W.Cohn first developed ion exchange chromatography procedure. The reversible exchange of ions in solution with ions electrostatically bound to some sort of insoluble support medium. The ion exchange chromatography in protein purification has very important role.
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What is ion exchange chromatography?
Ion exchange chromatography definition (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It can be used for almost any kind of charged molecule including large proteins, small nucleotides, and amino acids.
Principle of Ion-exchange Chromatography
Ion Exchange chromatography principle, Exchange of ions is the basic principle in this type of Chromatography. In this process, two types of ion-exchange chromatography. They are i.e., cationic and anionic exchangers can be used.
- Cationic exchangers possess negatively charged groups, and these will attract positively charged cations. These exchangers are also called “Acidic ion exchange” materials because their negative charges result from the ionization of acidic groups.
- Anionic exchangers have positively charged groups that will attract negatively charged anions. These are also called “Basic ion exchange” materials.
Types of Ion Exchange Resins
- Two main groups of materials are used to prepare ion exchange resins: Polystyrene and Cellulose.
- Resins made from both of these materials differ in their flow properties, ion accessibility and chemicals, and mechanical stability.
- Polystyrene resins are proposed by the polymerization reaction of styrene and divinylbenzene.
- A higher concentration of divinylbenzene produces higher cross-linkages.
- Polystyrene resins are very useful for separating small molecular weight compounds.
- Increasing the cross-linkage increases the rigidity, reduces swelling, reduces porosity & reduces the solubility of the polymeric structure.
- Sulfonic acids are strong acids with good proton dissociation ability. By sulfonation process, acidic functional groups are easily attached to nearly every aromatic nucleus.
- Resins substituted with sulfonic acid groups are strong cationic exchangers.
- To prepare weekly acidic exchanger, carbohydrate groups can be attached to the aromatic rings instead of a sulfonic acid group.
- If basic functional groups are introduced, the resin can exchange anions rather than cations. Strong anion exchangers are prepared with a tertiary amine, yielding a strongly basic quaternary ammonium group. Weak anionic exchangers can be prepared with secondary amines, yielding a weakly basic tertiary amine.
Cellulose resins have much greater permeability to macromolecular polyelectrolytes and possess a much lower charge density as compared to polystyrene exchangers.
- Carboxymethyl cellulose (CM-cellulose) – Cationic exchanger
- DEAE cellulose – Anionic exchanger
Preparation of the exchange medium
There are three steps are of absolute importance:
1) Swelling of medium: (Pre-cycling)
Swelling makes the functional groups to be exposed for ion exchange.
- The swelling of anion exchangers is usually carried out by treating it. first with an acid (0.5N HCl) and then with base (0.5N NaOH).
- Exactly the reverse is the case with cationic exchangers. The matrix can be treated with EDTA for impurity eliminations.
2) Removal of very small particles
These fines will decrease the flow rate and the unsatisfactory reaction. To remove fines, the exchanger is repeatedly suspended in a large volume of water and after the larger polymers have settled down, the slow sedimenting materials decanted.
3) Equilibration with counter ions
This is accomplished by washing the exchanger with different reagents depending upon the desired counter ion to be introduced.
- NaOH –> counter ion to be introduced is “Na+”
- HCl –> counter ion to be introduced is “H+”
- NaNO3 –> counter ion to be introduced is “NO3”
Choice of Buffers
- Anionic exchange Chromatography should be carried out with cationic buffers.
- Cationic exchange Chromatography should be carried out with anionic buffers.
- The pK of the buffer should be as near as possible to the pH at which the system is buffered. This results in high buffer capacity, which can withstand the local changes of pH in the column easily.
|Ammonium acetate||4 to 6|
|Ammonium formate||3 to 5|
|Pyridinium formate||3 to 6|
|Pyridinium acetate||4 to 6|
|Ammonium carbonate||8 to 10|
Ion-Exchange Chromatography Procedure
- Ion exchange separations are carried out mainly in columns packed with an ion-exchanger. These ionic exchangers are commercially available. They are made up of styrene and divinylbenzene.
- DEAE-cellulose is an anionic exchanger, CM-cellulose is a cationic exchanger. The choice of the exchanger depends upon the charge of the particle to be separated. To separate anions “Anionic exchanger” is used, to separate cations “Cationic exchanger” is used.
- First, the ion exchange chromatography column is filled with ion exchanger then the sample is applied followed by the buffer. The tris-buffer, pyridine buffer, acetate buffer, citrate and phosphate buffers are widely used.
- The particles which have a high affinity for ion exchanger will come down the column along with buffers. In the next step using the corresponding buffer separates the tightly bound particles.
- Then these particles are analyzed spectroscopically.
Ion exchange chromatography applications
- It is extremely used in the analysis of amino acids. The amino acid “Autoanalyzer” is based on in exchange principle.
- To determine the base composition of nucleic acids. Chargaff used this technique for established the equivalence of Adenine and Thymine; Guanine and Cytosine.
- This is the most effective method for water purification. Complete deionization of water (or) a non-electrolyte solution is performed by exchanging solute cations for hydrogen ions and solute anions for hydroxyl ions. This is usually achieved by the method is used for the softening of drinking water.
- protein purification by ion exchange chromatography is another successful technique.
- It is also used for the separation of many vitamins, other biological amines, and organic acids and bases.
One of the main disadvantages of ion-exchange chromatography is its buffer requirement: because binding to IEX resins is dependent on electrostatic interactions between proteins of interest and the stationary phase, ion exchange chromatography column must be loaded in low-salt buffers. The ion exchange chromatography for protein purification is most popular method in biochemical techniques.
For some applications, this restriction may require a buffer exchange step prior to ion exchange chromatography.