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Ion Exchange Chromatography (IEX)

Introduction

Ion-exchange chromatography (IEX) is based on the different affinities of analyte ions for oppositely charged ionic functional groups (ligands) bound to the stationary phase surface, and the ability of these analyte ions to displace counter ions associated with the stationary phase ligands.

The charge of the bonded stationary phase ligands determines whether the resin is an anion exchanger (positive ionic functional groups on the surface) or a cation exchanger (negative functional groups on the surface, Figure 1).

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Figure 1: Anion and cation exchange chromatography mechanism.

Mobile phase pH, ionic strength, and counter ion additives can all be altered to change the analyte solubility and, therefore, affect analyte retention.

In ion exchange chromatography, separation is dependent upon the different degrees of analyte interaction with the charged moieties bonded to the stationary phase.  Interactions are controlled by altering the charge state of either the analytes or the ionic functional groups on the stationary phase surface.

The pKa of any ionic functional group determines the pH at which it will be charged.

  • For a basic analyte to be charged (cationic, positively charged) the pH must be kept approximately two units below its pKa
  • For an acidic analyte to be charged (anionic, negatively charged) the pH must be kept approximately two units above its pKa (Figure 2)
Schematic representation of the 2 pH rule
Figure 2: Schematic representation of the 2 pH rule, describing the effects of pH on the degree of analyte ionization in solution.

The above considerations for ionic functional groups are valid not only in the case of analytes but also in relation to the stationary phase.

The charge state of functional groups within a molecule determines the optimal chromatographic conditions for the separation.  As a consequence, analyte retention in ion exchange chromatography can be controlled through correct modification of pH (Figure 3).

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Figure 3. Analyte and stationary phase ionization in relation to pH.
 
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