Ion-Exchange Chromatography

Ion-exchange chromatography involves the separation of molecules such as proteins based on their charge profile. It is often defined by process engineering texts as the reversible exchange of ions in solution with ions that are electrostatically bound to an insoluble matrix or a stationary phase. 

This technique is extremely useful in the separation of differently charged compounds. These can be proteins, nucleic acids, carbohydrates and so on. The technology is now so well developed that a protein which differs by one charged amino acid for example can be separated from another. It also means that only slight differences in the charge profile are needed for a separation.

Ion-exchange chromatography usually involves the binding of a substance of interest whilst the contaminants pass through. These are usually washed away. The reverse can happen where contaminants are bound to a stationary phase and are removed leaving a pure or cleaner material to be used elsewhere. It was in this situation that drinking water was first purified by the removal of debris leaving relatively pure water. This type of technology has been use in the 1800s.

Ion-exchange chromatography is often referred to simply as ion chromatography.

The Principle Of Ion-Exchange Chromatography

Ion exchange chromatography is based on an attraction between an oppositely charged stationary phase and components often called analytes which are usually in solution.

An ion-exchange system consists of an inert support medium usually called a stationary phase or a matrix. This is coupled by covalent bonding to either positively charged functional groups as with an anion exchanger or to negative functional groups as in a cation exchanger.

These covalently bound functional groups attract oppositely charged ions which are effectively bound. These are known as the mobile counter ions. These mobile counter ions become exchanged with similar charged ions present in the sample having a greater charge magnitude than the ions bound to the matrix.

For example, if an anion exchange chromatography operation is performed, negatively charged sample components interact more with the stationary phase and will be exchanged for like charged ions already bound to the matrix.   

Advantages Of Ion-Exchange Columns

• Cost effectiveness
• Easily manageable
• Low maintenance costs
• Efficient in technique
• rapid separation
• re-usable

Typical Applications

• water softening
• demineralization of water
• purification of proteins and nucleic acids
• concentration of metal ions

Factors Affecting Ion-Exchange Chromatography Separation

1. Ion-exchange resins: the swelling factor and degree of cross-linking is important for the effectiveness of separation. Cross-linking should be controlled as it affects the exchanger’s capacity. Swelling helps in the proper exposure of charged functional groups for exchange of ions.
2. The sample: The concentration and charge of the ions in the sample are critical.
3. The buffer: The pH of the buffer should impart the same charge to the sample ions as present in the column. In anion exchange chromatography it is  performed with cationic buffer and vice versa because buffer ions will indulge in ion exchange, which is of little value.

Stationary Phases

Resins for the stationary phase are made from a number of materials which help with liquid flow around them, accessibility for ions and large compounds, their chemical and mechanical stability. Chemical stability is such that the matrix must be stable enough to withstand washing by acid and alkaline solutions. All these factors are important for packing materials used in columns in other applications like gel filtration chromatography and affinity chromatography too.

The matrix should also be highly insoluble and hydrophilic. 

The selection of any type of resin depends on the type of compound to be separated too. The ion-exchange capacity must also be high enough for a large amount of the compound to be adsorbed. The matrix should also be inert in the sense there should be no nonspecific adsorption interactions because these are undesirable.

Matrices used for binding ligands include agarose, cellulose, dextran, polystyrene, polyacrylate

Polystyrene is often used. It is a resins prepared by polymerization of styrene and divinylbenzene. A higher concentration of divinylbenzene produces lighter cross linkages. Polystyrene resins are good for separating small molecular weight compounds but not for separating macromolecules such as proteins.

Matrices are defined by a charge value usually quoted as meq/g exchange capacity. 

Types of Anion Exchangers

There are two basic type of anion exchanger.

Ones that have weakly basic cations bound to them and include  aminoethyl- and diethylaminoethyl- groups.

Ones that are strongly binding include trimethylaminomethyl-, triethylaminoethyl-, and dimethyl-2-hydroxyethyl-aminomethyl- groups. All these cations are based on a range of amino groups.

A DEAE-ion exchanger is often used for protein separation and can be bought off the shelf as a coarse mesh analytical, preparative and large-scale chromatography material. Typical types have a value of 0,.95 meq/g exchange capacity.

Cation Exchangers

Cation exchangers have negatively charged ligands bound to a stationary phase. These are anions which are based on groups such as sulphonic acid and carboxylic acid. The sulphonic acid groups are strong cation exchangers whilst carboxylates are weak cation exchangers. The compounds they are binding are positively charged.

Preparing An Ion Exchange Column

There are three key steps in the preparation of an ion exchange column for purification.

1. The stationary phase is usually equilibrated in a process called swelling. With anion exchangers, this is done by treating it first with an acid such as 0.5N HCl and then with a base such as 0.5N NaOH. The reverse is the case for preparing cation exchangers.
2. Fine particles are removed: a large number of such finer particles will clog up a stationary phase which then decreases the flow rate and leads to improper resolution. Most ion-exchange resins  are repeatedly suspended in large volumes of water and the fines decanted away.
3. Addition of counter ions: this is achieved using a suitable reagent depending on which the counter ion is to be introduced. This could mean adding sodium hydroxide to provide sodium cations or  hydrochloric acid providing protons.

A typical process for preparing an ion-exchange column was described in the 1950s by Moore and Stein (1954) in the chromatographic determination of amino acids found in tomato juice. This was an application using 4% cross-linked sulphonated polystyrene resin and offers a good example of the type of column preparation needed and subsequent elution of the amino acids.

In this example the cation-exchange resin was treated with 4N hydrochloric acid until the filtrate appearing at the end of the column was colourless. The resin was then repeatedly washed with water and the counterion added in the form of 2N sodium hydroxide until the filtrate had an alkaline pH. Sodium ions were the counterion in this case. The sodium salt was suspended in 3 times its own volume of 1 normal sodium hydroxide and heated over a steam bath for 3 hours with stirring. This alkaline treatment was repeated 5 times.

The resin had  now been prepared with the counterion. The resin was finally washed to remove any remaining alkali before packing. Whilst this suggests extensive work to regenerate the resin, it illustrates a typical approach to the preparation of such resins.

Eluting Buffers

DEAE-cellulose is a classic example of an anionic ion-exchanger. In many cases elution is the process of selectively removing a protein bound to a column and the chemical and physical approaches are typical too of other types of chromatography.

A typical example might be the system used here for the purification of egg yolk proteins (McBee & Cotterill, 1979). A DEAE-cellulose resin was used in this case. The DEAE ion-exchange material was prepared and equilibrated to pH4.5 with 0.02M glycine as the first buffer. The samples were applied and eluted with buffers of increasing ionic strength and decreasing pH. Typical buffers were based on citric acid-sodium citrate, and hydrogen phosphates.

Once the sample from which the proteins to be purified has been applied it then remains for elution to occur. Typical buffers are prepared from 0.1M glycine, 1 M potassium dihydrogen phosphate, 1 M sodium chloride and 1M hydrochloric acid. 

A series of buffers will be applied in the pursuit of elution of the individual proteins.

Regeneration of Ion-Exchange Columns

Cation exchange resins are regenerated by treatment with acids and then washing with water.

Anion exchange resins are regenerated by treatment with NaOH, then washing with water.

References

Yamamoto, S., Nakanishi, K., Matsuno, R. (1988) Ion-Exchange Chromatography of Proteins. Marcel Dekker, Inc. New York & Basel.

Wilson, K., & Walker, J. (Eds.). (2010). Principles and techniques of biochemistry and molecular biology. Cambridge University Press.