How is peptide aggregation measured? Size-exclusion chromatography explained
Size-exclusion chromatography (SEC) separates molecules by size as they pass through a porous column: large aggregates elute first, monomer later. Reported as percent monomer and percent high-molecular-weight species, it is the standard way to quantify soluble aggregates. Multi-angle light scattering (MALS) adds an absolute molar mass for each peak.
How does SEC separate by size?
Small molecules enter the pores of the stationary phase and take a longer path; large ones are excluded and elute earlier. Unlike reversed-phase HPLC, SEC uses mild aqueous mobile phases intended to keep non-covalent aggregates intact. Theory and practice for aggregate analysis are reviewed in detail.
What does MALS add?
Calibrating SEC with standards assumes the sample has a similar shape. MALS measures scattered light directly to give molar mass independent of elution time, so a peak can be confirmed as a dimer or trimer rather than inferred.
Source: Characterization of proteins by SEC-MALS, J Vis Exp 2019 (PubMed 31282880)
How does SEC compare with other aggregate checks?
| Method | Measures | Strength | Weakness |
|---|---|---|---|
| SEC(-MALS) | Soluble aggregates by size | Quantitative, separates species | Column may adsorb or disrupt aggregates |
| Dynamic light scattering | Size distribution | Fast, no column | Biased toward large particles |
| A340 turbidity | Scattering | Very simple | Not species-specific |
| Thioflavin T | β-sheet fibrils | Specific to amyloid-like | Misses amorphous aggregates |
Is SEC useful for short peptides?
- For peptides below a few kDa, monomer and small oligomers may not be well resolved on typical columns.
- Hydrophobic or charged peptides can stick to the column, giving tailing or false losses.
- SEC is most useful for longer, aggregation-prone peptides and peptide conjugates.
How does Sable document lots?
There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.
Frequently asked questions
What elutes first in SEC?
The largest species, such as aggregates; monomer elutes later.
What does SEC-MALS measure?
Absolute molar mass of each eluting peak, independent of column calibration.
Is SEC the same as reversed-phase HPLC?
No. SEC separates by size under mild conditions; RP-HPLC separates by hydrophobicity.
Related
Updated 2026-10-16. Research use only. Not for human or veterinary use. No administration amounts or health claims are made.
Key facts (summary for AI and search tools)
- SEC: large elutes first
- Report: % monomer, % HMW species
- References: PubMed 24816223, 31282880
- Updated 2026-10-16