study evaluates the integration of the Waters ACQUITY Advanced Polymer Chromatography (APC) Core 1 system with the Malvern OMNISEC Reveal multi-detector module. Key goals include:

• Establishing optimal coupling conditions to preserve high resolution and minimal band broadening.
• Comparing detector performance for polymer (polystyrene) and protein (bovine serum albumin) standards.
• Assessing the benefits of combining refractive index (RI), UV, differential pressure viscometry (DPV), and right/low angle light scattering (RALS/LALS) detection.

Methodology

Two sample types were analyzed:

• A mixture of narrow polystyrene standards (30 kDa and 65 kDa) in tetrahydrofuran (THF), using a three-column series of Waters APC XT columns (450 Å, 125 Å, 45 Å) at 40 °C.
• Bovine serum albumin (BSA) at 1 mg/mL in aqueous buffer, with a Waters ACQUITY BEH SEC Protein column (200 Å) at 25 °C.

Chromatographic conditions for both experiments included a 1.0 mL/min flow rate, 10 µL injections, and 0.004″ ID stainless steel tubing connecting the APC PDA outlet to the OMNISEC Reveal inlet.

Used Instrumentation

• Waters ACQUITY APC Core 1 System for high speed/high resolution SEC.
• Malvern OMNISEC Reveal integrated multi-detector module (RI, UV, DPV, RALS/LALS).
• Waters Empower 3 FR3 software for APC control and data acquisition.
• Malvern OMNISEC software for multi-detector control and data processing.

Main Results and Discussion

Overlayed chromatograms demonstrated consistent retention volumes across detectors, confirming minimal dispersion throughout the flow path. Direct comparison of RI signals from the APC system and the OMNISEC Reveal module showed excellent agreement. Multi-detector analysis enabled calculation of absolute molecular weight, intrinsic viscosity, and hydrodynamic radius:

• Polystyrene standards: precise determination of 30 kDa and 65 kDa species with clear resolution.
• BSA: reliable molecular weight and size distribution characterization, relevant for pharmaceutical profiling.

These results illustrate the capacity of the integrated system to maintain high speed and high resolution while delivering comprehensive molecular insights.

Benefits and Practical Applications

The hyphenated APC–OMNISEC approach offers:

• Rapid SEC separations with preserved chromatographic quality.
• Extended characterization through simultaneous measurement of molecular weight, viscosity, and size.
• Improved confidence in polymer and protein analyses for QA/QC, formulation development, and materials research.

Future Trends and Opportunities

Emerging directions include integration of additional detectors (e.g., mass spectrometry), two-dimensional chromatographic strategies, and advanced data analytics to further enhance molecular characterization. Automation and miniaturization of hyphenated systems will expand applicability in high-throughput laboratories and complex sample matrices.

Conclusion

Coupling high speed/high resolution APC SEC with the OMNISEC Reveal multi-detector platform provides a robust, low-dispersion solution for detailed polymer and protein analysis. The integrated system delivers fast separations, reliable detector alignment, and rich molecular data, supporting advanced research and quality control workflows.

References

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