| Summary: | 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 = H - L (P-1) (1 + Kapp) 2x where H 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 E (incidence of plate height upon velocity) followed the well known trend in liquid crystallography 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 those 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 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 longstanding 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 10µg for analytical columns: larger loads lead to loss of efficiency and change in retention volume.
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