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For preparative columns larger particle sizes are often used. The material
properties also vary. Generally, the particles are composed of cross-linked
synthetic polymers such as (poly)acrylamide or (poly)styrene) or neutral
polysaccharides (chemically cross-linked dextran or agarose). For analytical
SEC the chromatography particles are usually based on synthetic
(hydrophobic) polymers. Particles intended for biomacromolecules usually
have a hydrophilic surface layer (covalently linked), otherwise many proteins
(in particular) would absorb irreversibly (by hydrophobic interactions)
When a mixture of macromolecules elute through a SEC column, smaller
molecules will to a larger extent than larger molecules diffuse into the pores
(the stationary phase). In fact, equilibrium between the mobile and the
stationary phase is established locally. Since the solvent inside the pores is
stagnant (stationary), molecules therein will not be transported along the
column until they diffuse out. Overall, molecules become delayed (retained),
and the retention depends on the size of the molecules. Small molecules are
retained more than larger molecules.
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