Size-exclusion chromatography (SEC) is a technique that separates analytes based on their hydrodynamic radius - their effective size in solution - rather than chemical properties or molecular weight. The column is packed with porous particles of defined pore size. Larger molecules are excluded from most pores and elute first, while smaller molecules enter more pores, travel a longer path, and elute later. Unlike other chromatographic methods that rely on chemical interactions, SEC acts as a molecular sieve, making it ideal for analyzing the size distribution of proteins, polymers, and other macromolecules.

Advancements in column technology, improved detectors (such as viscometry, light scattering, and chemical detectors), and two-dimensional coupling now allow SEC to analyze molecular weight distribution as well as the structure and composition of biomolecules in greater detail.

The principle of SEC involves using porous particles within a column to filter molecules by their size, geometry, or molecular weight. The column is filled with spherical beads containing pores of a specific size.

Smaller molecules penetrate the pores of the particles, slowing their transit through the column. Larger molecules are unable to enter the pores and are eluted in the column's void volume. Molecules are then separated by size and eluted in order of decreasing molecular weight. The smaller the molecule, the longer the retention time.

SEC differs from other chromatographic techniques because it classifies molecules based on size rather than interaction. SEC is used for protein fractionation and other water-soluble polymers. It can also determine molecular weight and particle size using standard proteins.

Depending on the type of analyte and the mobile phase, SEC can be categorized into:

Aqueous size exclusion
Aqueous size-exclusion chromatography is used for separating water-soluble macromolecules using columns that are packed with particles that are diol-based and utilize an aqueous mobile phase. This technique is used to analyze or characterize proteins, peptides, and other biomolecules (e.g., antibodies, immunoglobulins, protein complexes, protein aggregates) and for desalting.

Non-aqueous size-exclusion/ gel permeation chromatography (GPC)
Non-aqueous size-exclusion chromatography is a technique that is used for the purpose of separating analytes on the basis of their size. This is a technique in which larger analytes are eluted first, followed by smaller analytes, as the latter has more interaction time with the stationary phase. It is used for analyzing polymers such as adhesives, oils, plasticizers, plastics, resins, and rubbers/elastomers.

• Analyzing the structure and conformation of any intrinsically distorted proteins.
• Separating and quantifying high- and low-molecular-weight proteins in biopharmaceuticals (e.g., monoclonal antibodies, antibody-drug conjugates, bispecific antibodies).
• Ensuring regulatory compliance by evaluating size variants and aggregate levels for safety, potency, and pharmacokinetics.
• Analyzing antibody-drug conjugates to determine the drug-to-antibody ratio, drug load distribution, and levels of free drug/antibody.
• Monitoring chain association and verifying structural integrity in bispecific antibodies and other complex monoclonal antibody products.
• Characterizing plasmid deoxyribonucleic acid (e.g., distinguishing supercoiled and open-circular isoforms) and assessing mRNA quality (e.g., aggregate levels, polyadenylate [poly(A)] analysis).
• Investigating mRNA–lipid nanoparticle formulations for size, heterogeneity, and potential adsorption issues.

• Conceptual and experimental simplicity, especially when using a single concentration-sensitive detector such as a differential refractometer or ultraviolet/visible spectrophotometer.
• Ability to provide average molar mass and complete molar mass distribution in one run, eliminating time-consuming fractionations.
• Absolute molar mass determination by coupling with viscometry (universal calibration) or static light scattering, without needing separate calibration curves.
• Predictable elution window because the distribution coefficient for size-exclusion chromatography lies between 0 and 1.
• Broad dynamic range is achievable by combining columns with different pore sizes, allowing the separation of molecules across orders of magnitude in molar mass.
• Capability to separate analytes ranging from small oligosaccharides to macromolecules hundreds of nanometers in radius.
• Minimal enthalpic effects, enabling purely size-driven separations even for some diastereomers without temperature-dependent interactions.

Size-exclusion chromatography is limited to separating molecules by size without regard to other properties like charge or hydrophobicity. SEC may also struggle to separate molecules of similar sizes because they elute at nearly the same time. Additionally, the number of bands that can be used is limited because the chromatogram's time frame is short. SEC is best for larger molecules like proteins and polymers and may not be effective for very small molecules or those lacking size-based separation characteristics.

Other limitations include:

• No standard molecular weight marker exists for comparison.
• Only a limited number of baseline resolved chromatographic peaks can be accommodated because the time scale of the chromatogram is short.
• The load capacity is limited; no more than 5% of the column volume should be loaded.

Modern SEC methods are reliable, but problems may still occur. Key factors such as column dimensions, flow rate, sample volume, and mobile phase properties can influence separation efficiency and resolution.

Common issues include:

• Poor Resolution may be caused by overloading, incorrect flow rate, or inadequate column dimensions.
• Tailing Peaks may be caused by overloading, poor column packing, or interactions between the sample and the stationary phase.
• Unexpected Retention Times may be caused by interactions between the sample and the stationary phase or changes in the mobile phase composition.
• High Pressure: Scientists sometimes encounter problems dealing with too high pressure.
• Loss of Resolution: Scientists sometimes encounter problems such as loss of resolution.
• Drifting Baselines: Scientists sometimes encounter problems such as drifting baselines.
• Clogged Columns can lead to poor resolution and increased back pressure.
• Detector Sensitivity: If the detector is not sensitive enough, it may be difficult to detect low-concentration samples.

Matt Boag

Global Biopharmaceutical Marketing Manager

Matt Boag is the Global Biopharmaceutical Marketing Manager at Phenomenex, where he leads strategic marketing and product initiatives that empower scientists in the development and characterization of biotherapeutics. With over a decade of experience in the pharmaceutical and biopharmaceutical industries, Matt brings deep expertise in regulatory compliance, analytical chemistry, and process development. He holds a BSc in Biochemistry/Biotechnology and an MSc in Chemistry, combining scientific rigor with commercial insight to drive innovation and customer success.

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