Retention in IEC

The concentration of the counterion can be used to control the retention in IEC. It plays a role similar to that of the eluotropic strength of the eluent in RPLC or LSC, in that it affects retention much more than it does selectivity. The capacity factor can be related to the distribution coefficient of the solute (Dx) ... 

Metal complexation — One of the most insidious and widely occurrent sources of analytical variation in IEC is product complexation with metal ions. Most proteins can form complexes with metals, regardless of whether or not they are metalloproteins.1 Participant metal ions can derive from the cell culture production process, purification process buffers, or even stainless steel chromatography systems. Complexation can alter retention times, create aberrant peaks, and substantially increase peak width. To the extent that metal contamination of your sample is uncontrolled, so too will be the performance of your assay. 

Affinity complexation — Many proteins have affinities for other molecules that can be exploited to alter their retention characteristics in IEC. For example, some enzymes may be combined with synthetic substrates, cofactors, or products.1315 The same principle can be applied to other protein/receptor systems. One well-characterized example is the change in chromatographic behavior of fructose 1,6-diphosphatase in the presence of its negatively charged substrate... 

The retention of a sample by a column and the resultant column efficiency is principally dependent on the rate of diffusion of the analyte through the column. The diffusion rate is dependent upon both the size and porosity of the resin beads and the viscosity of the eluent. The mean free path of an analyte through a column is increased when the resin particle diameter is reduced and also if the stationary phase resin is more porous. The combination of these diffusion mechanisms is the rate determining step in IEC. 

The retention and selectivity in IEC are influenced by a number of parameters, which we will discuss below. 

The validity of eqn.(3.84) is demonstrated in figures 3.22a, b and c. In figures 3.22a and b the retention of some nucleotides in IEC is shown. The counterion is monovalent potassium dihydrogen phosphate. The figure shows a series of straight lines, the slopes of which are in good agreement with the predicted values from eqn.(3.84) 0.96 for the monophosphates (solutes 1 to 5), 1.85 for the diphosphate (solute 6) and 3.03 for the triphosphate (solute 7). 

From eqn.(3.84) and figures 3.22a, b and c we conclude that the concentration of counterions in IEC is a primary parameter which may be used to vary retention, i.e. to bring the capacity factor into the optimum range. Only the selectivity between solutes of different valencies will be affected considerably by changes in the concentration of the counterion. 

Figure 3.2S Experimental variation of the retention with pH for some nucleobases (a) and nucleosides (b) in IEC. Stationary phase Aminex A-28. Mobile phase 5 mM citrate — 5 mM phosphate buffer in 50-50 ethanol-water. Temperature 70 °C. Figure taken from ref. [371]. Reprinted with permission.

A number of parameters may affect retention and selectivity in IEC. Because of the subtle differences in selectivity between different ion-exchangers, it is often necessary to optimize the separation in a specific situation. The common way to do this [367] is by varying a single parameter at a time, keeping all the others constant. As we will see in chapter 3 (section 5.1.1), this is not the most appropriate way to approach the optimization... 

Figure 3.28 reduces to the simple mechanism of figure 3.27 if both Kx and K Y are very small. If Kx is small (i.e. the solute molecule is mainly in the mobile phase) but K Y is large (the pairing ion is mainly absorbed into the stationary phase), then the mechanism of retention in IPC becomes similar to that of IEC. Typical ion-pairing as well as typical ion-exchange mechanisms may play a role in practical IPC systems. 

As in 1EC, the counterion concentration has a considerable effect on the retention in IPC. In IPC the counterion is charged similar to the solute molecules, but opposite to the pairing ion. For example, for the separation of anionic solutes, the pairing agent may be a sodium sulfonate, in which the sulfonate is the pairing ion and sodium the counterion. The addition of a buffer salt (e.g. sodium phosphate) and a neutral salt (e.g. sodium bromide) may also contribute to the concentration of the counterion. Because of the similar retention mechanism, the counterion concentration has a similar effect on retention and selectivity in RP-IPC as in IEC. 

The temperature will affect both retention and efficiency in IPC, but not to the same extent as it does in IEC. IPC is usually a much more efficient technique (in terms of plate counts) than is IEC and therefore ambient temperatures usually yield satisfactory results. 

Figure 9.10. Effect of pH on retention time in IEC. Reprinted with permission of Alltech Associates/Applied Science Labs, Deerfield, IL.

Some kinds of chromatography require relatively little optimization. In gel permeation chromatography, for example, once the pore size of the support and number of columns is selected, it is only rarely necessary to examine in depth factors such as solvent composition, temperature, and flow rate. Optimization of affinity chromatography is similarly straightforward. In RPLC or IEC, however, retention is a complex and sensitive function of mobile phase composition column type, efficiency, and length flow rate gradient rate and temperature. 

In ion-exchange chromatography (IEC), the mobile phase modulator is typically a salt in aqueous solution, and the stationary phase is an ion-exchanger. For dilute conditions, the solute retention factor is commonly found to be a power-law function of the salt normality [cf. Eq. (16-27) for ion-exchange equilibrium]. 

Variations in sample composition other than those toward which an assay is directed may detract from the performance and reproducibility of HIC assays. Controlling these influences can significantly improve resolution and sensitivity as well as reproducibility. As with IEC, product complexation with other solutes can be an important source of aberrant retention behavior. 

In ion-exchange chromatography (IEC), species are separated on the basis of differences in electric charge. The primary mechanism of retention is the electrostatic attraction of ionic solutes in solution to fixed ions of... 

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