Ion Exchange Mechanism
Ion exchange separates components in a sample depending on their relative ionic strengths and interaction with the stationary phase. The stationary phase is a charged substance that selectively retains species with opposite charges.
This is an effective method for separating species with similar charges, as charged analytes compete to bind with the opposite charge on the column. In anion exchange HPLC, the stationary phase is positively charged and holds negatively charged ions, while cation exchange uses the opposite principle.
Sample elution in ion chromatography primarily depends on the pH and ionic strength of the mobile phase and, to a lesser degree, the operating temperature. The Hamilton ion exchange columns offer the advantage of operating across a wide pH range and at higher temperatures compared to silica-based supports. Ion chromatography reduces sample preparation significantly and enhances the accuracy and precision of results.
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What is Exchange Capacity?
Ion exchange capacity is a measure of how many positive (cations) or negative (anions) charges can bind to the resin, typically reported as singly charged ion equivalents per gram of resin. This capacity depends on the mobile phase pH. In anion exchange chromatography, as pH rises (acidity lowers), exchange capacity declines, while in cation exchange chromatography, the opposite occurs.
Controlling Retention Time in Ion Exchange Chromatography
Retention time and ion separation can be adjusted by altering eluent concentration and flow rate. Increasing the concentration of the mobile phase can shorten retention time but may also reduce resolution. However, decreasing eluent strength increases both retention and separation if greater resolution is required.
Ion Exchange Chromatography Detection
Conductivity and UV are the most commonly used methods for the detection of ions. The ion detection usually employs conductivity detection or UV methods. Both methods offer similar detection limits of 0.5 to 150 ppm per anion with a 100 μL injection volume. Mass spectrometers (MS) and electrochemical detectors are becoming more popular. The choice of detection depends on available instrumentation.
Conductivity Detection
Conductivity is often a preferred method of detection, as it is the most universal method, able to detect ionized samples against a poorly conducting mobile phase background. The use of ion exchange suppressors could greatly reduce the background conductivity of the eluent, thus improving the method sensitivity.
UV Detection
Some inorganic ions don't have significant absorbance of UV or visisble light. What is called indirect UV detection is employed by using a UV-adsorbing mobile phase. It works becausse inorganic ions absorb less light in the mid-UV range (280-350 nm) than mobile phase ions. Sample ions appear as negative peaks against the UV-absorbing background. If the detector output leads are reversed, these peaks appear positive.
Ion Exchange Chromatography Column Equilibration
Electrostattic interaction is cosidered slower for ion exchange than the reversed phase mechanism. Therefore, equilibration requires approximately 150 column volumes of mobile phase and is complete when analyte retention times are consistent.
Ion Exchange Chromatography Column Regeneration
Regenerating fouled columns can extend their lifespan. Over multiple sample injections, buildup on the column can degrade separation performance, and the column may need to be regenerated. Indications that this is needed include a drifting baseline and recurring artifact peaks during elution.
This information has been sourced, reviewed, and adapted from materials provided by Hamilton Lab.
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