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The theory behind SEC retention is not fully understood (despite lots of
literature), but for a specific polymers such as pullulan we find that log M
decreases linearly with V (elution volume), except near V
0
and V
T
, where the
curve asymptotically approaches a vertical line.
If we have a monodisperse sample of the same type as the standards, and
obtained under the same experimental conditions, then we can use the
calibration curve as standard curve.
Note that the calibration curve depends on the physical size (hydrodynamic
volume) of the molecules, not the molecular weight. The
calibration curve is therefore different for different polymers.
Thus, calibration based on another polymer (dextran or
pullulan standards) can lead to large errors (factor 2 or
more).
The figure (below) illustrates calibration lines determined for alginate and
pullulan on a certain column system. They differ by a factor of about 4
(vertically). If we use a calibration line for pullulan to estimate the molecular
weight of alginate it becomes 4 times overestimated. This is a common error
in many articles and reports.
SEC calibration curves (example)
10,000
100,000
1,000,000
10,000,000
18
20
22
24
26
Elution volume (ml)
Molecular weight (g/mol)
Pullulan
Alginat
e