CHROMATOGRAPHY · PDF file Adsorption chromatography Ion exchange chromatography Partition...

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Transcript of CHROMATOGRAPHY · PDF file Adsorption chromatography Ion exchange chromatography Partition...

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    CHROMATOGRAPHY it is technique for separating the components of the mixture based on the relative amount of each substance divided between the moving fluid stream, known as the mobile phase and the adjacent stationary phase. Explained/nav.htm?cid=10048919&locale=en_PL

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    Method used for separation of mixture of the compounds between two phases:

    ❖ Stationary phase ( solid or liquid located on neutral


    ❖ Mobile phase ( liquid or gas)

    Choice of the method depends on:

    ❖ the amount of analysed mixture

    ❖ kind of compounds

    ❖ complexity of separation process


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    Chromatographic separation involves placing the test mixture on the liquid or solid stationary phase and then passing the liquid or gaseous mobile phase through it or above it, that is elution of the mixture from a stationary phase.

    The mixture of components with different ratios of division are eluted (migrate) at different speed. These differences in the speed of migration lead to the separation of components in time and space.



    Stationary phase

    Mobile phase


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    Investigated compound

    is located in mobile

    phase(solvent) and

    migrates with it across

    stationary phase which

    is porous medium:

    adsorbent, ion

    exchanger or molecular



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    Illustration of Chromatography

    Component s

    Affinity to Stationary Phase

    Affinity to Mobile Phase

    Blue ✓✓✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

    white ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

    Red ✓ ✓ ✓ ✓ ✓ ✓ ✓

    Yellow ✓ ✓✓✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

    Mixture Components


    Stationary Phase

    Mobile Phase


  • Mobile phase or carrier - solvent moving through the column

    Stationary phase or adsorbents - substance that stays fixed

    inside the column

    Eluent - fluid entering the column

    Eluate - fluid exiting the column (that is collected in flasks)

    Elution - the process of washing out a compound through a

    column using a suitable solvent

    Analyte - mixture which individual components have to be

    separated and analyzed


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    1. According to physico – chemical forces:

    ▪ Adsorption chromatography

    ▪ Ion exchange chromatography

    ▪ Partition chromatography

    ▪ Gel (size exclusion) chromatography

    ▪ Affinity chromatography

    ▪ Capillary chromatography

    2. According to parameters of the proces:

    ▪ High Pressure/Performance Liquid Chromatography (HPLC)

    ▪ Fast protein liquid chromatography (FPLC)

    Classification of chromtographic methods


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    3. According to applied techniques:

    ◼ Column chromatography

    ◼ Planar chromatography (thin layer, paper)

    4. According to mobile phase type:

    ◼ Gas Chromatography, GC

    ◼ Liquid Chromatography, LC

    ◼ Supercritical Fluid Chromatography, SFC

    5. According to mobile and stationary phases type:

    ◼ Gas – liquid chromatography (GLC)

    ◼ Liquid – liquid chromatography (LLC)

    ◼ Gas – solid chromatography (GSC)

    ◼ Liquid - solid chromatography (LSC)

    Classification of chromtographic methods


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    Liquid Chromatography – separates liquid samples with a liquid solvent

    (mobile phase) and a column composed of solid beads (stationary phase)

    Gas Chromatography – separates vaporized samples with an inert carrier

    gas (mobile phase) and a column composed of a liquid or of solid beads

    (stationary phase)


    Types of chromatography

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    Types of chromatography

    Adsorption Chromatography uses solid material as stationary phase

    (adsorbents). The separation of the mixture is caused by a

    different affinity of individual components (adsorbates) of a

    mixture to the surface of stationary phase.

    Planar chromatography:

    • Paper Chromatography – separates liquid samples with a liquid

    solvent (mobile phase) on a paper strip (stationary phase)

    • Thin-Layer Chromatography – separates liquid samples with a

    liquid solvent (mobile phase). Glass plate is covered with a thin

    layer of aluminium oxide or silica gel (stationary phase)

  • •Adsorption chromatography

    If the stationary phase is solid, chromatography is called adsorption chromatography.

