Handbook of Size Exclusion Chromatography and Related ...

absence of secondary (i.e., non-SEC) retention mechanisms, the resulting
calibration curve is the well-known S-shaped curve containing a linear portion.
Thus, a column is selected for which the solutes of interest elute on the linear
portion of the curve. This method requires narrow distribution standards and
samples that have the same molecular conformation as the standards. Without
appropriate standards, the calculated molecular weight for an unknown can be in
error by a factor of 2 or 3 and up to an order of magnitude under the most
unfavorable conditions (45).
The effect of pore diameter upon KD values for globular proteins was
investigated by Gooding and Hagestam Freiser (11). For the same protein, the KD
value was approximately 0.2 units lower on a 100 A ˚ material vs. a 300 A ˚ material.
The slope of the linear portion of the calibration curve indicates the homogeneity
of the pore structure. The smaller the slope, the more pores there are of the same
size and the higher the potential for resolution of two solutes with similar
molecular weight (10, 19,33). The steeper the slope, the larger the variety of pores
of different size and the broader the range of molecular weights that can be
separated.
When no narrow molecular weight distribution standards are available, then
the single broad standard calibration or integral molecular weight distribution
method provides the most accurate molecular weight measurements. Reference 8
outlines this method, which requires knowledge of the complete molecular weight
distribution [i.e., weight- (MW) and number-averaged (MN) molecular weights] for
a single broad molecular weight polymer. Unlike narrow standard methods,
calibrations obtained by broad standard methods are affected by instrumental peak
broadening. Without corrections, this calibration error can cause errors in the
molecular weight analysis of polymer samples. The GPC calibration curve is
obtained by matching those molecular weight and elution volume values that
correspond to the same value of sample weight fraction on the molecular weight
distribution and GPC elution curves (8).
Approximate molecular weights can be obtained when the single broad
standard method or universal calibration method is not feasible (8,45). The
accuracy of this method depends upon the unknown polymer having the same
structure and molecular weight distribution as the standard.
The universal calibration method can be utilized for the molecular weight
determination of known polymers. This method is valid when polymer retention is
determined only by its hydrodynamic volume. In this case, a plot of the logarithm
of the intrinsic viscosity times molecular weight, log[h]MW vs. the elution
volume of the polymer provides a calibration curve that applies to all polymers.
The resulting universal calibration curve is approximately the same for all
polymers (random coil, rigid rod, or spherical). First, a peak position calibration is
performed for the molecular weight range of interest using narrow molecular
weight standards, such as polystyrene, providing a value for M2. After obtaining
© 2004 by Marcel Dekker, Inc.