Applications of Ion Chromatography in the Analysis of Pharmaceutical and Biological Products

Ion Chromatography in USP-NF
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Polystyrene is used as a medium for ion- exchange. It is made from the polymerization of styrene with the use of divinylbenzene and benzoyl peroxide. Such exchangers form hydrophobic interactions with proteins which can be irreversible. Due to this property, polystyrene ion exchangers are not suitable for protein separation. They are used on the other hand for the separation of small molecules in amino acid separation and removal of salt from water.

Polystyrene ion exchangers with large pores can be used for the separation of protein but must be coated with a hydrophillic substance. Cellulose based medium can be used for the separation of large molecules as they contain large pores. Protein binding in this medium is high and has low hydrophobic character. DEAE is an anion exchange matrix that is produced from a positive side group of diethylaminoethyl bound to cellulose or Sephadex. Agarose gel based medium contain large pores as well but their substitution ability is lower in comparison to dextrans. The ability of the medium to swell in liquid is based on the cross-linking of these substances, the pH and the ion concentrations of the buffers used.

Incorporation of high temperature and pressure allows a significant increase in the efficiency of ion chromatography, along with a decrease in time. Temperature has an influence of selectivity due to its effects on retention properties. Despite ion selectivity in different mediums, further research is being done to perform ion exchange chromatography through the range of 40— o C.

An appropriate solvent can be chosen based on observations of how column particles behave in a solvent. Using an optical microscope, one can easily distinguish a desirable dispersed state of slurry from aggregated particles.

Ion chromatography - Wikipedia

A "strong" ion exchanger will not lose the charge on its matrix once the column is equilibrated and so a wide range of pH buffers can be used. If the pH of the buffer used for a weak ion exchange column goes out of the capacity range of the matrix, the column will lose its charge distribution and the molecule of interest may be lost. In some experiments, the retention times of weak ion exchangers are just long enough to obtain desired data at a high specificity.

There are also special columns that have resins with amphoteric functional groups that can exchange both cations and anions. These two types of exchangers can maintain the charge density of their columns over a pH range of 5—9. In ion chromatography, the interaction of the solute ions and the stationary phase based on their charges determines which ions will bind and to what degree. When the stationary phase features positive groups which attracts anions, it is called an anion exchanger; when there are negative groups on the stationary phase, cations are attracted and it is a cation exchanger.

Each resin features relative selectivity which varies based on the solute ions present who will compete to bind to the resin group on the stationary phase. The selectivity coefficient, the equivalent to the equilibrium constant, is determined via a ratio of the concentrations between the resin and each ion, however, the general trend is that ion exchangers prefer binding to the ion with a higher charge, smaller hydrated radius, and higher polarizability, or the ability for the electron cloud of an ion to be disrupted by other charges.

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A sample is introduced, either manually or with an autosampler , into a sample loop of known volume. A buffered aqueous solution known as the mobile phase carries the sample from the loop onto a column that contains some form of stationary phase material. This is typically a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. Equilibration of the stationary phase is needed in order to obtain the desired charge of the column. If the column is not properly equilibrated the desired molecule may not bind strongly to the column. The target analytes anions or cations are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that displaces the analyte ions from the stationary phase.

For example, in cation exchange chromatography, the positively charged analyte can be displaced by adding positively charged sodium ions. A type of ion exchange chromatography, membrane exchange [33] [34] is a relatively new method of purification designed to overcome limitations of using columns packed with beads. Membrane Chromatographic [35] [36] devices are cheap to mass-produce and disposable unlike other chromatography devices that require maintenance and time to revalidate.

There are three types of membrane absorbers that are typically used when separating substances. The three types are flat sheet, hollow fibre, and radial flow.

The most common absorber and best suited for membrane chromatography is multiple flat sheets because it has more absorbent volume. It can be used to overcome mass transfer limitations [37] and pressure drop, [38] making it especially advantageous for isolating and purifying viruses, plasmid DNA, and other large macromolecules.

