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80 Tugcu
Figure 2B illustrates a typical affinity ranking plot for three displacers,
“A,” “B” and “C.” For this range of , “A” has a greater dynamic affinity than
“B” and “C.” However, the relative efficacies of “B” and “C” can change as
indicated by the plot. At values less than about 10, “B” has a higher dynamic
affinity than “C.” However, the order changes for values greater than 10.
A range of values could be picked, once the dynamic affinity lines for feed
components and displacer candidates are plotted using an affinity ranking plot.
If time or material is not available for the detailed screening described above,
then evaluating displacer candidates via linear elution chromatography could be
a replacement. In that case, the suggestion will be to pick the displacer with the
highest affinity (longest retention time) while making sure that a regeneration
protocol for this displacer on the specific resin is available (see Note 4).
2.4. Operating Regime Plots
Once a displacer has been selected and its corresponding was determined
based on its affinity to displace feed components as described above, the
next step would be calculating the corresponding displacer concentration at
a given mobile phase salt concentration. A detailed analysis of the displacer
concentration necessary for displacement of feed components as a function
of mobile phase salt concentration is done via use of operating regime plots
described later in this section. However, if the reader has already established a
salt concentration leading to relatively strong binding of the feed components
and the displacer, then once the is determined, the SMA isotherm (Eq. 1)
can simply be used to calculate the displacer concentration.
As mentioned previously, is a function of displacer and mobile phase salt
concentrations. Therefore, having an operating regime plot that shows as
a function of salt concentration would be invaluable. To create these plots, a
displacement line that separates the displacement and desorption regions should
be determined. It has been shown that low molecular weight displacers will
generally have a critical partition ratio () at which they cease to act as a
displacer and begin to act as a desorbent (44). D and P in these equations refer
to displacer and protein, respectively. The equation for the displacement line
is given by
C salt =
(
KD
) 1/D
− D + D C D (11)