The specification consists of a series of tests, one or more analytical procedure for each test, and the acceptance criteria for each procedure. General chapters provide procedures, sometimes with acceptance criteria and other requirements, in order to compile repetitive information in one location, which would otherwise appear in many monographs. In most of the other countries, pharmacopeia is part of the government. However, in the United States, the pharmacopeia USP is a non-government not-for-profit organization that supports itself from the sale of books USP-NF and other publications and reference standards.
It does not receive any financial support from federal, state or local governments or non-government organizations. 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.
Separation based on ion exchange has been used for a long time and is the predominant form of IC to-date.
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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.
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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. The choice depends upon the nature of the analyte molecules. However, traditionally, IC is associated with electrochemical detectors. Two types of electrochemical detectors are widely used in IC—conductivity suppressed and nonsuppressed and pulsed amperometry. When a constant voltage is applied across two electrodes between which the effluent from a column flows, a current is generated because the effluent contains ions or polar molecules.
The problem, however, is that the conductivities of mobile phases are significantly higher than the conductivities of the analytes, simply because the concentrations of ions in the former solutions are 10 4 5 higher than that of the analytes.
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The suppressed conductivity detection permits detection and quantitation of analytes at near zero background baseline conductivity of the mobile phases. Used typically in combination with high-performance anion-exchange chromatography HPAEC, originally introduced as high-pH anion-exchange chromatography , Pulsed Amperometry Detection PAD has proved to be a powerful tool in the detection of mono- and oligosaccharides, sugar alcohols, aminoglycosides, amino acids and other molecules that do not have a suitable chromophore, without requiring any sample derivatization.
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A detailed discussion on the mechanism of action of the suppressed conductivity and the pulsed amperometry detectors is beyond the scope of this article. The interested analysts are encouraged to read books and other publications that provide details of the principles of actions and suitability of selection for use in different types of IC-based chromatographic procedures [].
However, the number of monographs that include one or more IC-based test procedures increased dramatically in last 10 years. These principles are the reasons that ion exchange chromatography is an excellent candidate for initial chromatography steps in a complex purification procedure as it can quickly yield small volumes of target molecules regardless of a greater starting volume.
Comparatively simple devices are often used to apply counterions of increasing gradient to a chromatography column. Counterions such as copper II are chosen most often for effectively separating peptides and amino acids through complex formation. A simple device can be used to create a salt gradient. Elution buffer is consistently being drawn from the chamber into the mixing chamber, thereby altering its buffer concentration.
Generally, the buffer placed into the chamber is usually of high initial concentration, whereas the buffer placed into the stirred chamber is usually of low concentration. As the high concentration buffer from the left chamber is mixed and drawn into the column, the buffer concentration of the stirred column gradually increase.
Altering the shapes of the stirred chamber, as well as of the limit buffer, allows for the production of concave, linear, or convex gradients of counterion. A multitude of different mediums are used for the stationary phase. Successful packing of the column is an important aspect of ion chromatography. Stability and efficiency of a final column depends on packing methods, solvent used, and factors that affect mechanical properties of the column.
In contrast to early inefficient dry- packing methods, wet slurry packing, in which particles that are suspended in an appropriate solvent are delivered into a column under pressure, shows significant improvement. Three different approaches can be employed in performing wet slurry packing: 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.
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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. 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. 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. 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. 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.
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. 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. Using the information from results, correlation could be made about the diagnosis of patients and the kind of CK isoenzymes found in most abundant activity. From the finding, about 35 out of 71 patients studied suffered from heart attack myocardial infarction also contained an abundant amount of the CK-MM and CK-MB isoenzymes.
Findings further show that many other diagnosis including renal failure, cerebrovascular disease, and pulmonary disease were only found to have the CK-MM isoenzyme and no other isoenzyme. The results from this study indicate correlations between various diseases and the CK isoenzymes found which confirms previous test results using various techniques.
Studies about CK-MB found in heart attack victims have expanded since this study and application of ion chromatography. Since ion chromatography has been widely used in many branches of industry. The main beneficial advantages are reliability, very good accuracy and precision, high selectivity, high speed, high separation efficiency, and low cost of consumables.
The most significant development related to ion chromatography are new sample preparation methods; improving the speed and selectivity of analytes separation; lowering of limits of detection and limits of quantification; extending the scope of applications; development of new standard methods; miniaturization and extending the scope of the analysis of a new group of substances. Allows for quantitative testing of electrolyte and proprietary additives of electroplating baths.
Ions, catalysts, brighteners and accelerators can be measured.
Applications of Ion Chromatography for Pharmaceutical and Biological Products - Google Книги
Applications for such purposes have been developed, or are under development, for a variety of fields of interest, and in particular, the pharmaceutical industry. The usage of ion exchange chromatography in pharmaceuticals has increased in recent years, and in , a chapter on ion exchange chromatography was officially added to the United States Pharmacopia -National Formulary USP-NF.
Majority of these applications are primarily used for measuring and analyzing residual limits in pharmaceuticals, including detecting the limits of oxalate, iodide, sulfate, sulfamate, phosphate, as well as various electrolytes including potassium, and sodium. In total, the edition of the USP-NF officially released twenty eight methods of detection for the analysis of active compounds, or components of active compounds, using either conductivity detection or pulse amperometric detection.
There has been a growing interest in the application of IC in the analysis of pharmaceutical drugs. IC is used in different aspects of product development and quality control testing. For example, IC is used to improve stabilities and solubility properties of pharmaceutical active drugs molecules as well as used to detect systems that have higher tolerance for organic solvents.
IC has been used for the determination of analytes as a part of a dissolution test. For instance, calcium dissolution tests have shown that other ions present in the medium can be well resolved among themselves and also from the calcium ion. Therefore, IC has been employed in drugs in the form of tablets and capsules in order to determine the amount of drug dissolve with time. Detection of sugar and sugar alcohol in such formulations through IC has been done due to these polar groups getting resolved in ion column.
IC methodology also established in analysis of impurities in drug substances and products. Impurities or any components that are not part of the drug chemical entity are evaluated and they give insights about the maximum and minimum amounts of drug that should be administered in a patient per day. From Wikipedia, the free encyclopedia. Ballou, and Marilee Benore