The column is packed with microporous membranes with internal pores which contain adsorptive moieties that can bind the target protein. Adsorptive membranes are available in a variety of geometries and chemistry which allows them to be used for purification and also fractionation, concentration, and clarification in an efficiency that is 10 fold that of using beads. A more recent method involved the use of live cells that are attached to a support membrane and are used for identification and clarification of signaling molecules.

Ion exchange chromatography can be used to separate proteins because they contain charged functional groups.

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The solutes are most commonly in a liquid phase, which tends to be water. Take for example proteins in water, which would be a liquid phase that is passed through a column. The column is commonly known as the solid phase since it is filled with porous synthetic particles that are of a particular charge. These porous particles are also referred to as beads, may be aminated containing amino groups or have metal ions in order to have a charge. This is because slow diffusion of the solutes within the pores does not restrict the separation quality.

The amino acids that have negatively charged side chains at pH 7 pH of water are glutamate and aspartate. The beads that are negatively charged are called cation exchange resins, as positively charged proteins will be attracted.

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The amino acids that have positively charged side chains at pH 7 are lysine, histidine and asparagine. The isoelectric point is the pH at which a compound - in this case a protein - has no net charge. Using buffers instead of water for proteins that do not have a charge at pH 7, is a good idea as it enables the manipulation of pH to alter ionic interactions between the proteins and the beads.

Separation can be achieved based on the natural isoelectric point of the protein. Alternatively a peptide tag can be genetically added to the protein to give the protein an isoelectric point away from most natural proteins e. Elution by increasing ionic strength of the mobile phase is more subtle. It works because ions from the mobile phase interact with the immobilized ions on the stationary phase, thus "shielding" the stationary phase from the protein, and letting the protein elute.

Elution from ion-exchange columns can be sensitive to changes of a single charge- chromatofocusing. Ion-exchange chromatography is also useful in the isolation of specific multimeric protein assemblies, allowing purification of specific complexes according to both the number and the position of charged peptide tags. In ion exchange chromatography, the Gibbs—Donnan effect is observed when the pH of the applied buffer and the ion exchanger differ, even up to one pH unit. For example, in anion-exchange columns, the ion exchangers repeal protons so the pH of the buffer near the column differs is higher than the rest of the solvent.

This effect comes as a result of two similarly charged particles, one from the resin and one from the solution, failing to distribute properly between the two sides; there is a selective uptake of one ion over another. However, since the concentration of the sulphonic acid in the resin is high, the hydrogen of HCl has no tendency to enter the column.

This, combined with the need of electroneutrality, leads to a minimum amount of hydrogen and chlorine entering the resin. A use of ion chromatography can be seen in the argentation ion chromatography. This phenomenon has been widely tested on olefin compounds.

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The ion complexes the olefins make with silver ions are weak and made based on the overlapping of pi, sigma, and d orbitals and available electrons therefore cause no real changes in the double bond. This behavior was manipulated to separate lipids, mainly fatty acids from mixtures in to fractions with differing number of double bonds using silver ions. The ion resins were impregnated with silver ions, which were then exposed to various acids silicic acid to elute fatty acids of different characteristics.

Ion Exchange Resins IER have been widely used especially in medicines due to its high capacity and the uncomplicated system of the separation process.

Applications of Ion Chromatography for Pharmaceutical and Biological Products

One of the synthetic uses is to use Ion Exchange Resins for kidney dialysis. This method is used to separate the blood elements by using the cellulose membraned artificial kidney.


Another clinical application of ion chromatography is in the rapid anion exchange chromatography technique used to separate creatine kinase CK isoenzymes from human serum and tissue sourced in autopsy material mostly CK rich tissues were used such as cardiac muscle and brain. Mini columns were filled with DEAE-Sephadex A and further eluted with tris- buffer sodium chloride at various concentrations each concentration was chosen advantageously to manipulate elution. Human tissue extract was inserted in columns for separation.

All fractions were analyzed to see total CK activity and it was found that each source of CK isoenzymes had characteristic isoenzymes found within. Therefore, the isoenzymes found in each sample could be used to identify the source, as they were tissue specific. This is a comprehensive source of information on the application of ion chromatography IC in the analysis of pharmaceutical drugs and biologicals. John Wiley and Sons Ltd.

