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Transcript of Chromatography - 2018-10-12¢  separation methods include paper chromatography (PC), thin...

  • Part 3 Chromatography


    In 1903 M.S. Tswett described a technique for the separation of plant

    pigments. He called this technique “chromatography" (derived from

    the Greek word which means colour writing). Today chromatography

    encompasses a diverse group of methods, which permit the separation,

    isolation, and identification of the components in a mixture. These

    separation methods include paper chromatography (PC), thin layer

    chromatography (TLC), gas chromatography (GC) and liquid

    chromatography (LC).

    Basically, chromatography is a physical separation technique, which

    resolves the individual components of a mixture based on their

    distribution between two immiscible phases:

    a- Stationary phase (adsorbent). It is a solid porous media, which

    consists of the rigid porous particles, usually silica based, with the

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    specific surface properties (surface chemistry). It is non-moving and

    may exist in a variety of forms (e.g., bed, layer, column).

    B- Mobile phase (eluent): It is a liquid solvent or mixture of solvents, which

    is moving through the stationary phase or chromatographic column

    and carrying analytes. It may be either a gas (i.e., GC) or a liquid (i.e.


    The chromatographic process occurs as the result of the repeated

    sorption-desorption of the sample components as they move along the

    stationary phase. When the individual component favors the stationary

    phase, it is held longer and moves more slowly through the column. If

    the velocities and hence the distribution coefficients of samples

    components are different, the mixture could be resolved. The

    distribution coefficient (Ka) is defined as:

    𝐾𝑎 = Concentration of solute in stationary phase

    Concentration of solute in mobile phase

    The oldest form of liquid chromatography is the column

    chromatography. In classical column chromatography the columns

    were open tubes (e.g., burettes), which were individually packed with

    coarse material. The mixture to be separated is loaded onto the top of

    the column followed by more solvent. Different components in the

    sample mixture pass through the column at different rates due to

    differences in their partitioning behavior between the mobile phase

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    and the stationary phase. Compounds are separated by collecting

    aliquots of the column effluent as a function of time. Flow of the mobile

    phase was achieved by gravity feeding; and components were

    collected as colored fractions (Fig.1).

    In the 1960's researchers began to look for ways to improve liquid

    chromatography and developed high performance liquid

    chromatography (HPLC). HPLC: means high performance liquid

    chromatography, which refers to high speed, high-resolution

    separation. Initially, pressure was selected as the principal criterion of

    modern liquid chromatography and thus the name was "high pressure

    liquid chromatography" or HPLC. This was, however, an unfortunate

    term because it seems to indicate that the improved performance is

    primarily due to the high pressure. This is not true because naturally,

    pressure is needed only to permit a given flow rate of the mobile phase;

    otherwise, pressure is a negative factor not contributing to the

    improvement in separation.

    Figure 1 Schematic of

    a simple liquid



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    Comparison between classical and modern LC:

    Modern LC Classical LC

    1- Column:

    - Stainless steel columns

    - Small diameter (2-5 mm). Reusable.

    - Packing with very small (3, 5 and 10

    mm) particle stationary phase.

    Open tube ex: a burette

    -Large diameter (1-4


    - Prepared each time.

    -Packing with

    coarse particles.

    Stationary phase:

    - Continuous development of new

    substances to be used stationary


    - Only few materials are

    used as stationary phase

    ex: silica and alumina.

    2- Volume of sample:

    - Precise sample introduction (in ul)

    using micro-syringe

    -Large amounts of

    sample are loaded on

    top of column.

    4- Flow of mobile phase:

    - Relatively high inlet pressures and

    controlled flow of mobile phase.

    -Flow of mobile phase is

    achieved by specific


    5- Detection of sample:

    - Special continuous flow detectors

    capable of handling small flow rates

    and detecting very small amounts of

    samples. Then a final chromatogram is

    obtained with all the information data.

    -Coloured samples are

    washed out and

    collected in fraction,

    then analyzed


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    6- Resolution of sample:

    - High resolution.

    - Resolution is not good.

    7- Analysis:

    - Analysis is very rapid (in few minutes).

    - Analysis is very slow.

    8- Instrument:

    - Automated standardized instrument

    is used

    - It is just a simple


    II- Classification of chromatographic methods:

    1- According to the states of the two phases:

    Usually the mobile phase is named first

    (a) Liquid-solid chromatography (LSC).

    (b) Liquid-Liquid chromatography (LLC)

    (c) Gas- solid chromatography (GSC).

    (d) Gas- Liquid chromatography (GLC)

    2-According to the shape of stationary bed:

    (a) Plane or flat-bed chromatography: The stationary bed is

    coated on a flat surface. The two common types of plane

    chromatography are paper chromatography (PC) and thin

    layer chromatography (TLC).

    (b) Column chromatography: The stationary bed is contained in a

    column. Examples are open-column chromatography (OCC),

    gas chromatography (GC) and high pressure liquid

    chromatography (HPLC).

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    (c) 3- According to the mechanism of chromatographic


    According to the type of equilibration process involved between

    the mobile and the stationary phases, chromatographic methods

    are classified into:

    (a) Adsorption chromatography:

    This uses a solid stationary (like silica gel or any other silica based

    packing) where the sample components are adsorbed. The mobile

    phase may be a liquid (liquid-solid chromatography) or a gas (gas-solid

    chromatography). The components distribute between the two phases

    and the separation is based on repeated adsorption-desorption steps.

    Equilibration between the

    adsorbed state and the solution

    accounts for the separation of

    sample components, e.g. TLC,

    GSC, and LSC. (Fig.2) Two

    modes are defined depending

    on the relative polarity of the

    two phases (Fig.3). These are:

    Fig. 2: Adsorption


    1- The normal phase chromatography: in which the stationary bed

    is strongly polar in nature (e.g., silica gel), and the mobile phase is

    non polar (such as n-hexane or tetrahydrofuran). Polar samples

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    are thus retained on the polar surface of the column packing

    longer than less polar materials.

    Fig.3: Graphical illustration of normal and reversed-phase

    chromatography. Circles represent types of compounds present in

    the sample; their relative position to direction of mobile phase flow

    indicates their order of elution.

    2- The reversed-phase chromatography: which is the inverse of normal

    phase. The stationary bed is non polar (hydrophobic) in nature,

    while the mobile phase is a polar liquid, such as mixtures of water and

    methanol or acetonitrile.

    Here the more non polar the material is, the longer it will be retained.

    (b) Partition chromatography:

    -In which the stationary phase is a liquid supported on an inert solid. The

    mobile phase may be a liquid (liquid-liquid chromatography) or a gas

    (gas-liquid chromatography).

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