Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/Ion chromatography

Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.

History
Ion methods have been in use since 1850, when H. Thompson and J. T. Way, researchers in England, treated various clays with ammonium sulfate or carbonate in solution to extract the ammonia and release calcium. In 1927, the first zeolite mineral column was used to remove interfering calcium and magnesium ions from solution to determine the sulfate content of water. The modern version of IEC was developed during the wartime Manhattan Project. A technique was required to separate and concentrate the radioactive elements needed to make the atom bomb. Researchers chose adsorbents that would latch onto charged transuranium elements, which could then be differentially eluted. Ultimately, once declassified, these techniques would use new IE resins to develop the systems that are often used today for specific purification of biologicals and inorganics. In the early 1970s, ion chromatography was developed by Hamish Small and co-workers at Dow Chemical Company as a novel method of IEC usable in automated analysis. This later lead to the formation of Dionex Corp (Dow -Ion Exchange) who lead the market in IC equipment and developments. IC uses weaker ionic resins for its stationary phase and an additional neutralizing stripper, or suppressor, column to remove background eluent ions. It is a powerful technique for determining low concentrations of ions and is especially useful in environmental and water quality studies, among other applications.

Principle
Ion exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. The ionic compound consisting of the cationic species M+ and the anionic species B- can be retained by the stationary phase.

Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group:

$$\text{R-X}^-\text{C}^+\,+\, \text{M}^+ \, \text{B}^- \rightleftarrows \,\text{R-X}^-\text{M}^+ \,+\, \text{C}^+ \,+\, \text{B}^-$$

Anion exchange chromatography retains anions using positively charged functional group:

$$\text{R-X}^+\text{A}^-\,+\, \text{M}^+ \, \text{B}^- \rightleftarrows \,\text{R-X}^+\text{B}^- \,+\, \text{M}^+ \,+\, \text{A}^-$$

Note that the ion strength of either C+ or A- in the mobile phase can be adjusted to shift the equilibrium position and thus retention time.

The ion chromatogram shows a typical chromatogram obtained with an anion exchange column.

Typical technique
A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. A buffered aqueous solution known as the mobile phase carries the sample from the loop onto a column that contains some form of stationary phase material. This is typically a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions. The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance.

In order to control an IC system, a chromatography data system (CDS) is usually needed. In addition to IC systems, some of these CDSs can also control gas chromatography (GC) and HPLC systems.

Separating proteins


Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange chromatography separates proteins according to their net charge, which is dependent on the composition of the mobile phase. By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively-charged beads, whereas a negatively charged protein would not. By changing the pH so that the net charge on the protein is negative, it too will be eluted.

Elution by changing the ionic strength of the mobile phase is a more subtle effect - it works as ions from the mobile phase will interact with the immobilized ions in preference over those on the stationary phase. This "shields" the stationary phase from the protein, (and vice versa) and allows the protein to elute.