Handbook of Size Exclusion Chromatography and Related ...

60–120 A ˚ are large enough to be fully accessible for the molecular weight range
(below 2000 dalton) of most organic compounds analyzed by HPLC.
Unlike silica, polymer-based particles are readily available in smaller pore
sizes.Smallporesizesilicas,suchasMerck40orDavisil20,arenotcommercially
available in the 5–10 mm particle size range suitable for high-performance SEC.
Syloid 63, afood additive produced by WR Grace, is an irregular 9mm particle
size silica with 22 A ˚ pores and 0.4 mL/g pore volume. Its broad particle size
distribution does not make it readily suitable for high-performance SEC of small
molecules.
Table5liststwolinesofcommerciallyavailablesilica-basedgelpermeation
columns.TheselectionwaslimitedtotheZorbaxandLiChrosphersilicasbecause
these materials were specifically developed for gel permeation chromatography.
Zorbaxsilicahasa6mmparticlesizeforoptimumefficiency.Theporesizeswere
chosen such that alinear calibration curve is obtained when coupling columns of
different pore sizes. In addition to plain silicas, Zorbax silicas are also available
derivatized with trimethylchlorosilane, providing asurface that is less adsorptive
for certain organic soluble polymers. Several important water-soluble industrial
polymers,suchaspolyacrylamide, polyacrylic acid,andpolyvinylalcohol,donot
require deactivation of the silica surface to obtain ideal size exclusion behavior.
LiChrospher silicas are 10 mm in size; they vary in pore size from 100 to 4000 A ˚
to allow the separation of very large polymers.
Table 6 summarizes the most well-known silicas used in gel filtration
chromatography.Note that all the siicas are derivatized. The diol functionality,or
some variation thereof, is the most widely used. Because most proteins have
molecular weights well below 1million dalton, they can be separated on silicabased
SEC columns with pore sizes of 500A ˚ or less. Table 7 shows the
fractionation ranges for globular proteins in common buffers and under denaturing
conditions on TSK-GEL SW columns varying in pore size from 125 to 500 A ˚
(58). Table 7 also shows the fractionation ranges for double-stranded DNA
fragments (59). Note that globular proteins are more compact in solution than
double-stranded DNA fragments. Using acrylic-based TSK-GEL PWXL columns,
DNA fragments of up to 10 times this size can be separated (60).
2.6 Surface Area
Independent of other qualities, surface area is a crucial parameter in the
development of an adsorbent because it determines its capacity for purifying or
drying chemicals or for catalyzing a reaction. In contrast to the techniques used in
interactive chromatography or catalysis, an ideal size exclusion support is not
chemically or physically attractive to any sample component. Size exclusion
requires the presence of pores, and thus surface area is still a critical factor in the
design of SEC packing materials. A discussion of hydrodynamic size exclusion
© 2004 by Marcel Dekker, Inc.