23 Ion exchange chromatography

Dr. Varinder Kaur

Objectives: To study the basics of ion exchange chromatography and know the following  questions.

1. What is ion exchange chromatography?
2. How it works?
3. What are the ion exchangers?
4. Where ion exchange chromatography is applied?

1. Description

It is the chromatographic techniques, which is used to separate the charged molecules such as ions, large proteins, nucleotides and amino acids, etc. It was discovered by an agricultural chemist who worked on the adsoption of ammonium salts to soils and followed by several scientists for the respective separation of rare earth elements, anionic species, and proteins. With the more advancement, anion exchange and cation exchange chromatography came into existence. It is also known as ion chromatography. Nowadays, ion exchange chromatography is popular for potential applications in pharmaceutics, biotechnology, environment science, agriculture science and industries.

2. Principle

Ion exchange chromatography, the separation of charged analytes takes place on the basis of ionic (or electrostatic) interactions between analytes and the stationary phase consisting of ionic functional groups (ionic exchangers). The driving force for the separation of ions is competitive ionic binding and repulsion between the similarly charged analyte ions/stationary phase ions.

3. Theory of ion exchange

The basic steps and components of ion exchange chromatography are same as the liquid column chromatography i.e. mobile phase, stationary phase and eluent. However, the properties of stationary phase are specific in ion exchange chromatography. The stationary phases used in ion exchange chromatography consist of insoluble surfaces with exchangeable ions and are called as ion exchangers. The ionic functional groups fixed on the surface of the stationary phase are referred as fixed ions while the exchangeable ions with opposite charge are referred as counter ions. The counter ions have tendency to move under the influence of electric field, diffusion or via exchange with similarly charged external ions. The counter ions may include protons (H+), hydroxide groups (OH-), uni-positive/negative ions (Na+, K+, Cl-), double charged ions (Ca2+, Mg2+), and polyatomic ions (SO42-, PO43-), organic bases (NR2H+) and acids (COO-), etc. Therefore, the ion exchangers are classified into two categories; 1) Cation exchangers when fixed ion is negatively charged 2) Anion exchangers when fixed charge is positively charged.

To understand the mechanism of ion exchange, an illustration is given below. Suppose S+ is stationary material carrying a positively charged fixed ion and negatively charged counter ion E-. When it is exposed to mobile phase having negatively charged analyte A-, the counter ion E- moves into the mobile phase and analyte ion A- interacts with the stationary phase. This exchange between counter ion and analyte ion establishes equilibrium and at equilibrium both the A- and E- are distributed between S+ and mobile phase. However, co-ion of the analyte does not take part in the exchange process and electroneutrality of the solution is maintained by the flow of equal charges between the two phases.

The tendency of ions/charged molecules to interact with the charged stationary phase vary considerably due to their overall charge, charge density and surface charge distribution, which results in the variable retention time of each components and helps in their separation.

4. Configuration of ion exchange system

The ion exchange systems are of two types; classical system and modern system.

4.1 Classical ion exchange chromatographic system

The classical ion exchange system consisted of and open tubular column and mobile phase or eluent. The glass column (1-2 cm diameter) is filled with loosely bound ion exchanger material. The mobile phase consisting of a competing ion is passed through the column from the top to bottom to run under gravity. After the saturation of column with the mobile phase, sample solution containing components is poured into the column from the top and flow of eluent (mobile phase) is stopped for some time to allow the equilibration. Then, the flow of eluent is resumed and eluent is collected at the bottom at regular intervals of time. Each fraction of eluent is analyzed for the eluted components and parameters are evaluated.

The classical ion exchange chromatography is not much preferred because it involves flow of solvent under gravity which makes the separation process slow, and leads to poor resolution and poor efficiency.

4.2 Modern ion exchange chromatography

The modern ion chromatography is able to solve the problems associated with classical ion exchange chromatography. Like high performance liquid chromatographic technique, it involves use of pump to push the mobile phase at a high flow rate, rigid column filled with ion exchanger of high quality, and a detector to analyze the components continuously eluted with the eluent. The working and instrumentation of ion chromatography is same as HPLC, however the specific characteristics of the eluent (mobile phase), and stationary phase (ion exchanger) are discussed below.

4.2.1 Eluent (mobile phase)

The main component required for the ion exchange between the stationary phase and the mobile phase is competing ion. This ion helps in the elution of the analyte ion bound with the ion exchange material within suitable time period. Therefore, eluent in ion exchange chromatography consists of aqueous solution of a salt (or mixture of salts) with small percent fraction of organic solvent. An additional buffer is added to the eluent (if required) otherwise the salt mixture itself acts as a buffer in most of the cases. The separation process is governed by various factors associated with the eluent like nature and concentration of competing ions, pH and flow rate of the eluent and temperature of the eluent.

The separation of components is highly influenced by the nature and concentration of competing ions. The affinity of a competing ion to replace analyte ion from the stationary phase makes is determined in terms of selectivity coefficient of that particular competing ion. This selectivity coefficient varies with the nature of competing ion therefore separation is affected by the type of salt solution selected as an eluent. Secondly, the higher is the concentration of the competing ion in the eluent, higher is the displacement of the analyte ion from the stationary phase. This shifts the direction of equilibrium and elution of analyte increases.

