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A SEC chromatogram plots a detector signal against elapsed time or elution volume. At constant volumetric flow rate $Q$, the volume delivered after injection is $Q(t-t_{\mathrm{injection}})$; subtraction of extra-column transport volume is needed for a column-only $V_e$. Real peak widths include injection volume, column dispersion, and tubing/detector spreading. Peak position, width, and area answer different questions.

SEC molar mass calibration measures standards under the same column, solvent, and temperature conditions as the unknown. Over a suitable fractionation interval, a common empirical approximation iswhere $M_{\mathrm{ref}}$ specifies the molar mass unit and $b$ has inverse-volume units. Interpolation is conditional on the standards' size–molar mass relationship. SEC measures access associated with hydrodynamic size, so a branched polymer, an extended chain, and a compact protein need not share that relationship.

In this model the $80,000$, $40,000$, and $20,000\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$ standards have hydrodynamic radii $2\sqrt{2}$, $2$, and $\sqrt{2}\mathrm{nm}$ and elute at $7$, $9$, and $11\mathrm{mL}$. An unknown at $8\mathrm{mL}$ would be assigned $M_{\mathrm{app}}=40,000\sqrt{2}\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$ by this calibration. But a $40,000\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$ species that expands from $2$ to $2\sqrt{2}\mathrm{nm}$ elutes at $7\mathrm{mL}$ and is assigned $80,000\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$ without gaining any mass. The numbers demonstrate ambiguity, rather than predict a real unfolding transition.

Changes in conformational isomerism, protein folding, protein denaturation, and protein aggregation can therefore shift peaks. Association usually increases size; compaction can reduce size at unchanged molar mass. Adsorption creates a different ambiguity: delayed elution need not indicate a smaller species. A different solvent can change both polymer coil dimensions and interactions with the packing. The SEC protein conformation experiment tests these alternatives with additional detectors.

For ideal SEC, define the SEC partition coefficient byHere ${\overline{c}}_{\mathrm{pore}}$ averages solute concentration over all pore liquid, including inaccessible regions. $V_e$ is column elution volume. Pure steric exclusion gives $0\le K_d\le 1$; the endpoints mean exclusion and full access.^[ref]

A storage argument explains the relation. Assume a dilute stable solute, unchanged packing and solvent, constant volumetric flow rate $Q$, rapid local chemical equilibrium between pore liquid and interparticle liquid, negligible binding, and negligible transport through the pores by bulk flow. At interparticle concentration $c$, the column holds an amount of substance $n=c{V}_0+K_{d}c{V}_i$. Its exit transports $Qc$ per unit time. The ratio $n/(Qc)$ is the mean residence time in this equilibrium transport model; multiplying by $Q$ gives $V_e=V_0+K_d{V}_i$. For a narrow symmetric SEC chromatogram peak, the peak maximum approximates that mean; a strongly asymmetric peak needs an explicitly stated location convention.

The handbook also uses an operational normalizationThis denominator includes solid matrix, so full access gives $K_{av}<1$ when $V_{\mathrm{matrix}}>0$. It differs from the pore-liquid SEC partition coefficient. Read each source's volume convention before comparing coefficients.^[ref]

For an original numerical example, take $V_0=5\mathrm{mL}$, $V_i=8\mathrm{mL}$, and $V_{\mathrm{matrix}}=3\mathrm{mL}$. Then $V_t=13\mathrm{mL}$ and $V_c=16\mathrm{mL}$. Elution at $9\mathrm{mL}$ gives $K_d=1/2$ but $K_{av}=4/11$. Even a fully included tracer gives $K_{av}=8/11$, not $1$. These are stipulated volumes, not experimental measurements.