Ion-Exchange Chromatography

Ion-Exchange Chromatography

In ion-exchange chromatography (IEC) the stationary phase is a cross-linked poly- mer resin, usually divinylbenzene cross-linked polystyrene, with covalently attached ionic functional groups (Figure 12.33). The counterions to these fixed charges are mobile and can be displaced by ions that compete more favorably for the exchange sites. Ion-exchange resins are divided into four categories: strong acid cation ex- changers; weak acid cation exchangers; strong base anion exchangers; and weak base anion exchangers. Table 12.5 provides a list of several common ion-exchange resins.

Strong acid cation exchangers include a sulfonic acid functional group that re- tains its anionic form, and thus its capacity for ion-exchange, in strongly acidic so- lutions. The functional groups for a weak acid cation exchanger, however, are fully protonated at pH levels less then 4, thereby losing their exchange capacity. The strong base anion exchangers are fashioned using a quaternary amine, therefore re- taining a positive charge even in strongly basic solutions. Weak base anion exchang- ers, however, remain protonated only at pH levels that are moderately basic. Under more basic conditions, a weak base anion exchanger loses its positive charge and, therefore, its exchange capacity.

The ion-exchange reaction of a monovalent cation, M+, at a strong acid ex- change site is

The equilibrium constant for this ion-exchange reaction, which is also called the se- lectivity coefficient, is

where the brackets { } indicate a surface concentration. Rearranging equation 12.31 shows that the distribution ratio for the exchange reaction

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

Ion-exchange resins are incorporated into HPLC columns either as micron- sized porous polymer beads or by coating the resin on porous silica particles. Selec- tivity is somewhat dependent on whether the resin includes a strong or weak ex- change site and on the extent of cross-linking. The latter is particularly important because it controls the resin’s permeability and, therefore, the accessibility of the ex- change sites. An approximate order of selectivity for a typical strong acid cation ex- change resin, in order of decreasing D, is

Al3+ > Ba2+ > Pb2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+

> Ag+ > K+ > NH₄+ > Na+ > H+ > Li+

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

SO₄2– > I– > HSO₄– > NO₃– > Br– > NO₃– > Cl– > HCO₃– > CH₃COO– > OH– > F–

Again, ions of higher charge and smaller hydrated radius bind more strongly than ions with a lower charge and a larger hydrated radius.

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

Ion-exchange columns can be substituted into the general HPLC instrument shown in Figure 12.26. The most common detector measures the conductivity of the mobile phase as it elutes from the column. The high concentration of electrolyte in the mobile phase is a problem, however, because the mobile-phase ions dominate the conductivity. For example, if a dilute solution of HCl is used as the mobile phase, the presence of large concentrations of H₃O+ and Cl– produces a background conductivity that may prevent the detection of analytes eluting from the column.

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

H+(aq) + Cl–(aq) + Resin+–OH– < = = = = > Resin+–Cl– + H₂O(l)

replaces the ionic HCl with H₂O. Analyte cations elute as hydroxide salts instead of as chloride salts. A similar process is used in anion ion-exchange chromatography in which a cation ion-exchange resin is placed in the suppressor column. If the mo- bile phase contains Na₂CO₃, the exchange reaction

2Na+(aq) + CO₃2–(aq) + 2Resin––H+ < = = = = >  2Resin––Na+ +H₃CO (aq)

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

Ion suppression is necessary when using a mobile phase containing a high con- centration 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 can be minimized. Typically this is done by using a stationary phase resin with a low capacity for ion exchange and a mobile phase with a small concentration of ions. Because the background conductivity due to the mobile phase 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 also be used if the solute ions absorb ultravi- olet or visible radiation. Alternatively, solutions that do not absorb in the UV/Vis range can be detected indirectly 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 has found important applications in water analysis and in biochemistry. For example, Figure 12.34a shows how ion-exchange chromatography can be used for the simultaneous analysis of seven common an- ions in approximately 12 min. Before IEC, a complete analysis of the same set of anions required 1–2 days. Ion-exchange chromatography also has been used for the analysis of proteins, amino acids, sugars, nucleotides, pharmaceuticals, con- sumer products, and clinical samples. Several examples are shown in Figure 12.34.