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This book, with contributors from academia, pharma, the biotech industry, and instrument manufacturing, presents the different perspectives, experience, and expertise of the thought leaders of IC in a comprehensive manner. USP-NF standards are recognized not only in the Unites States but also in many other countries because they are authoritative, science-based, and are established through a transparent and credible process with established integrity.

The transparency and credibility of the monographs come through the open review and comment process that takes place when proposed monographs are published in the Pharmacopeial Forum PF. Although the Council of Experts of USP is the ultimate decision making body for the USP-NF standards, these standards are developed through public involvement and substantial interaction between USP and the stakeholders through consensus building. A stakeholder may be an institution or individual, domestic or international. Interested and knowledgeable stakeholders can provide scientific and regulatory comments to monographs published in PF.

The public participation in the monograph development and revision process results in consensus among many individuals and groups, including scientific and trade organizations. The members of the Expert Committees are unpaid volunteers who are experts in their respective fields and who participate in the USP process as individual scientists and not as representatives of their employers or any trade association, thereby eliminating the conflict of interest and providing unbiased authoritative and science-based standards.

A manufacturer sponsor submits a proposal for a new monograph or a revision to an existing monograph to USP Request for Revision. The information and data that need to be provided with a Request for Revision is available at the USP website www. USP staff review the proposals for the appropriateness and completeness, and, when satisfied, publish the proposal to PF as In-process Revision for public comment. After the public comment period, members of an appropriate Expert Committee together called Council of Experts reviews the proposal and the comments and approves or disapproves the request for official adoption.

However, it should be noted that the manufacturers are not required at any stage to submit proposals for a new monograph or a revision to an existing monograph. Such submission is discretionary to the manufacturers and they do so voluntarily to support the mission of the development of consensus standards for therapeutic products. The IC involves separation based on ionic interactions between ionic or polar analytes, ions present in the eluent, and ionic functional groups derivatized to the chromatographic support.

This can lead to two distinct mechanisms of separation— a ion exchange due to competitive ionic binding attraction to the chromatographic support column resin , and b ion exclusion due to repulsion between similarly charged analyte ions and the ions derivatized on the chromatographic support.

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Separation based on ion exchange has been used for a long time and is the predominant form of IC to-date. However, increasing applications of ion exclusion chromatography have been reported more recently. In addition, chromatographic methods in which the separation due to ion exchange or ion exclusion is modified by the hydrophobicity of the analytes and the chromatographic support materials, presence of the organic modifiers in the eluent or due to ion-pair agents, resulting in better resolution of analytes or separation that were not achieved before, have gained popularity recently due to increased applications of mixed mode columns.

Ion-exchange chromatography involves separation of ionic and polar analytes on a chromatographic support, which is derivatized with ionic functional groups that have charges opposite that of the analyte ions. Thus, a column used to separate cations, called a cation-exchange column, contains negative ions. Similarly, an anion-exchange column, which separates anions, is derivatized with positively charged ions. The separation is effected by repeated binding of the analyte ion to the ionic sites on the chromatographic support and desorption by the ions present in the mobile phase.

The ion-exchange method of separation is widely used in the analysis of anions and cations, including metal ions, mono- and oligosaccharides, sugar alcohols and other polyhydroxy compounds, aminoglycosides antibiotics , amino acids and peptides, organic acids, amines, alcohols, phenols, thiols, nucleotides and nucleosides, and other polar molecules. Ion-exclusion Chromatography uses strong cation- or anion-exchange chromatographic supports to separate ionic analytes from polar, weakly polar and neutral analytes, and has been used typically in the analysis of organic acids, alcohols, glycols, sugars, and other weakly polar compounds.

In contrast to the ion-exchange chromatography, the charge on the functional groups on the chromatographic support is same as the charge on the analyte ion. That is, to separate negatively charged or negatively polarized analytes, the chromatographic supports are derivatized with negatively charged functional groups. Similarly, analytes with positive charge or polarity are separated using a chromatographic support that carries positive charges.

Any suitable detector can be used for the detection and quantitation of analytes in IC.