Size Exclusion Chromatography

Size-exclusion chromatography (SEC) is utilized for the fractionation and analytical characterization of macromolecules but also for the separation of particles. The term gel-permeation chromatography (GPC) is used simultaneously in the literature with almost equal frequency. Other terms employed to describe this separation method are steric-exclusion liquid chromatography, steric-exclusion chromato-graphy, gel filtration, gel-filtration chromatography, gel chromatography, gel-exclusion chromatography, and molecular-sieve chromatography. Each reflects an effort to express the basic mechanism governing the separation but the appropriate choice is more a question of individual preference.

The historical origins of SEC date from the late 1950s and early 1960s. Using cross-linked dextran gels swollen in aqueous media, Porath and Flodin separated various proteins according to their sizes. The 'soft gel' column packing used in these experiments was applicable only at low pressure and, consequently, at low flow rates resulting in very long separation times. The first successful separation of a synthetic polymer by SEC was described by Vaughan who succeeded in separating low molar mass polystyrene in benzene on a weakly cross-linked polystyrene gel. Some years later, Moore described the separation of polymers on moderately cross-linked polystyrene gel column packings.

The first rigid macroporous packing, suited also for the separation of particles, was porous silica introduced in 1966 by De Vries and co-workers. This packing was fully compatible with both aqueous and organic solvents, exhibited a very good mechanical stability, but its use was restricted by strong nonsteric exclusion interactions between the silica surface and a number of separated species. In 1974, the appearance of the packings of small porous particles with a typical diameter around 10 |im, instead of 50-100 |im particle diameter used in conventional SEC columns, resulted in an important technological improvement in SEC. The high pressure technology, the lowering of the column volume due to the use of small particle diameter packings and the high efficiency of the columns allowed the separation time to be reduced from hours to minutes. Other porous silica microparticle packings, introduced by Kirkland, Unger, and others, were resistant to the high pressure and compatible with the quasi-totality of the solvents. The undesired interactions were suppressed by organic grafting or by organic coating of the porous silica.

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