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

Jun 30, 2012

For this month's installment, guest authors Barth and Saunders discuss the present state of size-exclusion chromatography (SEC) columns for the separation of synthetic polymers and biopolymers. A comprehensive review of commercially available standard columns is included along with coverage of specialty columns ranging from those for small molecules to ultrahigh-molecular-weight polymers. Newer products such as low-bleed and high-throughput columns are highlighted. Column selection criteria are suggested and future directions in the technology are explored.

Recently, we provided in-depth coverage of the basics of size-exclusion chromatography (SEC) (1). In this installment, we expand on that coverage and discuss the present state of affairs with respect to SEC columns for molecular size separation of synthetic polymers and biopolymers. A comprehensive review of commercially available SEC columns from a dozen leading chromatographic companies is included ,with an emphasis on individual pore-size columns, mixed-bed packings, and wide-pore packings. Also tabulated are specialty columns for the SEC analysis of small molecules and oligomers, ultrahigh-molecular-weight polymers, water-soluble polymers, cationic polyelectrolytes, polyolefins, polar and nonpolar polymers, and carbon nanotubes. "Low-bleed" columns for use with light-scattering detectors are considered, including columns of unusual dimensions for high-throughput and rapid analysis. For those who develop SEC methods, we also include an approach for column selection based on our combined decades of experience since the inception of the method. A final section on future directions and trends of SEC column technology concludes this review.

Historical Perspective

SEC is an entropically controlled separation process (2) in which molecules in solution are separated on the basis of molecular size differences, rather than by chemical composition as with enthalpic-based separations. Size separation was first recognized as a new type of "partition" process in 1956 by Lathe and Ruthen (3,4) when they reported the separation of peptides, proteins, oligosaccharides, and polysaccharides using an aqueous mobile phase and swollen starch granules. This group correctly inferred that biopolymer "retention" was caused by their differential penetration into starch granules; larger macromolecules were eluted first because they were not able to diffuse as deeply into the granules as compared to smaller macromolecules. The extent of gel penetration or pore-volume occupancy was dictated by the molecular size or molecular weight of a sample. Soon after this discovery, gel filtration got off the ground when Pharmacia (the formerly famous Swedish pharmaceutical company) introduced a series of cross-linked dextran media, called Sephadex, with controlled porosity for the separation of biopolymers (5).


Table I: Overview of major manufacturers of high-performance SEC columns for synthetic polymers and biopolymers
After years of failed attempts by polymer chemists, John Moore of Dow Chemical Company (6) was finally able to synthesize a series of cross-linked polystyrene resin particles of known porosities for the molecular size separation of synthetic polymers. Dow Chemical Company transferred the polymerization technology to Waters Associates, which was a small instrument company in Framingham, Massachusetts, at the time. Waters built and sold a much-needed flow-through refractometer, together with custom-made chromatographs and columns packed with cross-linked polystyrene that was synthesized in a building formerly used as a women's jail. As founder James Waters exclaimed (7), "GPC took off like a rocket!"It literally became an overnight success, like its Pharmacia counterpart.

Table I presents current major suppliers of SEC packings, including a brief summary of their offerings. Please note that this table does not include manufacturers of conventional large-diameter, low-efficiency packings used mainly for large-scale separations or purification, biopolymer desalting, and environmental sample cleanup. In addition to SEC products, all of these companies offer their own line of enthalpic high performance liquid chromatography (HPLC) columns, and many sell SEC chromatographs and related instrumentation.

We should recognize the early leaders and major players in the SEC arena that were the first to introduce SEC packings and through their R&D programs, helped launch and promote the technique: Waters, Polymer Labs, Pharmacia, Toyo Soda (now Tosoh Bioscience), Showa Denko, Dow Chemical Co., DuPont Co., and Synchrom Inc. See reference 8 for a list of those companies that have merged.