Ion, exchange chromatography

Ion exchange is a process wherein a solution of an electrolyte is brought into contact with an ion exchange resin and active ions on the resin are replaced by ions (ionic species) of similar charge from the analyte solution. 

Ion exchange resin materials are based on styrene-divinylbenzene polymers and polyacrylates (Table 4.3). The synthesis of these materials is easily controlled and gives materials of the necessary chemical and physical stability, in terms of uniformity of particle size and shape, porosity and chemical composition. These resins find wide application in demineralisation, water treatment and ion recovery from wastes. 

Sulphonic acid (SO3H) exchange units are introduced in a controlled fashion by reacting the solvent swollen resin with chlorosulphonic acid, which results in mainly para-substitution of the benzene rings. The sulphonic acid resin is described as a strong cation exchanger, is fully dissociated over a 

Cation exchange Strong acid -SO3H 1-14 4mmol H g  

Anion exchange Weak acid Strong base CO2H -CH2-i tR3 5 14 1-15 9-lOnimol H g 4mmoI OH g-  

To reduce retention time of nucleotides on reversed-phase columns, organic modifiers such as methanol or acetonitrile may be added to the buffer. Acetonitrile is usually more effective than methanol in the sense that less is required to elute a given nucleotide with a specific retention time. 

At the conclusion of each work day it is advisable to wash the salt buffer thoroughly from the both the pump heads and the column. A 0.02% sodium 

Ion-exchange chromatography is based on the equilibration of solute species between the solvent, or mobile phase, and charged sites fixed on the stationary phase. Anion-exchange columns have cationic sites immobilized, and anionic 

TABLE 14.5. Functional Groups on Representative Ion-Exchange Gels 

Anion Strong base Triethylaminoethyl (TEAE) OCH2CH2N+(CH2CH3)3 

The classical model of selectivity12 in ion-exchange chromatography is based upon stoichiometric exchange for example, the competition of Na+ and Li+ for sites on a cation-exchanger (R )  

The selectivity coefficient for this classical model is the thermodynamic equilibrium constant for this reaction  

Ion exchange chromatography is based upon the differential affinity of ions for the stationary phase. The rate of migration of the ion through the column is directly dependent upon the type and concentration of ions that constitute the eluent. Ions with low or moderate affinities for the packing generally prove to be the best eluents. Examples are hydroxide and carbonate eluents for anion separations. 

In ion-exchange chromatography (IEC), species are separated on the basis of differences in electric charge. The primary mechanism of retention is the electrostatic attraction of ionic solutes in solution to fixed ions of 

Chapter 2 Separations in High-Performance Liquid Chromatography 

If the analytes are charged cations or anions (strong or weak acids or bases), then IE may perform better. This methodology is discussed later at great length in Section 13.2. 

The process of removing absorbed ions is known as elution, the solution employed for elution is termed the eluant, and the solution resulting from elution 

If the ion exchange column is loaded with several ions of similar charge, B, C, etc., elution curves may be obtained for each ion by the use of appropriate eluants. If the elution curves are sufficiently far apart, as in Fig. 7.2, a quantitative separation is possible only an incomplete separation is obtained if the elution curves overlap. Ideally the curves should approach a Gaussian (normal) distribution (Section 4.9) and excessive departure from this distribution may indicate faulty technique and/or column operating conditions. 

The rate at which two constituents separate in the column is determined by the ratio of the two corresponding distribution coefficients, where the distribution coefficient is given by the equation 

The distribution coefficient can be determined by batch experiments in which a small known quantity of resin is shaken with a solution containing a known concentration of the solute, followed by analysis of the two phases after equilibrium has been attained. The separation factor, a, is used as a measure of the chromatographic separation possible and is given by the equation, 

An important relationship exists between the weight distribution coefficient and the volume of eluant (Vmax) required to reach the maximum concentration of an eluted ion in the effluent. This is given by the equation  

4 METHOD DEVELOPMENT AND OPTIMISATION OE CONDITIONS IN ISOCRATIC HPLC 

Developing an HPLC method requires a clear specification of the goals of the separation. The primary objective could be (1) resolution, detection and characterisation or quantitation of one or a few substances in a product, so that it is important to separate only a few sample components and complete separation of the sample is not necessary (2) complete resolution, characterisation and quantitation of all sample components (3) isolation of purified sample components for spectral identification or for other assays. Further points that should be considered include the required sensitivity (especially for trace analysis), accuracy, precision, character of sample matrices (which determines sample dissolution, extraction or pretreatment necessary for possible concentration of sample analytes or for removing interference), expected frequency of analyses and the HPLC equipment available. 

