Ion Exchange Chromatography

Ion Exchange Mechanism
 

Ion exchange is a separation mechanism based on the relative ionic strengths of the components in a sample mixture. The stationary phase is a charged material that will preferentially retain species with the opposing charge. This mechanism is useful for separating species of like charge, since charged species compete to pair with the opposing charge of the column. Anion exchange HPLC has a positively charged stationary phase that retains negatively charged species. The opposite is true for cationic exchange. Elution in ion chromatography is affected by mobile phase pH and ionic-strength, and to a lesser extent, operation temperature. The ability to use the full pH range and elevated temperatures are distinct advantages of Hamilton ion exchange columns compared to silica-based supports. Ion chromatography greatly reduces sample pretreatment and improves the accuracy and precision of results.

What is Exchange Capacity?

Ion exchange capacity is a measurement of the number of positive charges (cations) or negative charges (anions) that the exchange resin can bind to and is reported in singly charged ion equivalents per 1 gram of resin. Exchange capacity is dependent upon the pH of the mobile phase and in anion exchange chromatography; as mobile phase acidity decreases (pH increases), the exchange capacity decreases. In cation exchange chromatography, the inverse is true.

Controlling Retention Time in Ion Exchange Chromatography
 

Ion retention and analysis time can be altered by changing eluent strength and flow rate. When the mobile phase concentration is increased ion retention is shortened and some resolution is lost. Conversely, if the analysis requires more resolution and greater sample retention, then a decrease in eluent strength is more appropriate.

Ion Exchange Chromatography Detection

Detection of ions is generally via electronically suppressed conductivity or indirect UV. Selecting a detection method is often dictated by the available equipment. Both conductivity and indirect UV provide comparable detection limits, 0.5 to 150 ppm per anion at a 100 μL injection volume.

Conductivity Detection

Conductivity detection is often the preferred mode because it is the most universal; ionized samples are detected against the poorly conducting mobile phase ions.

Indirect UV Detection

Indirect UV detection works because inorganic ions absorb less in the mid UV (280-350 nm) than mobile phase ions. Sample ions appear as negative peaks in the UV absorbing background. When the leads from the detector to the recorder are reversed, the peaks appear as positive.

Ion Exchange Chromatography Column Equilibration

Equilibration of ion exchange columns requires approximately 150 column volumes of mobile phase. Equilibration is complete when analyte retention is reproducible.

Ion Exchange Chromatography Column Regeneration

The ability to regenerate badly fouled columns allows you to extend column life. After numerous injections, it is possible that materials may have built up on the column and caused column performance to deteriorate. An upward drifting baseline and the appearance of reproducible artifact peaks during elution are good indications that the column needs to be regenerated. The regeneration protocol for anion exchange HPLC columns can be found here.