Handbook of Size Exclusion Chromatography and Related ...

the cross-sectional area of the column regardless of particle size. They determined
1 and 8mg, respectively, for 60 cm 7.5 mm (10 g packing)
and 60 cm 21.5 mm (80 gpacking) TSKgel G3000SW columns. Freiser and
Gooding (114) reported loads of 2–4mg without band broadening on a
300 7.8 mm SynChropak GPC 100 column. For best resolution, it is
recommended that samples be 0.01–0.5% (wt/vol). However, very dilute
samples (,10 mg) sometimes lead to skewed peaks and/or poor recovery (58).
For preparative protein purification, loads are usually 10–20 mg/mL (15). The
concentration dependence of polymers is aspecial case and is discussed next.
For macromolecules, the sample size may be limited by viscosity. As a
rule of thumb, the sample injected should have aviscosity no greater than
twice the viscosity of the mobile phase. For proteins, this equals 70 mg/mL in
adilute aqueous mobile phase (9). Thus, viscosity of the sample is seldom an
issue with proteins, although it can be aproblem when glycerol or sucrose is
used as stabilizing agent or ethylene glycol is present to prevent protein
adsorption. Increasing viscosity causes restricted diffusion and irregular flow
patterns, which lead to broad and tailing peaks (115). With high molecular
weight synthetic polymers, asample concentration 0.1% is often required to
eliminate undesirable effects on both molecular coil dimensions and sample
viscosity (8). As the sample load increases, the polymer elutes at higher elution
volumes (116). The concentration dependence can be attributed to contraction
of polymer coils with increasing concentration. It may also be accounted for by
the combined effects of coil contraction and sample viscosity in the interstitial
pore volume. The viscosity effect can be operative to different extents,
depending upon the column system. Viscosity can drastically affect retention
volume and peak width (for molecules that elute within the interstitial pore
volume), accounting for 80% of the total concentration effect. With other
systems, coil contraction can account for 50–80% of the total concentration
effect (116).
For small volumes, peak height increases with increasing sample volume,
but retention time and resolution are not affected. At some critical volume, a
noticeable decrease in retention time occurs (see Fig. 15), as well as loss of
resolution and efficiency. Theoretically, the maximum injection volume for
protein SEC is equal to the separation volume between two proteins of interest,
but in practice, microturbulence, nonequilibrium between stationary phase and
mobile phase, and long diffusion lead to additional band broadening (115). As a
general rule, the maximum injection volume is 1–2% of the total column volume
(for example, 265–530 mL for a 60 cm 7.5 mm column), which agrees with
the data shown in Fig. 15. Injection volumes less than 1% of the column volume
do not necessarily improve resolution. The manufacturer of TSK-GEL SW
columns recommends injection volumes up to 0.5% of the analytical column
volume (58).
© 2004 by Marcel Dekker, Inc.