Size-exclusion chromatography (SEC) separates species purely by
hydrodynamic size: bigger aggregates elute first (less time inside
the porous resin beads), the correctly-folded monomer elutes next,
and small clipped fragments elute last. The trace shows a detector
response (UV absorbance) as the sample passes through — the area
under each peak is proportional to how much material is in that
size class. The rotating model shows the same molecule's higher-order
structure (HOS): more aggregate pulls extra domains into a clumped,
less-native shape.
%purity = Area(monomer) / Area(total) × 100
Rs = 2·(RT2 − RT1) / (width1 + width2) ← peak resolution
- Aggregate level — fraction of high-molecular-weight species; raises the early "aggregate" peak and visibly clumps the protein model.
- Fragment level — fraction of clipped/degraded species; raises the late shoulder peak.
- Column resolution — how cleanly adjacent peaks separate; low resolution merges neighbouring peaks, which is exactly the comparability problem analytical scientists fight in real assays.
- Assay mode — Purity (SEC trace), Identity (a peptide-map-style fingerprint of many small peaks) or Potency (a sigmoidal dose–response curve used to confirm the molecule is still biologically active).