Fig 1. Cytiva Superdex 75 Size Exclusion Chromatography (SEC) column
Separation of molecules is performed using Size Exclusion Chromatography (SEC), which separates molecules based on size as they pass through a porous resin. Widely used for both preparative and analytical purposes, SEC plays a key role in the purification and characterization of proteins, enzymes, polysaccharides, nucleic acids, and other biological macromolecules. Advances in SEC technology continue to expand its capabilities, enabling higher resolution, enhanced molecular profiling, and more efficient workflows across a range of complex applications.
Principles of gel filtration and size exclusion chromatography
Multi-Detection Size Exclusion Chromatography
Traditional SEC typically relies on single-point detectors such as refractive index (RI) or UV/Vis absorbance to monitor eluting samples. While these methods effectively estimate concentration or detect specific chromophores, they provide limited structural or compositional insight. Increasingly, a more comprehensive characterization is required, driving the use of more sophisticated detection tools that utilize multiple detectors.
- Multi-angle light scattering (MALS) detectors measure the light scattered by a sample at many angles to generate a Debye plot from which absolute molecular weight can be determined without the need for molecular standards. For non-uniform, anisotropic samples, where light scattering varies at different angles, the radius of gyration can also be calculated from the scattering angle to give molecule size.
- Viscometer detectors can determine a sample's intrinsic viscosity, reflecting its molecular volume and conformation in solution. Viscometry is particularly useful for studying structural properties such as polymer branching, protein folding, or aggregation behavior.
- Multi-detector SEC typically combines MALS, viscometer, RI, and UV detectors to deliver a robust profile of each analyte. This enables researchers to determine molecular weight, size, concentration, and conformation in a single analytical run, greatly enhancing the depth and reliability of SEC-based analysis.
Fig 2. Schematic drawing of separation principles in chromatography purification
2D-LC Approaches Involving SEC
Two-dimensional liquid chromatography enhances separation power by combining orthogonal techniques. Separating analytes based on multiple parameters, in addition to size, can further resolve a sample and increase purification. Common pairings include:
SEC × Reversed-Phase (RP) Chromatography: SEC can be used before RP protein separation based on hydrophobicity. This technique is a powerful tool for analyzing complex samples such as therapeutic products or protein aggregates.
SEC × Ion Exchange Chromatography (IEC): The development of biopharmaceuticals, particularly monoclonal antibodies, relies on understanding the variation in charge across proteins in a sample, which can significantly impact stability and efficacy. Combining SEC with IEC, which separates a sample based on charge, provides effective charge variant analysis.
SEC for Non-Traditional Analytes
Size Exclusion Chromatography is traditionally used to separate proteins and other biological macromolecules. However, SEC techniques have been adapted for analyzing larger, more complex structures.
Cytiva demonstrated a scalable workflow for the isolation of exosomes from a sample using SEC combined with tangential flow filtration that is significantly more gentle than typical ultracentrifugation techniques. Elsewhere, SEC has been used in 2D-LC approaches to isolate nanoparticles for analysis based on size and other biologically relevant properties.
In materials science, complex synthetic macromolecules often exhibit broad polydispersity and branching. When combined with multi-detector systems, SEC can provide detailed information on molecular weight distribution, conformation, and branching, critical for materials development and regulatory compliance.
Cytiva: A Legacy of Innovation
From the development of Whatman™ filter paper to the introduction of the first gel filtration resin Sephadex™, Cytiva has led the way in advancing chromatography technologies. As molecular characterization demands grow increasingly complex, Cytiva continues to innovate, expanding the boundaries of what chromatography can achieve.
Check out the Size Exclusion Chromatography Handbook from Cytiva to see how you can boost your chromatography capabilities and achieve more precise, insightful results in your workflows.
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