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    Column Chromatography

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    Nofrizal
    The document discusses the principles of chromatographic separation. Chromatography separates compounds based on how they distribute between a mobile phase and stationary phase. Separation is determined by each compound's distribution coefficient (Kd) which represents the concentration of the compound in the stationary phase versus the mobile phase. There are different types of chromatography depending on the physical state of the mobile phase and analytes, including gas chromatography and liquid chromatography. Liquid chromatography can operate via various equilibriums like adsorption, partition, ion exchange, or affinity.

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    Column Chromatography

    Uploaded by

    Nofrizal
    7 views18 pages
    The document discusses the principles of chromatographic separation. Chromatography separates compounds based on how they distribute between a mobile phase and stationary phase. Separation is determined by each compound's distribution coefficient (Kd) which represents the concentration of the compound in the stationary phase versus the mobile phase. There are different types of chromatography depending on the physical state of the mobile phase and analytes, including gas chromatography and liquid chromatography. Liquid chromatography can operate via various equilibriums like adsorption, partition, ion exchange, or affinity.

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    The document discusses the principles of chromatographic separation. Chromatography separates compounds based on how they distribute between a mobile phase and stationary phase. Separation is determined by each compound's distribution coefficient (Kd) which represents the concentration of the compound in the stationary phase versus the mobile phase. There are different types of chromatography depending on the physical state of the mobile phase and analytes, including gas chromatography and liquid chromatography. Liquid chromatography can operate via various equilibriums like adsorption, partition, ion exchange, or affinity.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    7 views18 pages

    Column Chromatography

    Uploaded by

    Nofrizal
    The document discusses the principles of chromatographic separation. Chromatography separates compounds based on how they distribute between a mobile phase and stationary phase. Separation is determined by each compound's distribution coefficient (Kd) which represents the concentration of the compound in the stationary phase versus the mobile phase. There are different types of chromatography depending on the physical state of the mobile phase and analytes, including gas chromatography and liquid chromatography. Liquid chromatography can operate via various equilibriums like adsorption, partition, ion exchange, or affinity.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 18

    Chromatography

    Chromatographic separation is based on


    distribution of separated compound
    (analyte) between mobile phase and
    stationary phase

    Richard Vytášek 2009 Presentation is only for internal purposes of 2nd Medical
    The principle of separatation
    Concentration of separated compound in these
    phases (stationary and mobile) is
    determined by distribution coeficient Kd
    Kd = c s / c m
    Compounds are sepatared when their
    distribution koeficients in choosen mobile
    and stationary phase are different
    Chromatography and mobile phase

    • gas chromatography (mobile phase and


    analytes are gaseous)
    • liquid chromatography (mobile phase is
    liquid and analytes are disolved in it)
    Liquid chromatography
    1. an adsorption equilibrium (between
    statinary solid phase and mobile liquid
    phase) - adsorption chromatography,
    hydrophobic chromatography
    2. a partition equilibrium (between
    stationary liquid phase and mobile liquid
    phase) - partition chromatography,
    reversed-phase liquid chromatography
    Liquid chromatography
    3. an equilibrium between liquid phase trapped
    inside pores of stationary porous material and
    mobile liquide phase - permeation
    chromatography or molecular exclusion
    chromatography
    Liquid chromatography
    4. an ion-exchange equilibrium (between
    stationary ion exchanger and mobile
    electrolyte phase) - ion-exchange
    chromatography
    5. an affinity equilibrium (between
    stationary immobilised ligand and mobile
    liquid phase) - affinity chromatography
    ( e.g. immunoaffinity chromatography,
    lectin affinity chromatography, dye-ligand
    chromatography)
    Affinity chromatography
    Models of liquid chromatography
    • column chromatography - stationary phase attached
    to suitable matrix (insoluble support) is packed in
    glass or metal column and mobile phase is passed
    through column by gravity or pump
    • planar chromatography - suitable matrix is coated
    in thin layer onto a glass, plastic or metal plate
    (special case is a filter paper) and mobile phase is
    passes across the thin layer by capillary action -
    thin-layer chromatography, paper chromatography
    Column
    chromatography
    Planar chromatography
    Development modes of chromatography
    Sample is dissolved in a suitable solvent and applied to
    stationary phase as a narrow dicrete band. Sample
    component is called analytes.
    • elution (zonal) development - mobile phase called
    elluent flows continously over the stationary phase and
    the analytes with higher solubility in the mobile phase
    move along the stationary phase more rapidly.
    Analytes are eluted when they have been removed
    from column.
    • displacement (affinity) development - mobile phase
    contains specific solutes with higher affinity for
    stationary phase than separated analytes
    Development modes of chromatography
    Theory of chromatographic separation
    tR retention time

    hP height of peak

     - standard deviation
    of
    Gaussian peak
    Void volume V0 -
    volume of mobile phase
    in column (analyte that
    does not interact with
    stationary phase is
    eluted in this volume)

    Dead time tM - the time taken to pass


    throuhg void volume of column
    Theory of chromatographic separation -
    capacity and separation factor
    Capacity factor k´
    factor expressing proportion of mass of the
    analyte in the stationary and mobile phase k
    ´ = MS/MM

    Selectivity (separation factor) 


    ratio of capacity factors of two analytes
    Theoretical plate
    zone with sufficient space for for complete
    equilibration of analyte between the two
    phases. The length of column containing
    one theoretical plate is refered as the plate
    height (height of theoretical plate) H
    H = 2/x
    Number of theoretical plates in whole column
    is given by
    N = L/H
    Theoretical plate
    When peak of analyte is
    emerging the column then
    x = L. For symetrical
    gaussian peaks is standard
    deviation  equal to half
    of width of peak in point
    of inflexion (wi) or one
    quarter of width of peak at
    its base w :
    N = 4 (tr/w)2
    Resolution RS
    is ratio of the difference in retention times between two peaks to
    mean of their base width

    Influence of number of theoretical plates N, selectivity factor 


    and capacity factors k´ on resolution of two compounds

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