Ion exchange chromatography

Anil ShresthaMsc Medical microbiology

Principle

• The principle feature is the attraction between oppositely charged particles.

• Many biological materials have ionisable group, they may carry a net +ve or –ve charge.

• The net charge exhibited by such compounds is dependent on the pH of the solution and isoionic point of the compound.

• Carried out in column mode.• Packed with ion exchange resins.

Types of ion exchanger

• Cation exchanger which posses negative charge and attract positively charged molecules.

• Anion exchanger which posses positive charge and attract negatively charged particle.

Ion exchange mechanism

It is thought to be composed of five distinct steps:

1)Diffusion of the ion through the matrix surface. This occurs very quickly in homogeneous solutions.

2)Diffusion of the ion through the matrix structure of the exchanger to the exchanger site. This is dependent upon the degree of cross-linkage of the exchanger and the concentration of the solution. This process is thought to be the feature that controls the rate of the whole ion-exchange process.

3)Exchange of ions at the exchanger site. This is thought to occur instantaneously and to be an equilibrium process.Cation exchanger:

RSO3-………..Na+ + R’-NH3

+ RSO3-…….NH3

+-R’ + Na+

(Exchanger) (Counter ion) (Charged Bound molecular ion Exchanged ion Molecule to be Separated) Anion exchanger:(R4)N+………….Cl - + R’-COO_ (R4)N+………_OOC-R’ + Cl-

(Exchanger) (Counter ion) (Charged Bound molecular ion Exchanged ion Molecule to be Separated)

The more highly charged the molecule to be exchanged, the tighter it binds to the exchanger and less readily it is displaced by other ions.4)Diffusion of the exchanged ion through the exchanger to the surface.5)Selective desorption by the eluent and diffusion of the molecule to the external eluent. The selective desorption of the bound ion is achieved by changes in pH and /or ionic concentration or by affinity elution. In affinity elution the ion having greater affinity for the exchangers than for the bound molecule is introduced into the system.

Ion exchange material• Most are polymeric resins. The resins incorporate

various functional group, strong or weak acids or bases, to provide the ion exchange capacities. All exchangers are generally supplied with an appropriate counter ion, normally Na+ or Cl-.

Type Polymer Functional group

Cation exchange (Weakly acidic)

Agarosecellulose

-COO-

-CH2CCOO-

Cation exchange (Strongly acidic)

CelluloseDextran

-SO3-

-CH2SO3-

Anion exchanger (Weakly basic)

AgaroseCellulose

-CH2CH2NH3+

-CH2CH2NH+(CH2CH3)2

Anion exchanger (Strongly basic)

Polystyrene Callulose

`-CH2N+(CH3)3

-CH2CH2-N+(CH2CH3)2

CH2CH(OH)CH3

Table: List of ion-exchange resin

• Low pressure ion-exchange chromatography can be carried out using a variety of matrices and ionic group. Matrices used include polystyrene, cellulose and agarose. Functional ionic groups include sulphonate (-SO3

-) and quaternary ammonium(-NR3), both of which are strong exchangers because they are totally ionized at all normal working pH values. Carboxylate (-COO-) and diethylammonium (HN(CH2CH3)2),both of which are termed weak exchangers because they are ionized over only a narrow range of pH values.

• All exchangers are characterized by a total exchange capacity, which is defined as the number of milliequivalents of exchangeable ions available, either per gram of dried exchanger or per unit volume of hydrated resins. Thus the exchange capacity of Bio-rex 70 is 3.3 meq.cm-3, DEAE-Sephadex A-25 is 0.5 meq.cm-3.

Exchanger Selection• The choice of ion exchanger depends upon the stability of the

sample components, their relative molecular mass and the specific requirements of the separation.

• Many biological compounds, especially proteins, are stable within only a fairly narrow pH range so the exchanger selected must operate with in this range. Generally, if the sample is most stable below its isotonic point, giving its net positive charge, a cation exchanger should be used, whereas if it is most stable above its isotonic point, giving it a net negative charge, an anion exchanger should be used. Compounds that are stable over a wide range of pH may be separated by anion or cation exchanger.

• Although the degree of cross-linking af an exchanger does not influence the ion-exchange mechanism, it does influence its capacity. Resins with a low degree of cross linking are more permeable to high molecular weight compounds. The meshsize of polystyrene resins determines the flow rate.

• The pH of the buffer used should be at least one pH unit above or below isoionic point of the compounds being separated. In general, cationic buffers, eg, TRIS, pyridine, alkyl amines are used in conjugation with anion exchangers; anionic buffers, eg, acetate, barbiturate and phosphate are used in conjugation with cation exchangers. Initially the buffer pH and ionic strength should be such as to just allow the binding of the sample components to the exchanger. A buffer of the lowest ionic strength should initially be used for the subsequent elution of the components.

• The amount of the sample that can be applied to a column is dependent upon the size of the column and the capacity of the exchanger.

APPLICATION

• Water demineralization• Recovery of antibiotics from fermentation

broth• Recovery of vitamins from fermentation broth• Separation of amino acids• Water softening• Recovery of uranium from acid leach solution

Application in amino acid separation

• The separation of amino acids (in a protein hydrolysate) is usually achieved using a strong acid cation exchanger. The sample is introduced on top the column at a pH 1-2 thus ensuring complete binding. Gradient elution using increasing pH and ionic concentration results in the sequential elution of the amino acids. The acidic amino acids( aspartic and glutamic ) are eluted first, followed by the neutral amino acids(glycine and valine). The basic amino acids( lysine and arginine) retain their net positive charge upto pH 9-11 and are eluted last. Automatic aminoacid analyzers use these principles.

• The gist of ion-exchange chromatography is that ions of one types will be attracted to the charges on the resins beads more or less strongly than ions of a different type. Upon elution, least attracted ion will emerge from the bottom of the column before the others. So solutions of different ions can be collected in different flasks.