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Ion Exchange Chromatography BCH 332 lecture 11 1
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Transcript of Ion Exchange Chromatography - KSUfac.ksu.edu.sa/sites/default/files/bch_332_lecture_11.pdf•Ion...

  • Ion Exchange Chromatography

    BCH 332 lecture 11

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  • • Ion exchange chromatography is a form of adsorption chromatography in which charged proteins or other biomolecules are exchanged for small ions of like charge originating in salts (e.g. Na+, Cl−).

    • These ions are attached to chemical structures on the surface of the stationary phase called ion exchange groups or ion exchangers.

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  • • Two types of ion exchanger are common in biochemistry;

    • Anion exchangers (which exchange negatively charged ions or anions) and

    • Cation exchangers (which exchange positively charged ions or cations).

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  • • Some ion exchangers are regarded as weak, that is functioning best over a comparatively narrow pH range, while others are strong, that is functioning over a wider pH range. Exchangers with quaternary amino or sulfonic acid groups behave as strong anion and cationexchangers, respectively, while aromatic/aliphatic amino and carboxylic acid groups are weak anion and cation exchangers, respectively.

    • Choice of ion exchange stationary phase is heavily influenced by knowledge of the pI of the protein of interest.

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  • • Desalting is carried out before ion exchange either by gel filtration chromatography , by dialysis or by centrifugal filtration.

    • Elution of bound proteins is achieved by reversing the process of binding and, again, exchanging a counterion for protein.

    • This is usually carried out by applying a large excess of a salt (e.g. NaCl) containing the counterion in the mobile phase.

    • Because proteins have different net charge, they may bind to an ion exchanger at a given pH with a variety of strengths, that is some proteins may bind strongly whilst others bind weakly or not at all.

    • We can take advantage of this to separate proteins by applying salt in a continuous gradient. Weakly bound proteins will elute first from such a system while strongly bound proteins elute later.

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  • • Mobile phase Acids, alkalis, buffer.

    • Stationary phase The ionic compound consisting of the cationic species (M+) and the anionic species (B-)

    • Elution - Components of mixture separate & move down the column at different rates depending upon the affinity of the ion for ion exchanger. The eluates are collected at different stages

    • Analysis of the eluate (Spectrophotometric)

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  • Selection of an Ion Exchanger

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  • Selection of an Ion Exchanger

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  • • Below the isoelectric pH the protein has a positive charge and binds to a cation exchanger.

    • Above the isoelectric pH the protein has a negative charge and binds to an anion exchanger.

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  • Choice of Buffer

    • Cationic buffer should be used with anion exchanger.

    • Anionic buffer should be used with cation exchanger.

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  • • The basic process of chromatography using ion exchange can be represented in 4 steps:

    1. Equilibration

    2. Sample application and wash

    3. Elution

    4. Regeneration

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  • APPLICATIONS

    • For extraction of enzymes from tissues.

    • Separation of sugars, amino acids and proteins.

    • Ion exchange column in HPLC.

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