Advancing HPLC/ UHPLC Analysis with Multi-Angle Light Scattering Technology

Significance of the topic

The integration of multi-angle light scattering (MALS) with size exclusion liquid chromatography (SEC and UHPLC-SEC) enables absolute determination of macromolecular properties such as molecular weight, size, conformation, and aggregation state without reliance on reference standards or column calibration curves. This capability addresses critical challenges in protein and polymer analysis, including non-ideal column interactions, shear sensitivity in high-pressure systems, and complex sample heterogeneity.

Objectives and study overview

This compendium brings together insights from three applications-focused articles and interviews: an overview of next-generation SEC-MALS instrumentation (Daniel Some, Wyatt Technology), case studies of analytical light scattering in a biophysics core (Ewa Folta-Stogniew), and the extension to UHP-SEC-MALS for synthetic polymer analysis (Daniel Some). The primary objectives are to highlight technological advancements, demonstrate real-world applications across protein and polymer systems, and compare performance between traditional HPLC-SEC-MALS and UHPLC-SEC-MALS.

Methodology and Used Instrumentation

Online coupling of SEC/UHPLC with multi-detector arrays provides a rich, orthogonal data set:

• MALS provides direct measurement of molecular weight (M) and radius of gyration (Rg) from first-principles light scattering equations.
• Embedded dynamic light scattering (DLS) modules offer hydrodynamic radii (Rh) and insight into molecular conformation.
• Refractive index (RI) and UV absorption detectors quantify sample concentration and enable stoichiometry analysis for complexes or copolymers.

Used Instrumentation:

• Wyatt DAWN MALS detectors with 18-angle static scattering and optional WyattQELS dynamic module.
• Optilab refractive index detectors (Optilab UT-rEX).
• ViscoStar differential viscometry modules.
• Waters Acquity UPLC with APC SEC columns (125 Å and 450 Å) for UHP-SEC.
• ASTRA software for data acquisition and analysis.

Results and Discussion

Case studies illustrate the power of MALS-based detection:

• Protein–DNA complexes: SEC-MALS-UV-RI resolves unexpected stoichiometries and concentration-dependent dimerization of FIR with DNA.
• Membrane proteins in detergent: Triple-detector analysis differentiates bound detergent masses and corrects elution artifacts for LamB and alpha-hemolysin.
• Oligomer morphology: PopZ scaffolding protein variants display non-globular asymmetry; combined static and dynamic scattering yields Rg, Rh, and molecular weight to reveal high-aspect-ratio assemblies.
• Dimerization constants: SEC-MALS determines monomer–dimer equilibria and salt-dependent stability for proteins such as NUCB1 and SecA mutants.
• UHP-SEC-MALS vs. HPLC-SEC-MALS: Linear polystyrene standards show perfect weight-average molar mass and size agreement, confirming minimal shear under UHP conditions.
• Non-linear polymers: Stiff rod-like epoxy resins and branched PMMA deviate from standard calibration but are accurately characterized by absolute light scattering.

Benefits and practical applications

SEC/UHPLC-MALS enables:

• Absolute molar mass and size determination without calibration standards.
• Robust analysis of non-ideal, heterogeneous, and non-globular macromolecules.
• Reduced run times and solvent/sample consumption via UHP-SEC.
• Enhanced productivity through real-time system health monitoring and modular detector platforms.

Future trends and opportunities

Continued integration of online detectors promises deeper structural insights and higher throughput, including:

• Expansion of column chemistries and customizable flow cells for problematic separations.
• Automation of multi-detector diagnostics and predictive maintenance.
• Broader adoption in quality control, biopharma, polymer R&D, and nanomaterials characterization.
• Coupling with orthogonal techniques (e.g., mass spectrometry, light scattering imaging) for multimodal analytics.

Conclusion

The advent of next-generation MALS detectors and their application in both HPLC and UHPLC contexts represents a step change in macromolecule analysis. By delivering absolute, reference-free measurements of molecular weight, size, conformation, and interactions, SEC-MALS and UHP-SEC-MALS have become indispensable tools for research and quality assurance in biotechnology and polymer science.

Reference

• Some, D. Advancing HPLC/UHPLC Analysis with Multi-Angle Light Scattering Technology. LCGC June 2019.
• Folta-Stogniew, E. Applications of Analytical Light Scattering in a Biophysics Core Facility. LCGC June 2019.
• Some, D. UHP-SEC-MALS: Absolute Characterization of Polymers with Light Scattering and UHP-SEC. LCGC June 2019.