Handbook of Size Exclusion Chromatography and Related ...

solvent. Here again, solvent–solvent interactions play an important role. It should
alsobenotedthatamixtureoftwononsolventsforapolymermaytogetherforma
good solvent for it (co-solvency phenomenon). The opposite situation may also
sometimes appear, when amixture of twogood solvents will not dissolve agiven
polymer (co-nonsolvency phenomenon). With rising temperature, solubility of a
polymer in aliquid may either improve (upper critical solubility temperature) or
(sometimes) also deteriorate (lower critical solubility temperature).
From the viewpoint of HPLC, interactions between macromolecules and
column packing have apractical sense only in the presence of amobile phase.
Enthalpic interactions between column packing and polymer analytes are
sometimes expressed by means segmental interaction energy parameter 1. The
value of 1strongly depends on the eluent strength (Sec. 3.2).
3.1 Entropic Retention Mechanism
Changes of entropy within an HPLC column separating polymers result not only
from mixing phenomena but also from large conformation and possibly also from
orientation changes of macromolecules that get confined in the pores or excluded
from the pores and/or from the outer column packing surface. The conformational
contribution to the DS term in Eq. (3) is very large for polymer analytes and
therefore important entropic effects accompany all HPLC separations of
macromolecules. Evidently, the term “nonexclusion HPLC of polymers” is not
appropriate, although the nonexclusion, enthalpic retention mechanisms dominate
in some HPLC separation procedures. On the contrary, enthalpic interactions
between macromolecules and column packing can be successfully suppressed
(1 0). This is the case of “ideal” size exclusion chromatography.
Retention of macromolecules in liquid chromatographic systems is often
analysed from the point of viewof rules that are valid for low molar mass substances.
However, already, a brief comparison of macromolecular and low molecular bulk
static systems reveals important differences in their behavior. Neglecting these
differences makes it difficult to understand retention behavior of macromolecules.
The entropic retention mechanism that is the entropic partition of
macromolecules in porous systems forms a base for size exclusion
chromatography and hydrodynamic chromatography (HDC). Differences in
entropy changes for macromolecules of different sizes, which take place in
stationary phase regions (mainly in the pores of different sizes and shapes) are
responsible for different retention volumes of eluted polymer species. This results
in separation of macromolecular analytes according to their size.
To date SEC is performed mainly with columns packed by porous particles.
It is anticipated that improved technology of monolithic columns (improved
control of sizes and volumes of separation pores) will allow their application also
in SEC. HDC is carried out either with capillaries or with columns packed with
© 2004 by Marcel Dekker, Inc.