For most SEC applications involving polydisperse macromolecules using high-efficiency columns and standard calibration methods,
the relative molecular weight accuracy caused by column peak broadening is about 1%, which is quite acceptable considering
other sources of extracolumn peak broadening. Some of these other sources of error, such as injection volume, can be easily
reduced. Furthermore, broadening and skewing corrections can be applied to further increase molecular weight measurement accuracy.
If greater resolution is required for the SEC analysis of small molecules and oligomers, enthalpic separations should be considered
because of its higher peak capacity. Furthermore, for monodisperse biopolymers, MS is the obvious choice.
We have been suggesting for a number of years that a combination detector cell would certainly be advantageous and a major
step forward in the advancement of SEC. We envision a combination detector in which a single low-volume cell is used for four
simultaneous measurements: 1. light scattering (integral or dynamic) for molecular weight and size measurements; 2. refractive
index as a concentration detector; 3. UV absorbance for either concentration or for chemical heterogeneity; and 4. FT-IR for
short-chain branching or chemical heterogeneity. One possibility would be to use a charge-coupled device for multiangle and
multiwavelength light scattering detection. The elimination of interdetector volume would greatly reduce peak broadening and
Decreasing the particle size of packings, of course, is fundamental to high-performance separations. Although polymers with
large hydrodynamic volumes and elongated conformation will fragment at high linear velocity, as discussed in some detail earlier
(1,14), we must realize that most polymers of commercial interest fall below the roughly 1 × 106 limiting molecular weight value. Regardless, at least one column vendor has addressed this issue by supplying columns of
larger particle size and larger frit sizes, specifically for the analysis of large macromolecules (see Table IV).
The pore size of packings controls the molecular weight operating range over which a separation is achieved. Some packings
are able to separate molecules ranging from monomers to an estimated (that is, extrapolated) molecular weight value of up
to ~107 or ~108 depending on the standards used. (Note that compact polymers, like branched polysaccharides, synthetic dendrimers, and globular
proteins have much higher exclusion limits.) This range obviously covers all possible applications, so it is unlikely that
there will be any major developments in this area. For single-pore-size packings (Table II), a tight pore-size distribution
would be very beneficial, provided that a range of pore sizes are available that have similar pore volumes for a given packing
Reduced-Diameter Columns and Small-Bore Columns
Low-dispersion HPLC instrumentation and MS require the use of narrow-bore SEC columns, rather than the standard analytical
internal diameters of 6–8 mm. However, total pore volume becomes critically small and extracolumn peak broadening will severely
limit applicability of small bore-size SEC columns. The future trend involving SEC column internal diameter actually is in
the opposite direction; large internal diameter columns offer more advantages.