The first step in method development is selecting an adequate HPLC mode for the particular sample. This choice depends on the character of the sample compounds, which can be either neutral (hydrophilic or lipophilic) or ionic, low-molecular (up to 2000 Da) or macromolecular (biopolymers or synthetic polymers). Many neutral compounds can be separated either by reversed-phase or by normal-phase chromatography, but a reversed-phase system without ionic additives to the aqueous-organic mobile phase is usually the best first choice. Strongly lipophilic samples often can be separated either by non-aqueous reversed-pha.se chromatography or by normal-phase chromatography. Positional isomers are usually better separated by normal-phase than by reversed-phase chromatography and the separation of optical isomers (enantiomers) requires either special chiral columns or addition of a chiral selector to the mobile phase. 

Macromolecules can be separated on column packings with large pores, either 

2 Effects of experimental HPLC conditions on chromatographic resolution 

In preparing an ion-exchange column, the resin should first be fully hydrated with deionized water in a beaker because if it is put on the column dry, the swelling pressure when water is added may burst the column. The 

The sample is applied to the top of the column in much the same way as in the previous section, except that the solvent is generally water or a buffered aqueous solution. Elution is brought about by passing more solvent through the column. 

Type and exchange group Bio-Rad Labs Dowex Duolite Amberhte (England) (U.S.A.) Nalcite  

Strongly acidic, phenohc type R-CH2S03 H+ Bio-Rex 40 C-3 Zeocarb 215  

Weakly acidic chelating resin, polystyrene type  

Column and Ion-exchange Chromatography.— The separation of carbohydrates using column chromatography has been reviewed.  

The mixture could be eluted by decreasing the pH of the eluent. However, it is usually easier to elute by increased salt concentration since this is simpler to control. 

If the ionic strength of the sample solution is much higher than that of the starting conditions the mixture will not bind. A simple dilution can save a lot of lost time and reduce frustration. 

When purifying oligonucleotides it is particularly useful to use sample self displacement chromatography since the required component of the mixture is generally the later eluting moiety. With this approach the column loading is increased to such a point that the more strongly retained component displaces the 

One of the drawbacks of ion exchange chromatography is the need for a secondary technique to remove inorganic salts from the purified product. Desalting can often be performed by ultrafiltration, solid phase extraction or by gel filtration. The latter mode of separation is described briefly in Section 3.5. 

Up to 30 oligomers of polyoxyethylene have been separated on a K form cation exchange resin [231]. 

The separation of TcO, formed by neutron bombardment of molybdenum, from its molybdenum matrix, was accomplished by means of the Ambcrlite anion exchange resin IRA-400. Irradiated molybdenum was dissolved in NH4OH + H2O2 and molybdate was eluted from the anion exchanger with a mixture of potassium oxalate and potassium hydroxide, while the elution of pertechnetate was achieved with 0..5 M NH4SCN. 80 to 93 % of the calculated Tc could be recovered from the irradiated molybdenum [167]. 

The cationic thiourea complex of technetium is adsorbed from nitric and perchloric acid on the cation-exchanger Dowex 50WX4. In dilute HNO3 and HCIO4 (pH 1) distribution coefficients of 10 and 10 , respectively, were observed. Technetium was eluted with 8 M HCl or a mixture of 5 M HNO3 and 10 % H2O2 [169]. 

The addition of a compatible organic solvent may also influence selectivity, particularly when the stationary phase has a polymeric substrate. With these types of phases, the solute can undergo both hydrophobic and ion-exchange interactions. The addition of an organic solvent will result in increased 

A large number of stationary phases are now pressure-resistant and capable of high performance in ion-exchange chromatography also, so the scope of HPLC can be extended into this field complex mixtures can be separated over a short period of time. 

The principle of ion-exchange chromatography is not unlike that of adsorption chromatography. In the latter case, the adsorbent bears active sites which interact with molecules in their vicinity to a more or less specifically defined extent. Sample and solvent molecules compete with each other for adsorption. 