    Separation of the mixture is caused by different affinities of mixture components (adsorbates) to the surface of the stationary phase known as adsorbent.

    Adsorption of analysed sample particles, can be induced by:

    physical forces (physical adsorption) chemical influence (chemisorption)


  • The effectiveness of adsorption depends on:

    • kind of adsorbent and its properties

    • used solvent

    • properties of adsorbed molecules


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    Classification of adsorbents according to:

    1. Adsorption activity:

    ▪ Weak ( starch, saccharose, talc) ▪ Middle ( calcium carbonate, sodium carbonate) ▪ Strong ( aluminium oxide, activated silica acid)

    2. Chemical properties:

    ▪ acidic ( SiO2) ▪ basic ( CaO) ▪ neutral ( activated carbon) ▪ amphoteric ( Al2O3)

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    Classification of adsorbents

    3. Chemical nature:

    ▪ Organic ( carbon, starch, saccharose, polyamides )

    ▪ Inorganic ( Al2O3, MgO, natural and synthetic silicates )

    ▪ Mixed (talc + saccharose, CaCO3 + talc )

    ▪ Specific (silica gel with specific pores )

    4. Polarity :

    ▪ Highly polar ( aluminium oxide, silica oxide)

    ▪ Weak polar (calcium carbonate, MgO)

    ▪ Non-polar ( activated carbon, talc, graphite)


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    Solvents used in chromatogrphy are arranged in an eluotropic series,

    depending on the ability of adsorption with respect to the substances

    dissolved in them and the ability of elution.

    They are used to develop the chromatogram and to elute the separate components.

    1. Hexane 2. Carbon tetrachloride (CCl4) 3. Benzene 4. Diethyl ether 5. Acetone 6. Chloroform 7. Ethyl acetate 8. Ethanol 9. Methanol 10. Water


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    Properties of adsorbed molecules


    Adsorption ability of adsorbed molecules depends on::

    ➢ Size and spatial orientation

    ➢ Number and location of double bonds

    ➢ Number of polar and nonpolar substituents

    ➢ Number and kind of functional groups

    The degree of adsorption of the adsorbate increases with:

    ➢ Number of double bonds

    ➢ Number of functional groups

    ➢ Number of substituents of the same type

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    Thin Layer Chromatography – TLC (partition


    Separation of the mixture depends on the difference in partition

    coefficient of the mixture’s components between two non miscible

    phases, from which one is a liquid coated on a medium (stationary

    phase), and a second one is a mobile phase (liquid,gas).

    Partition Coefficient is equal to the ratio of the concentration of the

    same substance in the stationary phase and the mobile phase.

    Nernst law of partition:

    K = Cs / Cm

    K – partition coefficient at equilibrium state, depends only on the

    temperature and properties substance-forming solutions and does not

    depend on the amount of solute.

    Cs – concentration of particular compound (solute) dissolved in stationary


    Cm - concentration of solute dissolved in mobile phase ©

  • Thin-Layer Chromatography (TLC)

    This is a fast method for the initial

    orientation in an amount and relative

    contributions of the components in the

    mixture of organic compounds.

    Advantages of TLC:

    ◼ a short time to develop the chromatogram

    ◼ simple methods of process control

    ◼ minimum consumption of materials

    ◼ ease to perform and low costs

    Application of TLC:

    • separation of :

    • amino acids

    • peptides

    • saccharides

    • nucleotides

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    Chromatography application for qualititive analysis – Rf coefficient.

    Substance location on chromatogram is characterized by Rf values.

    (ratio of fronts) = retention factor

    Distance from START line to the middle of substance spot - A

    Rf = Distance from START line to the End line of solvent – B

    Rf - has characteristic value for particular substance in specific process conditions






    Invastigated sample

    Height reached by solvent

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    Preparation of chromatographic plates

    • Gl