The time spent by the analyte on the stationary phase and displacement of the analyte by competing ion directly depends upon the flow rate. Therefore, flow rate of the eluent should be appropriate as high flow rates give rise to less resolution and low flow rates make the process time consuming. In conrast, temperature has less effect on the separation process. Sometimes, increase in temperature increases the diffusion within matrix and therefore, increases the elution volumes.

4.2.2 Ion exchangers (stationary phase)

A stationary phase in chromatographic techniques remains fixed. In case of ion exchange chromatography, the stationary phase is termed as ion exchanger due to its ability to change the ion with mobile phase. The ion exchangers possess two main structural components; 1) a polymeric substrate possessing a fixed ion, 2) exchangeable ion. The fixed substrate is insoluble in the solvents, however, the its exchangeable ion is mobile in nature and can be exchanged with other ions having similar charge. The fixed polymeric network may be of organic or inorganic origin and can be exist naturally or may be synthesized. However, on the basis of exchangeable ions attached to the fixed substrate, ion exchangers can be classified into cation exchangers or anion exchangers.

5.  Characteristics of ion exchangers

5.1 Ion exchange capacity

The ion exchange capacity of an ion exchanger determines the number of ions displaced on the ion the exchanger. It depends upon the number of functional groups present per unit weight of the substrate. It is determined both for the dry state as well as wet state of the exchanger. The determination of ion exchange capacity involves saturation of known amount of ion exchanger with a particular ion followed by the washing and quantification of the displacement of exchangeable ion from the material. The ion exchange capacity is reported in terms of milliequivalent per gram or milliequivalents per litre.

5.2 Swelling characteristics

The ion exchangers of organic origin show swelling when come in contact with the mobile phase. These materials are highly cross-linked and have folded structures. In the presence of mobile phase, the polymeric networks unfold and tend to accommodate the solvated ions. As a result the water molecules enter into the spaces and the polymeric substrate shows swelling. The swelling property decides the mechanical strength of the exchanger.

5.3 Ion exchange selectivity

It determines the affinity of an ion exchanger for a particular ion. It depends upon the nature of polymer.

The main factors which affect the ion exchange selectivity are as follows:

1). Charge on the analyte ion

2). Size of the analyte ion

3). Degree of cross-linking of the exchanger

4). polarizability of the analyte ion

5). Ion exchange capacity of the exchanger

6). Interaction between the exchanger and the analyte ion

7). functional group on the exchanger

For example; ion exchange selectivity of strong acid cation exchanger decreases with the decrease in the charge on the cation.

Pu⁴⁺>La³⁺>Ce³⁺>Pr³⁺>Eu³⁺>Y³⁺>Sc³⁺>Al³⁺>Ba²⁺>Pb²⁺>Sr²⁺>Ca²⁺>Ni²⁺>Cu²⁺>Co2+>Zn²⁺>Mg²⁺>Tl⁺>Ag⁺> Cs⁺>Rb⁺>K⁺>NH4⁺>Na⁺>H⁺>Li⁺

6. Buffer

The pH of the eluent is very important parameter for the separation of charged components on the ion exchange chromatography. Therefore, buffers are added to the mobile phase or salts/mixture of salts are selected which can act as buffer to maintain a constant pH throughout the experiment. The pH variation can occur due to the co-ions released from the analyte or the exchangeable ions released from the stationary phase during equilibration. The buffers having high buffering capacity, wide working pH, good solubility, high purity and low cost are suitable for ion-exchange chromatography. The buffer should be inert towards the functional groups of the exchanger and should have negligible conductivity. Some shortcomings of using buffer solutions include the interactions of buffer with stationary phase or mobile phase, precipitation of the mobile phase, etc. The best conditions for ion exchange chromatography are slightly acidic to slightly alkaline conditions i.e. pH range 6.0-8.5. Some common buffers include phosphate buffer (anionic buffer) and ethanolamine, Tris and Tricine (as cationic buffers).

7. Detection

The detection process is common as used in HPLC. However, conductivity detector is the best detector in ion exchange chromatography. It offers excellent sensitivity when the conductance of the eluted solute ion is measured in an eluent of low background conductance. In addition, UV-Vis detectors are also used with ion exchange chromatography.

8. Various stages in exchange chromatography

In brief, the system is saturated with the mobile phase and sample is introduced into the system manually or using an autosampler. The mobile phase is selected according to the nature of analytes present in the sample. Usually it is a buffered aqueous solution and it carries the sample from the loop to stationary phase. The stationary phase is also selected on the basis of the nature of analytes. Then, the retained analytes (anions or cations) are eluted by increasing the concentration of a similarly charged species. These ions displace the analyte ions from the stationary phase, which are delivered to the detector for detection. The main stages for the interaction between analyte and stationary phase are summarized below.

9. Disadvantages of ion exchange chromatography

Apart from the various advantages, ion exchange chromatography has some disadvantages.

1.       It requires buffers in the mobile phase, which require proper washing making the process lengthy.

2.       The efficiency of column decrease because buffers left in the column may contaminate the column.

3.       Sometimes, it is difficult to achieve control over selectivity and resolution by ion exchange chromatography.

10. Applications of Ion Exchange Chromatography

1.   It is used for the separation and analysis of amino acids, lanthaides, nucleic acids, proteins, vitamins, organic acids and bases etc.

2.   It is used as preconcentration techniques for trace analysis.

3.   It is used to remove interferences due to radicals.

4.   This is most effective method for water purification and softening of drinking water.

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