An instrument for high performance gel permeation chromatography
(HPGPC) has been assembled with equipment designed for
high performance liquid chromatography separations. The pump was
a syringe-type giving uniform pulseless flow from 0.05 to 6.0 cm3
min-1 at pressures up to 3000 p.s.i.. Low dead-volume septum and
septumless injection heads have been used for single columns and
multiple column sets. Two ultraviolet detectors and a refractometer having low cell volumes were used to detect the low weights
of solute which were separated.
Narrow distribution silica gels of varying particle size and
porosity, as supplied by A.E.R.E. Harwell, have been packed into
columns. Efficiencies of permeating and non-permeating polystyrenes
determined with this HPGPC instrument gave an assessment
of chromatogram broadening due to mass transfer as a function of
eluent flow rate and polystyrene molecular weight. The results
show that fast separations can be obtained in several minutes and
that the most precise measurements of polydispersity are accomplished at very slow rates. A further assessment of chromatogram
broadening was made by deactivating the surface of the silica
particles with a hydrophilic bonded phase and examining the column
efficiency and polydispersity of proteins in aqueous buffer.
Results show that even after surface modification the composition
of the aqueous eluent used must be adjusted, e.g. by careful
selection of pH and ionic strength, to minimize interactions.
When interactions occur, they can be explained in terms of a
thermodynamic representation of a mixed mechanism. A comparison
of retention data for proteins, which do not participate in
interaction effects with the stationary phase, dextrans, which are
considered as "non-ionic" macromolecules, and polystyrenes suggest
that solute diameter is a reasonable universal size parameter for
the representation of macromolecules separating by a steric
exclusion mechanism operating close to equilibrium conditions.
A Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy of Loughborough University.