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6.3. SIZE-‐EXCLUSION CHROMATOGRAPHY
(SEC) OF
BIOPOLYMERS
6.3.1. General
Many biopolymers – polysaccharides in particular – are polydisperse. A full
description of the distribution of different molecular weights (or chain lengths)
requires fractionation and quantification of the relative amount of each of the
molecular weight classes. This can be achieved experimentally using classical
separation methods such as chromatography, electrophoresis, equilibrium
centrifugation, mass spectrometry etc.
A widespread method is column chromatography, where separation is based
on differences in the effective (hydrodynamic) size of the molecules. The
method is referred to as either size-exclusion chromatography (SEC), gel
permeation chromatography (GPC) or simply gel filtration.
6.3.2. SEC
separation mechanism
The separation mechanism is the passive diffusion of macromolecules into
porous particles (the stationary phase), where the distribution of pore sizes
should match those of the molecules to be separated. A wide range of such
particles is commercially available, with a relatively wide range of pore sizes.
For HPLC, which is the most common system for analytical purposes,
columns particles are typically in the range 10-30
µ
m in diameter, and with
uniform particle size distributions (essentially monodisperse particles). An
example is given in the figure below (Figure 2).
PC
Column
Solvent/eluent
(buffer)
HPLC-
Detector
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