Handbook of Size Exclusion Chromatography and Related ...

at 1008C under vacuum. The hydrophilic nature of the “polymaltose” phase
allowed the exclusion of all but the most basic proteins. The chemistry of
the popular TSK-GEL SW columns has not been described in the open literature.
The SW stationary phase has been referred to as a “glycol ether-type bonded
phase” similar in nature to the diol phase (82), containing the structure
CH2C(OH)HCH2O (14).
3 CALIBRATION
As mentioned in the introduction, in high-performance gel filtration chromatography,
silica- rather than resin-based packing materials are more widely used for
biopolymer separations. This is true for peptides, proteins, and possibly also for
nucleic acids, although size exclusion is not a common technique for determining
the molecular weight or for isolating this class of compounds. Polymer-based
packings are the material of choice for most other water-soluble polymers,
including oligo- and polysaccharides and the many examples of natural and
synthetic polymers discussed in other chapters.
GPC is routinely used for determining the average molecular weight of an
organic soluble polymer and the distribution of the molecular weights around this
mean. Although desirable, it is often not possible to obtain a reliable value for the
molecular weight of a protein by GFC. Despite elaborate bonding procedures, all
available silica-based (and polymer-based) packings show some deviation from
ideal size exclusion behavior for proteins. Unreacted and accessible silanol groups
are responsible for secondary retention mechanisms, resulting in inaccurate MW
estimates. This section discusses calibration curves for proteins and other
biopolymers. A review of the various parameters responsible for nonideal elution
behavior follows.
Under ideal SEC conditions, all solutes elute at a retention volume VE that is
larger than the interparticle volume Vi but smaller than the mobile-phase volume
VT (which is the sum of Vi and the pore volume VP). The distribution coefficient
KD for elution by ideal SEC is given by Eq. (13), in which KD varies from zero for
a fully excluded solute to 1 for a small molecular weight solute capable of
penetrating all the pores:
VE ¼ Vi þ KDVP
(13)
The selectivity curve of a packing material is obtained by plotting the elution
volume, or some function of VE, vs. an expression of the solute size. It is known
that the size for a random coil of a linear polymer is correlated with its molecular
weight. Thus, for polystyrene standards of known molecular weight, a unique pore
diameter can be assigned at which the polymer is excluded from the pores of a
packing material. With dextrans, the relative volume of the random coil is smaller
© 2004 by Marcel Dekker, Inc.