The State of the Art and Future Trends of Size-Exclusion Chromatography Packings and Columns - - Chromatography Online
The State of the Art and Future Trends of Size-Exclusion Chromatography Packings and Columns

LCGC North America
Volume 7, Issue 30, pp. 544-563


There have been studies on the use of SEC columns for performing both size exclusion and hydrodynamic chromatography (HDC) in a single run (15). In HDC, excluded macromolecules are separated within the interstitial volume of the packed bed by sampling different velocity streamlines. For example, larger macromolecules, on average, spend more time in the faster velocity streamlines than smaller-size macromolecules do. Smaller macromolecules can get closer to the walls of adjacent particles and can sample, on average, slower moving stream lines. The net result is that high-molecular-weight components are eluted first, followed by smaller-sized chains; the same elution order as SEC.

Although the dual separation mechanism is an interesting concept, HDC suffers from two of the same limitations as SEC does: HDC has a small elution volume range, in effect, the interstitial volume is close to the pore volume. Furthermore, macromolecules are exposed to the same elongational strain rates as in SEC; however, elongational strain at pore openings is absent with HDC. As a result, there is probably less shear rate degradation of macromolecules during HDC compared to SEC (14). Nevertheless, the single benefit HDC has over SEC is its lower peak broadening caused by the absence of nonequilibrium effects, that is, elimination of mass transfer of macromolecules into and out of pores, especially if nonporous particles are used for HDC. It is important to realize that HDC of higher-molecular-weight macromolecules is possible, only if extremely low linear velocities are used.

It is of interest to realize that the HDC mechanism is probably present to some extent in all SEC separations, but probably goes unnoticed since polymer peaks elute in the same order as with SEC. Dedicated SEC–HDC columns have yet to be commercialized for polymer separations, although specialized columns consisting of larger-diameter packings for particle-size analysis are available.

Nonporous Packings

Although pellicular particles offer improved flow properties and appear to have higher efficiency than porous SEC packings, their limited interstitial volume places more emphasis on using SEC systems with exceptionally low extracolumn volume. Furthermore, they have rather steep calibration slopes, thus they seem to be of limited interest unless used in the HDC mode.

Monolithic Columns

Monolithic columns have received more attention recently, but currently lack pores of the correct dimensions for size separations that would compete with traditional SEC columns (16). It is anticipated that work will continue in this area, but results may not be of commercial relevance.

High-Throughput Analysis

The usual approach for rapid analysis is to use short or narrow-bore columns, preferably packed with small particles for increased efficiency. However, the difficulty inherent in using these approaches for SEC is pore-volume reduction, which will magnify errors caused by extracolumn volume, and flow-rate and temperature variability, all of which will affect baseline drift and detector noise. Considering these difficulties, SEC instrumentation has been introduced that has exceptional temperature control and flow-rate stability, as in the case of Tosoh Bioscience's recently introduced EcoSEC GPC system (17) and Agilent's PL-GPC 220 system for dedicated SEC operation at high temperature.

Still another way of performing high-speed SEC separations, put forward by Polymer Standards Service, is to use large-diameter, short columns operated at high flow rates. These short and fat asymmetric columns have excellent resolution because of their large pore volume.

Related to high-throughput separations for the analytical laboratory setting is the need for on-line, at-line, or off-line SEC instrumentation, specifically designed for process control of polymerizations or quality assurance of polymers, which appears to have taken a back seat. There is no reason why SEC instrumentation, equipped with all necessary safety features, should not be an integral part of plant control laboratories involved with polymer production or biopolymer isolation and purification.

Packing Composition

One of the more vexing problems encountered in SEC used to be the limited availability of packings for the analysis of water-soluble polymers, specifically polyelectrolytes and especially cationic polymers that are very adsorbent. As shown in Table IV, there are now a number of packings available for these types of polymers.

Even though it appears that advancements in SEC column development have leveled off, there is always room for improvement, especially for stable, high-porosity structures with sufficient pore volume. The new types of hybrid packings introduced by Waters (9), for example, appear to be a promising approach for the development of new packings.


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