The chromatography which allows the separation of ions and polar molecules based on their affinity to the ion-exchanger is called Ion-exchange chromatography. It can be used for almost any kind of charged molecule including large proteins, small nucleotides, and amino acids. The solution to be injected is usually called a sample, and the individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.
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 side groups, whereas lysine, arginine and histidine have positively charged basic side groups. Protein molecules, therefore, possess both positive and negative charges, largely due to the presence of varying amounts of these seven amino acids. (N-terminal amino groups and the C-terminal carboxy groups also contribute to overall protein charge characteristics.) The net charge exhibited by any protein depends on the relative quantities of these amino acids present in the protein, and on the pH of the protein solution. The pH value at which a protein molecule possesses zero overall charge is termed its isoelectric point (pI). At pH values above its pI, a protein will exhibit a net negative charge, whereas proteins will exhibit a net positive charge at pH values below the pI.
Ion-exchange chromatography is based upon the principle of reversible electrostatic attraction of a charged molecule to a solid matrix that contains covalently attached side groups of opposite charge. Proteins may subsequently be eluted by altering the pH or by increasing the salt concentration of the irrigating buffer. Ion-exchange matrices that contain covalently attached positive groups are termed anion exchangers. These will adsorb anionic proteins, e.g. proteins with a net negative charge. Matrices to which negatively charged groups are covalently attached are termed cation exchangers, adsorbing cationic proteins, e.g. positively charged proteins. Positively charged functional groups (anion exchangers) include species such as aminoethyl and diethylaminoethyl groups. Negatively charged groups attached to suitable matrices forming cation exchangers include sulfo- and carboxy-methyl groups.
During the cation-exchange process, positively charged proteins bind to the negatively charged ion-exchange matrix by displacing the counter ion (often H ), which is initially bound to the resin by electrostatic attraction. Elution may be achieved using a salt-containing irrigation buffer. The salt cation, often Na of NaCl, in turn displaces the protein from the ion-exchange matrix. In the case of negatively charged proteins, an anion exchanger is obviously employed, with the protein adsorbing to the column by replacing a negatively charged counter ion.
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 purifi cation. 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, it leads to a concentration of the protein of interest. It is also one of the least expensive chromatographic methods available. At physiological pH values most proteins exhibit a net negative charge. Anion-exchange chromatography, therefore, is most commonly used.
Typical Ion-exchange Instrumentation
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 side groups, whereas lysine, arginine and histidine have positively charged basic side groups. Protein molecules, therefore, possess both positive and negative charges, largely due to the presence of varying amounts of these seven amino acids. (N-terminal amino groups and the C-terminal carboxy groups also contribute to overall protein charge characteristics.) The net charge exhibited by any protein depends on the relative quantities of these amino acids present in the protein, and on the pH of the protein solution. The pH value at which a protein molecule possesses zero overall charge is termed its isoelectric point (pI). At pH values above its pI, a protein will exhibit a net negative charge, whereas proteins will exhibit a net positive charge at pH values below the pI.
Design of an Ion-exchanger
Ion-exchange chromatography is based upon the principle of reversible electrostatic attraction of a charged molecule to a solid matrix that contains covalently attached side groups of opposite charge. Proteins may subsequently be eluted by altering the pH or by increasing the salt concentration of the irrigating buffer. Ion-exchange matrices that contain covalently attached positive groups are termed anion exchangers. These will adsorb anionic proteins, e.g. proteins with a net negative charge. Matrices to which negatively charged groups are covalently attached are termed cation exchangers, adsorbing cationic proteins, e.g. positively charged proteins. Positively charged functional groups (anion exchangers) include species such as aminoethyl and diethylaminoethyl groups. Negatively charged groups attached to suitable matrices forming cation exchangers include sulfo- and carboxy-methyl groups.
An Ion-exchange coloumn used to purify proteins
During the cation-exchange process, positively charged proteins bind to the negatively charged ion-exchange matrix by displacing the counter ion (often H ), which is initially bound to the resin by electrostatic attraction. Elution may be achieved using a salt-containing irrigation buffer. The salt cation, often Na of NaCl, in turn displaces the protein from the ion-exchange matrix. In the case of negatively charged proteins, an anion exchanger is obviously employed, with the protein adsorbing to the column by replacing a negatively charged counter ion.
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 purifi cation. 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, it leads to a concentration of the protein of interest. It is also one of the least expensive chromatographic methods available. At physiological pH values most proteins exhibit a net negative charge. Anion-exchange chromatography, therefore, is most commonly used.
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