Introduction

SEC columns separate components according to their molecular size in solution, larger molecules eluting first. Separation is achieved by the differential exclusion or inclusion of components within the packing material particles. In addition to the separation of discrete components, the technique is used for characterizing the molecular weight distribution of polymers. 

Base Material

Silica based SEC materials generally exhibit higher resolving power than polymer based materials. However, polymer based materials show greater stability for use with high pH eluents. Polymeric packing materials are generally available in larger particle sizes, which may be more practical for large-scale preparative separations.

Size Exclusion Chromatography Phases Modes of Operation

Gel permeation chromatography (GPC) refers to the SEC separation of organic soluble polymers using an organic solvent as the eluent. Gel filtration chromatography (GFC) refers to the separation of water soluble polymers in aqueous eluents. SEC separations exhibit lower resolving power and capacity compared with adsorptive HPLC techniques.

Applications

SEC analyses do not normally result in the denaturation of samples, making the technique a suitable choice for biological samples where activity must be retained. A wide range of bio-molecules and organic polymers are separated by SEC. For samples of wide molecular weight distribution, it can be useful to use a mixed pore size phase or to couple columns of one or more pore sizes in series.

Size Exclusion Chromatography Phases

Polyvinyl alcohol
Styrene -divinylbenzene Polyhydroxy methacrylate Silica
Polyhydroxy
methacrylate
Polystyrene¬divinylbenzene
Polystyrene¬divinylbenzene with polyhydroxyl functionality Silica with hydroxyethylaspartamide coating
Silica
Polymethacrylate
Polystyrene¬divinylbenzene
Hydrophilic polyvinyl
Zirconia-clad silica
Silica (silanized)
Silica (non-silanized)
Mode
GFC/ GPC
GPC
GFC GFC
GFC
GPC
GFC
GFC
GFC
GFC
GPC
GFC/ GPC GFC
GPC GFC/ GPC
Pore Size (Å)
120, 200, 600
50, 100, 500, 1000, 10,000, 100,000, MIXED
-
60, 100, 200, 300, 500, 1000, 1500
125, 250, 450
<100, 125, <200, 200, 500, 1000, >1000 15, 20, 30, 75, 200, 650
-
150, 300
60, 300, 1000
60, 300, 1000, 3000
Typical Applications
Hydrophilic and
hydrophobic compounds

Polymers, plastics

Water soluble samples

Proteins
Proteins, peptides,
enzymes and other
biomolecules
Oils, oligomers, high
MW synthetic polymers,
starches, polystyrenes,
resins
Surfactants,
polysaccharides,
polyacrylamides,
starches, gum
Peptides, proteins,
carbohydrates,
small molecules
Proteins, antibodies,
enzymes, nucleic acids

Water soluble organic
polymers,
polysaccharides, DNA

Oligomers, polymers

and polymer additives

Organic and water

soluble polymers

Proteins, peptides
Non-polar to polar
polymers
Organic and water
soluble polymers