Handbook of Size Exclusion Chromatography and Related ...

industrial application of SEC for protein fractionation (81), followed by the
fractionation of HSA proteins (82).
The term preparative SEC encompasses all forms and scales of SEC
depending on requirements for the product. Preparative protein SEC has been
categorized by the scale of the separation (83), which include the following.
1. Preparative–analytical: analytical columns (diameter ,1cm), single
injection, microgram to milligram quantities prepared.
2. Semi-preparative: analytical columns (diameter 0.7–2cm), multiple
injections, milligram quantities prepared.
3. Standard–preparative: preparative columns (diameter 2–20cm), single
or multiple injections, milligram to gram quantities prepared.
4. Large-scale preparative: large preparative column (diameter 20cm),
automated injections, gram to kilogram quantities prepared.
The complexities of large-scale applications arise from the absolute
requirements for optimal productivity (gram product/cm 2 /hour), cost effectiveness,
and product purity. There are many technical factors that affect these issues.
Evaluating these factors for a given application is paramount to successfully
utilizing SEC at the industrial scale.
Column diameter and length are primary factors affecting the scale of
preparative SEC. For preparative separations, it is most cost-effective to operate at
the highest sample loading and flow rate possible without loss of adequate
resolution. In general, both the sample size and the flow rate can be increased
proportionally to the column’s cross-sectional area (Pharmacia). However, with
soft gels, bed compression is a major factor for large-diameter columns, even at
moderate flow rates (.50cm/h). This compression imposes an additional
physical limitation, beyond that of resolution, on the throughput that can be
attained in scaling up from analytical columns using soft resins. Column length is
also a major factor affecting productivity. The chromatographic resolution (Rsc) is
weakly affected by the length (Rsc1 ffiffiffi p
L),
so doubling the bed length will only
increase the Rsc by 40%. However, doubling the bed length will double the overall
backpressure at a given flow rate. Moreover, Rsc is a weak inverse function of
linear velocity, and in some preparative applications it may even be advantageous
to the overall productivity to actually shorten the bed length and run at higher flow
rates (84). This approach may be taken to a point of diminishing returns or to the
physical flow limitations described above. In general, this optimum must be
determined empirically for each resin and protein sample.
Sample loading is also important to the overall productivity of SEC.
Different loadings are recommended for desalting (ffi30% bed volume), and
protein fractionation ( 5% bed volume). These loadings are low compared to
other forms of chromatography, and tend to limit the use of SEC to the final (more
© 2004 by Marcel Dekker, Inc.