A stationary phase capable of ion exchange, on the other hand, has electric charges on its surface. Ionic groups such as SOs , COO NH3 or NR3 are incorporated in the resin or gel. Charges are neutralized by mobile counter ions. The mobile phase contains ions, and ionic sample molecules compete with these for a place on the surface of the stationary phase. 

Practical High-Performance Liquid Chromatography, Fifth edition Veronika R Meyer 

Under these circumstances, how can competition between ions of the mobile phase and those of the sample, which is the essence of chromatographic separation, be stimulated The analyst must ensure optimum conditions by carefully selecting  

Practical High-Performance Liquid Chromatography, Fourth edition Veronika R. Meyer 2004 John Wiley Sons, Ltd ISBN 0-470-09377-3 (Hardback) 0-470-09378-1 (Paperback) 

It is not normally possible to capture antibodies direct from culture supernatant because the concentration of competing salt is too high. A preliminary buffer exchange step is necessary. A wide range of buffers might be suitable and buffer exchange is usually achieved by dialysis or diafiltration. Some antibodies precipitate under these conditions which can reduce the overall yield. Several options might be explored in such a case (1)  

For antibodies which are fully soluble in the binding buffer 

Dialyse or diafilter the culture supernatant against the binding buffer. Extensive changes are not necessary. 

Remove any visible precipitate by centrifugation (e.g. 3000 g fiar 30 min). Check that it does not contain antibody. 

Wash with 2 CV binding buffer, or until there is no protein detectable by the UV trace. 

The isoelectric points (pi) of most CGTases are about 9, and the pK of DEAE is 9.5, so the DEAE-cellulose column, using DEAE as the exchange agent, is mostly used for the separation of CGTase. This method is often used in the previous preparation process. 

Several of the 20 amino acids that constitute the building blocks of proteins exhibit charged side chains. At pH 7.0, aspartic and glutamic acids have overall negatively charged acidic 

The vast majority of purification procedures employ at least one ion-exchange step it represents the single most popular chromatographic technique in the context of protein purification. Its popularity is based upon the high level of resolution achievable, its straightforward scale-up (for industrial application), together with its ease of use and ease of column regeneration. In addition, 

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Stahlberg has presented models for ion-exchange chromatography combining the Gouy-Chapman theory for the electrical double layer (see Section V-2) with the Langmuir isotherm (. XI-4) [193] and with a specific adsorption model [194]. 

Schematics showing the basis of separation in (a) adsorption chromatography, (b) partition chromatography, (c) ion-exchange chromatography, (d) size-exciusion chromatography, and (e) eiectrophoresis. For the separations in (a), (b), and (d) the soiute represented by the soiid circie ( ) is the more strongiy retained.

A column used to minimize the conductivity of the mobile phase in ion-exchange chromatography. 

To minimize the mobile phase s contribution to conductivity, an ion-suppressor column is placed between the analytical column and the detector. This column selectively removes mobile-phase electrolyte ions without removing solute ions, for example, in cation ion-exchange chromatography using a dilute solution of HCl as... 

Ion-exchange chromatography in which conditions are adjusted so that an ion-suppressor column is not needed. 

Examples of the application of ion-exchange chromatography to the analysis of (a) inorganic anions, (b) inorganic cations, (c) antifreeze, and (d) vitamins. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Ohta and Tanaka reported a method for the simultaneous analysis of several inorganic anions and the cations Mg + and Ca + in water by ion-exchange chromatography. The mobile phase includes 1,2,4-benzenetricarboxylate, which absorbs strongly at 270 nm. Indirect detection of the analytes is possible because their presence in the detector leads to a decrease in absorbance. Unfortunately, Ca + and Mg +, which are present at high concentrations in many environmental waters, form stable complexes with 1,2,4-benzenetricarboxylate that interfere with the analysis. 

The resuspended and formulated Fraction II precipitate normally contains some aggregated IgG and trace substances that can cause hypotensive reactions in patients, such as the enzyme prekail ikrein activator (186). These features restrict this type of product to intramuscular adininistration. Further processing is required if products suitable for intravenous adininistration are required. Processes used for this purpose include treatment at pH 4 with the enzyme pepsin [9001-75-6] being added if necessary (131,184), or further purification by ion-exchange chromatography (44). These and other methods have been fiiUy reviewed (45,185,187,188). Intravenous immunoglobulin products are usually suppHed in the freeze-dried state but a product stable in the solution state is also available (189). 

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