Handbook of Size Exclusion Chromatography and Related ...

To use Eqs (8) and (9) effectively, one must decide if, in the context of a
given experiment, V0 or Vt may be determined less ambiguously. Himmel and
Squire assumed that in most cases Vt may be less accurately determined than the
void volume because of adsorptive effects experienced with most small solutes
and hence recommended the use of F0 v . However, Noll et al. have recently shown
(37) that the elution of deuterium oxide can be used as a reliable marker for Vt and
re-evaluation of the use of Eq. (9) may be in order. A further benefit of Eqs (8) and
(9) is that the values C and A can be accurately calculated from the limiting
chromatographic conditions, that is, at F0 v ¼ 1, M 1=3 ¼ A1=3 , and at
F0 v ¼ 0, M 1=3 ¼ C1=3 . The calculation of the column parameters C and A for a
series of similar columns, in different laboratories, is shown in Table 1.
The method of Himmel and Squire (38) has been applied to a wide range of
native protein SEC conditions, including TSK columns (39), Waters I125 columns
(40), as well as denatured protein SEC using Sephadex w (41). An important
extension to the method based on Eq. (8) was proposed by Bindels and Hoenders
(42), where Fv was plotted against (Mn) 1=3 . These workers found that this
approach gave better results than plots of M 1=3 or log M.
Assuming that the left-hand side of Eqs (8) and (9) provides an adequate
description of the column pores in SEC, then the predictive power of this method
may be improved by enhancing the picture of the solute during SEC beyond MW.
Although proteins are indeed roughly spherical, they can usually be more
accurately described as ellipsoids of revolution, either prolate or oblate, with axial
ratios normally ranging from 1.0 to 6 (35). And, as found by Bindels and
Hoenders, the correct SEC molecular radius must consider other factors. A
thorough treatment of proteins and nonflexible chain polymers as SEC solutes has
been contributed by Potschka (43). In this study, the parameters considered
included the equivalent (or effective) hydrodynamic radius, Re, the Stokes radius,
Rs, the root-mean-square radius of gyration, Rg, and the root-mean-square end-toend
distance, rrms. In an important recent contribution by Dubin and Principi (44),
Table 1 Calibration Constants for Toyo Soda TSK SW Series SEC Columns
TSK column support type A (daltons) C (daltons)
G2000 SW 940 91,000
G3000 SW 2460 340,000
G3000 SW 3900 330,000
G3000 SW a
3100 284,000
G4000 SW 550 3.4 10 6
a From this study.
Source: Adapted From Ref. 38.
© 2004 by Marcel Dekker, Inc.