Peak Broadening

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 skewing.

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).

Pore Size

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 type.

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.