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Purification of Proteins Using Displacement Chromatography 79
salt conc. (mM)
200
150
100
Displacement
Region
Region of Elution
by Induced Gradient
elution line
displacement line
50
Region of Desorption
by Displacer
0
0 100 200 300
displacer conc. (mM)
Fig. 2. (A) Dynamic affinity plot for “A,” “B” and “C.” Parameters: A ( =5,K =
50), B ( =8,K = 12) and C ( =2,K = 1), = 10. (B) Affinity ranking plot for “A,”
“B” and “C.” Parameters: A ( = 10, K =20),B( = 15, K =1)andC( =5,K = 3).
(C) Operating regime plot.
value of , “A” has a greater dynamic affinity than both “B” and “C” while
“C” has a lower dynamic affinity than “B.” Therefore, in a displacement train,
the order of elution will be “C” followed by “B” and then by “A.”
Displacer affinity ranking plots (42), on the other hand, serve a different
purpose. These plots enable the ranking of the relative efficacies of displacers.
In contrast to the dynamic affinity plot which is constructed for a specific
(operating condition), this type of ranking plot can show the variation of the
dynamic affinity of a molecule over a range of values. Thus, these plots
provide a means of comparing the affinity of various displacers over a range
of operating conditions and give a realistic understanding of the efficacy of
a molecule as a displacer. Displacer affinity ranking plots originate from the
rearrangement of Eq. 8 as follows:
log = 1 logK − 1 log (10)
Thus, a plot of log() versus log() (see Fig. 2B) can be constructed
using the linear SMA parameters, K and . On these plots, higher values of
correspond to lower values of displacer or salt concentrations. Thus, lower
values of result in higher values of the dynamic affinity ().