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Size Exclusion Chromatography Calibration Curves

To assess the size of analytes as they elute from the SEC column, a calibration curve is required. SEC calibration curves rely on measuring the elution volume (VR) of markers with known molecular weights, and comparing the elution volume of analytes against this.

This typically produces a third order polynomial line, with a linear central section that can be used for assessment of unknowns. Analytes and markers must fall within the exclusion and permeation limits of the column; the exclusion limit defines the size of molecules too large to fit in any of the stationary phase pores, and therefore, travel through the column with no retention at all; the permeation limit is set by the smallest analytes that are able to penetrate all pores and elute with no separation from each other.

A low calibration slope will have larger elution volume differences between similarly sized analytes than steeper slopes, and so produces more accurate molecular weight information.

SEC calibration curve

Figure 1: SEC calibration curve. Sample molecules with molecular weights above a certain value are totally excluded from the packing pores and have a retention volume VR = V0 (equal to the interparticle volume within the column). Molecules with molecular weights less than a certain value have complete access to the pores (total permeation) and have a retention volume (V0 + Vi) = Vm.

When selecting markers for SEC calibration, it is important to remember that molecule structure and hydration also affect elution volumes. Therefore, calibration standards should be as similar as possible to the analytes being assessed, and any necessary correction factors (such as molecular weight per unit chain length, Q-factor) should be applied where required to ensure results are accurate.

 
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