Size Exclusion Chromatography

Separate species by pore access and hydrodynamic behavior in the selected buffer.

Size-exclusion chromatography, panels A–D. A: Larger blue species are excluded from pores that smaller orange species can access. B: The excluded species travel ahead through the column. C: They elute earlier. D: Sequential fractions sample the separated bands. This idealized example assumes negligible binding; elution position alone does not establish molecular mass or identity.
Size-exclusion chromatography: four-panel overview. A: Larger blue species are excluded from pores that smaller orange species can access. B: The excluded species travel ahead through the column. C: They elute earlier. D: Sequential fractions sample the separated bands. This idealized example assumes negligible binding; elution position alone does not establish molecular mass or identity. Schematics and traces are conceptual, not measured data; a.u. means arbitrary units. Dashed gradient lines, where shown, indicate direction only. Open full-size figure.

Principle

SEC columns are packed with porous beads. Species that access more pore volume generally elute later, while larger hydrodynamic species generally elute earlier. The observed behavior also depends on shape, hydration, conformation, oligomerization, buffer, and non-ideal interactions with the medium.

SEC is often selected for relatively mild, isocratic handling, but it does not itself demonstrate native structure or activity. An early-eluting feature can be consistent with aggregate or higher-order assembly, but it can also reflect anomalous hydrodynamics or interactions; confirm the assignment with a method suited to the question.

Porous bead cross-section and SEC elution orderLeft: a bead cross-section with wide and narrow pores. Large molecules remain outside, intermediate molecules enter only the wide pores, small molecules enter all pores. Right: absorbance against elution volume with the large species at V0, the intermediate species between, and the small species near Vt, just before Vc.bead matrixlarge: excludedintermediate: partialsmall: fullV0VtVcsize-exclusion windowElution volume (schematic, fraction of Vc)Absorbance (a.u.)0
Figure 1. Separation by pore access (schematic). Species too large for any pore stay in the liquid between beads and elute at the void volume V0; intermediate species enter only wider pores; small species reach nearly all pore liquid and elute near the total liquid volume Vt, slightly before the geometric bed volume Vc. Ideal characteristic elution volumes lie between V0 and Vt, but broadened peak tails can extend beyond that interval. Unexpectedly late characteristic retention can indicate interactions with the medium; anomalously early retention can warrant checking for channelling. Shape and hydration matter, so size here means hydrodynamic size, not mass.

Running conditions

Select a column pore range and operating window from that column’s current instructions. Buffer, flow, sample volume, and load are method-development variables: assess resolution, recovery, stability, and unwanted interactions on the actual sample rather than applying a universal numeric recipe.

Reading the chromatogram

A typical protein SEC trace shows:

  • Early material — species with lower pore access; test any aggregate/assembly assignment.
  • Target-associated peak — a candidate pool, not an identity assignment.
  • Later material — species with greater pore access, which may include small components.

Peak shoulders can arise from partial resolution, overload, heterogeneity, or non-ideal interactions. They are hypotheses to investigate, not automatic assignments of oligomeric or conformational state.

SEC chromatogram simulator

Two proteins on an idealized column. Elution position follows a globular-protein selectivity curve; peak width comes from plate number and sample volume. Change one control at a time and watch Kav, peak widths and Rs.

0510152025V0VtABElution volume (mL)Absorbance (a.u.)solid: sum; dashed: A, dotted: B
Kav A / B
0.247 / 0.614
Ve A / B
11.35 / 17.51 mL
Width 4σ A / B
1.20 / 1.83 mL
Rs
4.06
V0 / Vt
7.2 / 22.1 mL
Sample volume
240 µL

Idealized model: Kav from a logistic curve in log Mr for globular proteins (5% and 95% of the permeation plateau at the range limits), clamped between exclusion (V0) and total permeation (Vt); Ve = V0 + Kav(Vc − V0); σ² = Ve²/N + Vs²/12; w = 4σ; Rs = 2ΔVe/(w1 + w2). Equal amounts give equal peak areas. N is an effective protein plate number, assumed equal for both proteins; it is much lower than a column’s acetone-test specification. The Gaussian with added injection variance approximates a flat-topped band at larger sample volumes. Real peaks tail, non-globular shape changes elution, and interactions with the medium can delay species beyond Vt. Sources: Cytiva™, Size Exclusion Chromatography: Principles and Methods (Kav, Vt/Vc, Rs, sample volume); Groskreutz & Weber 2015 (variance additivity).

Column calibration

Choose standards appropriate to the column pore range and question, then build a calibration only under the stated method conditions. Kav = (Ve − V0) / (Vc − V0), where Ve is the analyte elution volume, V0 the void volume, and Vc the geometric bed volume (some texts write Vt for it; check the convention before comparing values). A log(molecular mass) versus Kav curve yields an apparent molecular mass relative to those standards—not an absolute mass, numerical size measurement, or identity assignment. Use an orthogonal method such as SEC-MALS, analytical ultracentrifugation, MS, or a validated reference method when the decision requires it.

SEC calibration: Kav against log molecular massA sigmoidal curve falls from a plateau just below Kav 1 at low molecular mass to Kav 0 at high molecular mass. Five standards lie on the near-linear middle section. A dashed line from the unknown's Kav meets the curve and drops to the log Mr axis, marked apparent Mr.00.51total permeation (Kav at Vt)exclusion (elutes at V0)fractionation rangeunknown Kavapparent Mrstandardslog Mr (increasing →, schematic)Kav
Figure 2. Calibration concept (schematic, no specific column). Standards of similar shape give a sigmoidal Kav versus log Mr curve that is close to linear inside the fractionation range (amber) and flattens toward exclusion (Kav → 0) and toward total permeation. Because Kav uses the geometric bed volume Vc, the permeation plateau sits below 1. An unknown’s Kav read across to the curve gives an apparent Mr relative to those standards, valid only if it behaves like them.

Sources and scope

IUPAC Gold Book: distribution constant (exclusion) is the authoritative definition reference; direct access was limited in this review. Cytiva™ Superdex™ 200 Increase instructions for use (official, accessed 2026-09-25) illustrates method-specific operational and performance instructions. Cytiva™, Size Exclusion Chromatography: Principles and Methods (official handbook, read 2026-09-25) is the basis for the figures and simulator conventions. Consult the exact column manual for ranges and operating limits.

Try it on a trace

Open a two-column chromatogram in the workbench, or load the synthetic example. No account needed.

Open the workbench

Send feedback

What is it about?

At least 5 characters.0 / 4000

So we can reply. Leave it empty to stay anonymous.

Page: /learn/techniques/sec