This thesis describes the mechanism of exclusion chromatography
in terms of conventional liquid chromatography terminology.
The various theories for sample retention are discussed and
it is concluded that elution under ordinary conditions with a rigid
pore structure is governed by the equilibrium theory of steric
exclusion. Using the steric exclusion mechanism, calibration curves
were computed for various pore geometries and spherical macromolecules
which gave an exceptionally good fit to experimental data.
In exclusion chromatography, band broadening or dispersion of
a polymer sample arises from both the polydispersity, P, of the sample
and from the kinetic processes occurring within the column. It is
shown that the plate height due to kinetic processes within the column,
H, is given by
H = Hₐₚₚ - (L (P - 1) (1 + α))/x²
where Hₐₚₚ is the apparent plate height calculated from the
experimental peak, L is the column length, a is a function of (P -1)
and x is a measure of the relative molecular mass range covered by
the packing material.
By varying the column length and extrapolating to zero length,
the true plate height (due to kinetic effects) was obtained as a
function of velocity. This dependence of plate height upon
velocity followed the well known trend in liquid chromatography
giving a minimum plate height of around 2 particle diameters. This
was verified by measuring the plate height for monodisperse polymer
fractions obtained by adsorption chromatography.
By manipulation of this plate height versus velocity data
to account for longitudinal diffusion and flow effects, the
stationary phase mass transfer coefficients were obtained.
Interpretation of these coefficients in terms of the non -equilibrium
theory of chromatography leads to the conclusion that within the pores
of the material, diffusion of molecules is restricted with the degree
of restriction increasing as molecular size approaches the pore
size. However, this restriction is small enough and mass transfer
fast enough so as not to negate the near -equilibrium assumption of
chromatography which is shown by the narrow and symmetrical peaks
obtained for monodisperse polymers.
These results finally confirm that the theory of retentive
chromatography applies with only minor adjustments to exclusion
chromatography, thereby solving a long standing argument as to
the nature of the processes occurring in exclusion chromatography.
Sample loading is discussed and it is shown that sample loads
must be kept below about 10 pg for analytical columns: larger
loads lead to loss of efficiency and change in retention volume.