28.6: Ion-Exchange Chromatography

In ion-exchange chromatography (IEC) the stationary phase is a cross-linked polymer resin, usually divinylbenzene cross-linked polystyrene, with covalently attached ionic functional groups (see Figure 28.6.1 and Table 28.6.1 ). The counterions to these fixed charges are mobile and are displaced by ions that compete more favorably for the exchange sites. Ion-exchange resins are divided into four categories: strong acid cation exchangers; weak acid cation exchangers; strong base anion exchangers; and weak base anion exchangers.

Figure 28.6.1 . Structures of styrene, divinylbenzene, and a styrene–divinylbenzene co-polymer modified for use as an ion-exchange resin are shown on the left. The ion-exchange sites, indicated by R and shown in blue, are mostly in the para position and are not necessarily bound to all styrene units. The cross-linking is shown in red. The photo on the right shows an example of the polymer beads. These beads are approximately 0.30–0.85 mm in diameter. Resins for use in ion-exchange chromatography typically are 5–11 μm in diameter.

Table 28.6.1 . Examples of Common Ion-Exchange Resins

type	functional group	examples
strong acid cation exchanger	sulfonic acid	\(-\text{SO}_3^-\) \(-\text{CH}_2\text{CH}_2\text{SO}_3^-\)
weak acid cation exchanger	carboxylic acid	\(-\text{COO}^-\) \(-\text{CH}_2\text{COO}^-\)
strong base anion exchanger	quaternary amine	\(-\text{CH}_2\text{N(CH}_3)_3^+\) \(-\text{CH}_2\text{CH}_2\text{N(CH}_2\text{CH}_3)_3^+\)
weak base anion exchanger	amine	\(-\text{NH}_4^+\) \(-\text{CH}_2\text{CH}_2\text{NH(CH}_2\text{CH}_3)_3^+\)

Strong acid cation exchangers include a sulfonic acid functional group that retains it anionic form—and thus its capacity for ion-exchange—in strongly acidic solutions. The functional groups for a weak acid cation exchanger, on the other hand, are fully protonated at pH levels less then 4 and lose their exchange capacity. The strong base anion exchangers include a quaternary amine, which retains a positive charge even in strongly basic solutions. Weak base anion exchangers remain protonated only at pH levels that are moderately basic. Under more basic conditions a weak base anion exchanger loses a proton and its exchange capacity.

The ion-exchange reaction of a monovalent cation, M⁺, exchange site is

\[-\mathrm{SO}_{3}^{-} \mathrm{H}^{+}(s)+\mathrm{M}^{+}(a q)\rightleftharpoons-\mathrm{SO}_{3}^{-} \mathrm{M}^{+}(s)+\mathrm{H}^{+}(a q) \nonumber \]

The equilibrium constant for this ion-exchange reaction, which we call the selectivity coefficient, K, is

\[K=\frac{\left\{-\mathrm{SO}_{3}^{-} \mathrm{M}^{+}\right\}\left[\mathrm{H}^{+}\right]}{\left\{-\mathrm{SO}_{3}^{-} \mathrm{H}^{+}\right\}\left[\mathrm{M}^{+}\right]} \label{12.1} \]

where we use curly brackets, { }, to indicate a surface concentration instead of a solution concentration.

We don’t usually think about a solid’s concentration. There is a good reason for this. In most cases, a solid’s concentration is a constant. If you break a piece of chalk into two parts, for example, the mass and the volume of each piece retains the same proportional relationship as in the original piece of chalk. When we consider an ion binding to a reactive site on the solid’s surface, however, the fraction of sites that are bound, and thus the concentration of bound sites, can take on any value between 0 and some maximum value that is proportional to the density of reactive sites.

Rearranging Equation \ref{12.1} shows us that the distribution ratio, D, for the exchange reaction

\[D=\frac{\text { amount of } \mathrm{M}^{+} \text { in the stationary phase }}{\text { amount of } \mathrm{M}^{+} \text { in the mobile phase }} \nonumber \]

\[D=\frac{\left\{-\mathrm{SO}_{3}^{-} \mathrm{M}^{+}\right\}}{\left[\mathrm{M}^{+}\right]}=K \times \frac{\left\{-\mathrm{SO}_{3}^{-} \mathrm{H}^{+}\right\}}{\left[\mathrm{H}^{+}\right]} \label{12.2} \]

is a function of the concentration of H⁺ and, therefore, the pH of the mobile phase.

