 SEC molar mass calibration

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.