D

The breakthrough volume V for a ligand is corrected by the breakthrough volume of the ligand in the absence of the binding event, V0. This is a difficult number to obtain in practice so a structurally related nonligand is often used to assess V0. Dt refers to the dynamic capacity of the affinity column for the ligand, [A]0 the infusion concentration of the ligand and Kd the dissociation constant for the interaction. We may recognize this formalism as one example of a nonlinear convex binding isotherm. Figure 6.3 shows the basic elements that comprise a break-

Equilibrium Plateau

Equilibrium Plateau

Frontal Chromatograms

Fig. 6.3 A dissection of the frontal chromatogram [31]. The breakthrough curve is represented by the thick line. The two gray/hatched surfaces on the left side (A!, A2) represent the mass of compound in the extra- and dead-column volumes. Area A3 represents the mass of the compound adsorbed to the stationary phase. Adapted with permis^ sion from Elsevier.

Fig. 6.3 A dissection of the frontal chromatogram [31]. The breakthrough curve is represented by the thick line. The two gray/hatched surfaces on the left side (A!, A2) represent the mass of compound in the extra- and dead-column volumes. Area A3 represents the mass of the compound adsorbed to the stationary phase. Adapted with permis^ sion from Elsevier.

Fig. 6.4 The effects of ligand concentration on the FAC chromatogram [8]. A given ligand experiences an accelerated breakthrough as its concentration increases. Under linear chromatographic conditions ([A]0 « Kd), there is a direct relationship with zonal chromatography, where the breakthrough curve is coincident in retention time with the

Fig. 6.4 The effects of ligand concentration on the FAC chromatogram [8]. A given ligand experiences an accelerated breakthrough as its concentration increases. Under linear chromatographic conditions ([A]0 « Kd), there is a direct relationship with zonal chromatography, where the breakthrough curve is coincident in retention time with the zonal peak (short dashed trace). At higher ligand concentration, the breakthrough shifts to earlier elution times (i.e. lower elution volumes) and exhibit a noticeable ''sharpening'' of the curve (long-dash trace, dot-dash trace). Adapted with permission from the American Chemical Society.

through curve, and Fig. 6.4 the effect ofligand concentration on both the appearance of the curve and the breakthrough volume. At high dilution relative to the Kd of the particular interaction, the breakthrough volume is insensitive to slight changes in ligand concentration and has actually achieved its maximum value. Under these dilute conditions, FAC operates in the linear region of the binding function.

Was this article helpful?

0 0

Post a comment