An ion-exchange resin’s selectivity is somewhat dependent on whether it includes strong or weak exchange sites and on the extent of cross-linking. The latter is particularly important as it controls the resin’s permeability, and, therefore, the accessibility of exchange sites. An approximate order of selectivity for a typical strong acid cation exchange resin, in order of decreasing D, is

Al³⁺ > Ba²⁺ > Pb²⁺ > Ca²⁺ > Ni²⁺ > Cd²⁺ > Cu²⁺ > Co²⁺ > Zn²⁺ > Mg²⁺ > Ag⁺ > K⁺ > \(\text{NH}_4^+\) > Na⁺ > H⁺ > Li⁺

Note that highly charged cations bind more strongly than cations of lower charge, and that for cations of similar charge, those with a smaller hydrated radius, or that are more polarizable, bind more strongly. For a strong base anion exchanger the general elution order is

\(\text{SO}_4^{2-}\) > I^– > \(\text{HSO}_4^-\) > \(\text{NO}_3^-\) > Br^– > \(\text{NO}_2^-\) > Cl^– > \(\text{HCO}_3^-\) > CH3COO^– > OH^– > F^–

Anions of higher charge and of smaller hydrated radius bind more strongly than anions with a lower charge and a larger hydrated radius.

The mobile phase in IEC usually is an aqueous buffer, the pH and ionic composition of which determines a solute’s retention time. Gradient elutions are possible in which the mobile phase’s ionic strength or pH is changed with time. For example, an IEC separation of cations might use a dilute solution of HCl as the mobile phase. Increasing the concentration of HCl speeds the elution rate for more strongly retained cations because the higher concentration of H⁺ allows it to compete more successfully for the ion-exchange sites.

From Equation \ref{12.2}, a cation’s distribution ratio, D, becomes smaller when the concentration of H⁺ in the mobile phase increases.

An ion-exchange resin is incorporated into an HPLC column either as 5–11 μm porous polymer beads or by coating the resin on porous silica particles. Columns typically are 250 mm in length with internal diameters ranging from 2–5 mm.

Measuring the conductivity of the mobile phase as it elutes from the column serves as a universal detector for cationic and anionic analytes. Because the mobile phase contains a high concentration of ions—a mobile phase of dilute HCl, for example, contains significant concentrations of H⁺ and Cl^– ions—we need a method for detecting the analytes in the presence of a significant background conductivity.

To minimize the mobile phase’s contribution to conductivity, an ion-suppressor column is placed between the analytical column and the detector. This column selectively removes mobile phase ions without removing solute ions. For example, in cation-exchange chromatography using a dilute solution of HCl as the mobile phase, the suppressor column contains a strong base anion-exchange resin. The exchange reaction

\[\mathrm{H}^{+}(a q)+\mathrm{Cl}^{-}(a q)+\mathrm{Resin}^{+} \mathrm{OH}^{-}(s)\rightleftharpoons\operatorname{Resin}^{+} \mathrm{Cl}^{-}(s)+\mathrm{H}_{2} \mathrm{O}(l ) \nonumber \]

replaces the mobile phase ions H⁺ and Cl^– with H₂O. A similar process is used in anion-exchange chromatography where the suppressor column contains a cation-exchange resin. If the mobile phase is a solution of Na₂CO₃, the exchange reaction

\[2 \mathrm{Na}^{+}(a q)+\mathrm{CO}_{3}^{2-}(a q)+2 \operatorname{Resin}^{-} \mathrm{H}^{+}(s)\rightleftharpoons2 \operatorname{Resin}^{-} \mathrm{Na}^{+}(s)+\mathrm{H}_{2} \mathrm{CO}_{3}(a q) \nonumber \]

replaces a strong electrolyte, Na₂CO₃, with a weak electrolyte, H₂CO₃.

Ion-suppression is necessary when the mobile phase contains a high concentration of ions. Single-column ion chromatography, in which an ion-suppressor column is not needed, is possible if the concentration of ions in the mobile phase is small. Typically the stationary phase is a resin with a low capacity for ion-exchange and the mobile phase is a very dilute solution of methane sulfonic acid for cationic analytes, or potassium benzoate or potassium hydrogen phthalate for anionic analytes. Because the background conductivity is sufficiently small, it is possible to monitor a change in conductivity as the analytes elute from the column.

A UV/Vis absorbance detector can be used if the analytes absorb ultraviolet or visible radiation. Alternatively, we can detect indirectly analytes that do not absorb in the UV/Vis if the mobile phase contains a UV/Vis absorbing species. In this case, when a solute band passes through the detector, a decrease in absorbance is measured at the detector.

Ion-exchange chromatography is an important technique for the analysis of anions and cations in water. For example, an ion-exchange chromatographic analysis for the anions F^–, Cl^–, Br^–, \(\text{NO}_2^-\), \(\text{NO}_3^-\), \(\text{PO}_4^{3-}\), and \(\text{SO}_4^{2-}\) takes approximately 15 minutes (Figure 28.6.2 ). A complete analysis of the same set of anions by a combination of potentiometry and spectrophotometry requires 1–2 days. Ion-exchange chromatography also is used for the analysis of proteins, amino acids, sugars, nucleotides, pharmaceuticals, consumer products, and clinical samples.

Figure 28.6.2 . Ion-exchange chromatographic analysis of water from Big Walnut Creek in Putnam County, Indiana. Data provided by Jeanette Pope, Department of Geosciences, DePauw University.