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    Column Chromatography

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    brilly cahyo
    Ion exchange chromatography separates charged biological molecules like proteins based on their net charge. The charge depends on the protein's isoelectric point and the pH of the environment. Positively charged proteins bind to an anion exchange resin, while negatively charged proteins bind to a cation exchange resin. Proteins are then eluted by changing the pH or salt concentration to alter binding. Affinity chromatography also separates proteins based on specific binding interactions between a ligand and protein. Size exclusion chromatography separates proteins of different sizes, with smaller proteins entering the pores of beads in the column and taking longer to elute.

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    Column Chromatography

    Uploaded by

    brilly cahyo
    35 views11 pages
    Ion exchange chromatography separates charged biological molecules like proteins based on their net charge. The charge depends on the protein's isoelectric point and the pH of the environment. Positively charged proteins bind to an anion exchange resin, while negatively charged proteins bind to a cation exchange resin. Proteins are then eluted by changing the pH or salt concentration to alter binding. Affinity chromatography also separates proteins based on specific binding interactions between a ligand and protein. Size exclusion chromatography separates proteins of different sizes, with smaller proteins entering the pores of beads in the column and taking longer to elute.

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    Ion exchange chromatography separates charged biological molecules like proteins based on their net charge. The charge depends on the protein's isoelectric point and the pH of the environment. Positively charged proteins bind to an anion exchange resin, while negatively charged proteins bind to a cation exchange resin. Proteins are then eluted by changing the pH or salt concentration to alter binding. Affinity chromatography also separates proteins based on specific binding interactions between a ligand and protein. Size exclusion chromatography separates proteins of different sizes, with smaller proteins entering the pores of beads in the column and taking longer to elute.

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    35 views11 pages

    Column Chromatography

    Uploaded by

    brilly cahyo
    Ion exchange chromatography separates charged biological molecules like proteins based on their net charge. The charge depends on the protein's isoelectric point and the pH of the environment. Positively charged proteins bind to an anion exchange resin, while negatively charged proteins bind to a cation exchange resin. Proteins are then eluted by changing the pH or salt concentration to alter binding. Affinity chromatography also separates proteins based on specific binding interactions between a ligand and protein. Size exclusion chromatography separates proteins of different sizes, with smaller proteins entering the pores of beads in the column and taking longer to elute.

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    Column Chromatography

    Ion Exchange Chromatography

    • Ion exchange chromatography is commonly used to separate charged


    biological molecules such as proteins, peptides, amino acids, or nucleotides.

    • The amino acids that make up proteins are zwitterionic compounds that contain
    both positively and negatively charged chemical groups.

    • Depending on the pH of their environment, proteins may carry a net positive


    charge, a net negative charge, or no charge. The pH at which a molecule has
    no net charge is called its isoelectric point, or pI.
    • In a buffer with a pH GREATER than the
    pI of the protein of interest, the protein
    will carry a net negative charge;
    therefore, a positively charged ANION
    EXCHANGE resin is chosen to capture
    this protein.
    • In a buffer with a pH LOWER than the pI of
    the protein of interest, the protein will carry a
    positive net charge; thus a negatively-
    charged CATION EXCHANGE resin is chosen.
    Ion exchange chromatography

    • After loading an impure protein sample


    onto an ion exchange chromatography
    column, the column is washed to
    remove undesired proteins and other
    impurities, and then the protein(s) of
    interest is eluted using either a salt
    gradient or a change in pH.

    • To elute proteins from an anion exchange


    resin, a decreasing pH gradient is chosen,
    while an increasing pH gradient is chosen
    for elution from cation exchangers.
    Affinity Chromatography

    • Affinity chromatography is a separation method based on a specific


    binding interaction between an immobilized ligand and its binding partner.

    • Affinity chromatography offers high selectivity, resolution, and capacity in most protein
    purification schemes. It has the advantage of utilizing a protein's biological structure or
    function for purification. As a result, purifications that would otherwise be time consuming
    and complicated, can often be easily achieved with affinity chromatography.

    • A commonly used metaphor to illustrate affinity binding is the lock and key analogy. A
    unique structure present on the surface of a protein is the key that will only bind to the
    corresponding lock, a specific ligand on a chromatographic support.
    ELUTION

    • pH elution
    A change oh pH alters the degree of ionization of charged groups on the ligand and/or bound
    protein. This change may affect the binding sites directly, reducing their affinity , or cause indirect
    changes in affinity by alterations in conformation
    • Chaotropic eluents
    Chaotropic agents such as guanidine hydrochloride or urea can be used to alter the structure of
    proteins, but these eluents may cause permanent or temporary damage to the ligand.

    • Competitive elution
    Selective eluents are used to separate substances on group specific or when the binding affinity of
    the ligand/target protein interaction is relative high. The eluting agents competes either for binding
    to the target of protein or for binding to the ligand. The concentration of competing compound
    should be similar to the concentration of coupled ligand.
    Size exclusion chromatography

    • There are great diversity of proteins in the body and they all vary in size. One common
    purification method that separate proteins based on their size is gel filtration or size exclusion
    chromatography.

    • The setup consists of long column that contains special gel beads. These beads are insoluble
    but porous. They typically consist of a hydrabed polymer such as dextran.

    • SEC only works if there is relatively large differences size protein in the mixture that we want to
    separate it.
    Size exclusion chromatography

    A porous column acts


    as a “molecular
    sieve”, in which
    smaller molecules get
    stuck in pores and
    take longer to travel
    through the column ,
    but the larger
    molecules pass quickly
    to the bottom.
    Size exclusion chromatography

    • The small proteins enter


    the porous beads while
    the larger proteins
    cannot fit into the
    internal volume of the
    beads

    • therefore the larger


    protein reach the
    bottom first while the
    others emerge last.

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