the separation of many metals as halide complexes on anionic resins and the separation of amino-acids
with citrate buffers. The use of pressurized systems for complex mixtures is likely to become more
widespread in the future.
4.3.6—
Size Exclusion Chromatography
Summary
Principles
Separation of materials according to molecular size and shape by passage of a solution through a
column or across a surface consisting of a polymeric gel.
Apparatus and Instrumentation
Glass columns for separations by gravity flow; glass, metal or nylon tubing for pressurized systems;
fraction collector, detector and recorder.
Applications
Separation and desalting of high-molecular weight materials; determination of molecular weights.
Disadvantages
Gravity flow separations slow; resolution of low molecular weight compounds poor or non-existent.
Molecules that differ in size can be separated by passing the sample solution through a stationary phase
consisting of a porous cross-linked polymeric gel. The pores of the gel exclude molecules greater than a
certain critical size whilst smaller molecules can permeate the gel structure by diffusion. The process is
described as gel-permeation, gel-filtration or size exclusion chromatography. Excluded molecules pass
through the system more rapidly than smaller ones which can diffuse into the gel. Diffusion within the
gel also varies with molecular size and shape because pores of different dimensions are distributed
throughout the gel structure in a random manner. These smaller molecules are eluted at rates dependent
upon their degree of permeation into the gel, and components of a mixture therefore elute in order of
decreasing size or molecular weight.
Structure and Properties of Gels
Gels used for the stationary phase can be hydrophilic, for separations in aqueous and other polar
solvents, or hydrophobic, for use in non-polar or weakly-polar solvents. Agar, starch, polyacrylamide
and cross-linked dextrans possess hydroxyl or amide groups and are thus hydrophilic. They swell