Handbook of Size Exclusion Chromatography and Related ...

3 PROTEIN PARTITIONING IN SEC
3.1 General Retention Mechanisms
Retention mechanisms for SEC are generally given on both hydrodynamic
(actually hydraulic) or thermodynamic grounds. The validity of interpreting SEC
behavior in terms of thermodynamic generalities has been well expressed and
defended by Yau et al. (22–24), and will not be stressed here. The hydrodynamic
description of the SEC process, especially when describing well-behaved protein
systems, has been reasonably rewarding in its ability to converge theory and
predictive elution. Fundamentally, Ve is the sum of the void volume occupied
by all solutes and a portion of the internal pore volume defined by the size
exclusion differential equilibrium constant, KSEC, and a portion of the surface
of the column packing defined by the distribution coefficient describing
interactions between the column and solute, KLC. This condition leads to the
general equation
Ve ¼ V0 þ KSECVi þ KLCVS
In the execution of SEC procedures it is usual and desirable, however, to reduce
adsorptive effects as much as possible using appropriate packing materials,
buffers, or detergents so that the last term in Eq. (1) is reduced to insignificance.
While solute partitioning in other forms of liquid chromatography involve
primarily the solute/stationary phase interactions, solute partitioning in SEC can
be described loosely as an entrapping effect, where solute molecules lose
configurational freedom upon entering the gel pores, a process that results in
entropic changes with the occupation of different column volumes (25). This
explanation, then, represents the basis for thermodynamic characterization of
KSEC. KSEC may also be explained in terms of column compartmentalization and
geometry, however.
3.2 Protein Elution Calibration
We now understand that two parameters must be understood before such a tool
could be usable: the correct description of the solute (protein) exposed to the SEC
process, and the physical description of the internal pore spaces seen by the eluting
species, usually as some function of Ve. The correct physical or hydrodynamic
description of the protein solute and the column packing material exists as a
challenge today.
3.2.1 Column Partitioning Effects: Pore Geometries
The early work of Andrews (20) is typical of the approach used to first study the
elution of proteins from SEC columns. Here the volume, V, passing through the
© 2004 by Marcel Dekker, Inc.
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