Handbook of Size Exclusion Chromatography and Related ...

insoluble crosslinked polydextran gel and was originally described by Porath and
Flodinin1959(3).Soonafterthisinitialbreakthrough,GranathandFlodinclearly
demonstrated the relationship between the elution of fractionated dextrans and
proteinsandsomefunctionofthemolecularsizeofthesolute(4).Infact,theearly
work showed atendency for elution in reverse order of molecular weight. This
observation then stimulated interest in finding asimple relationship between the
absolute molecular weights of macromolecules and their elution volumes in the
hope that such arelationship might be useful as apredictive analytical tool for
unknown systems. The early uses of Sephadex w were broadly reviewed by Porath
(5) in 1967; however, the popularity of these packing materials diminished with
the availability of stronger, more efficient preparations.
The success of size exclusion chromatography (SEC) for protein separation
is undeniable and has been well chronicled. Milestone reviews of protein SEC
present treatments of applications and theory and, in chronological order, include
the works of Bly (6), Yau et al. (7), Barth (8), Giddings (9), Regnier (10), Dubin
and Principi (11), Gooding and Regnier (12), and Barth et al. (13). Column and/or
packing material selection guidelines have also been well described by Montelaro
(14), Unger and Kinkel (15), Makino and Hatano (16), and Gooding and Freiser
(17). Protein SEC in detergents has been recently reviewed (14,18). In the present
review, we shall explore fundamental partition parameters appropriate to protein
SEC and SEC theory, and then focus on several important aspects of protein SEC
that are not well and widely treated. These topics are column/elution calibrations,
non-SEC partitioning, and industrial-scale protein SEC.
2 COLUMN COMPARTMENTALIZATION
The volume elements found in the chromatography column filled with porous
media are usually defined in a manner that follows the first suggestions by Porath
(19) and later modified by Andrews (20). Here, the total geometrical volume of the
SEC column, Vg, is defined as the sum of the total mobile phase volume, Vt, and
the volume of the packing material or stationary phase, Vs. The mobile phase
volume is further defined as the sum of the volume external to the pores in the
packing material or void volume, V0, and the volume occupied by the “stagnant”
mobile phase found in the internal pore structural elements, Vi. V0 has been shown
to be near 0:2595 Vg for columns of rigid SEC packing materials (21) by
approximating the gel bed as an assembly of hexagonal closest-packed spheres.
It is thought that the differential solute distribution between the volumes internal
and external to the pores results in the separation of the solutes. The volume of
elution of these solutes is known as Ve.
© 2004 by Marcel Dekker, Inc.