Introduction to Chromatography - Dr. Introduction to Chromatography General Chemistry (CHMA1001)...

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Transcript of Introduction to Chromatography - Dr. Introduction to Chromatography General Chemistry (CHMA1001)...

  • Introduction to Chromatography

    General Chemistry (CHMA1001) February 25, 2019

  • Chromatography

    Ø Known as color writing. A lab technique that is based on PHYSICAL ELUILIBRIA.

    (Chemical equilibria – Keq and Ka )

    Ø Involves the separation of mixtures into individual components by passing a fluid (gas or liquid) along a stationary phase.

    Ø Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple blend of two entantiomers to a multi component mixture containing widely differing chemical species

    http://slideplayer.com/slide/9274099/28/images/2/Separation +of+plant+pigments+by+thin-layer+chromatography.jpg

  • Chromatography allows for:

    1. Qualitative analysis – What is

    present?

    Rf; tR; type of color (hue)

    2. Quantitative analysis – How much is

    present?

    Size of spot and intensity of the color

    TLC), size of peak (GC; HPLC); expressed

    in terms of amounts (ppm; mg, intensity of

    color)

  • Physical Equilibria

    • The distribution of analytes between phases can often be described as an analyte in equilibrium between the two phases;

    Amobile Astationary

  • Physical Equilibria • Components physically separate

    themselves by distributing themselves between two phases 1) Stationary phase - which can be a

    solid or a liquid supported on a solid; and

    2) Mobile phase – which can be a gas or a liquid that continuously flows through the stationary phase.

  • Mechanism of Physical Equilibra used in the separation process are

    • Adsorption – “stick to”

    • Absorption – “dissolve into”

  • Adsorption Chromatography • Each component interacts

    with its environment differently under the same conditions.

    • Components move at a different rate, depending upon their interaction with the stationary phase.

    • Adsorption is dependent upon the nature of the component, nature of the adsorbent and the temperature.

    http://www.rpi.edu/dept/chem-eng/Biotech- Environ/CHROMO/be_types.htm

  • Absorption Chromatography • The solute molecules

    distribute themselves between two immiscible liquid phases, the stationary phase and the mobile phase according to their solubilities.

    • The two phases must have different polarities (only for LC).

    http://www.rpi.edu/dept/chem-eng/Biotech- Environ/CHROMO/be_types.htm

  • Adsorption vs Absorption

    • ADsorption – Sticks to – Example

    of Kleenex sticks to a sweater. When a lint roller is used the lint has a stronger attraction (affinity for) the lint roller

    – Only moves when in the mobile phase

    • ABsorption – Dissolves into -

    Example of eating a cookie and how it dissolves into.

    – Like has the greatest affinity for like (polar to polar or non polar to non polar)

    – Only moves when in the mobile phase

  • Elution The process of moving the solutes through and out of the chromatographic system is called elution. Solutes having left the chromatographic system are said to have been eluted. The mobile or moving phase is sometimes referred to as the eluting phase.

    In a multi-component sample separation is based on their attraction for (ability to adsorb or absorb) the stationary phase

    http://biotech.matcmadison.edu/resources/proteins/labManual/chapter_4/section4_4.htm

  • Diffusion Solutes when contained in a fluid naturally diffuse and spread driven by their concentration gradient. In a chromatographic column a discrete solute band will diffuse in the gas or liquid mobile phase.

    http://www.chromatography-online.org/Principles/Peak-Dispersion/Longitudinal-Diffusion.html

  • Main Types of Chromatography Used in Labs

    1) Column Chromatography (CC) 2) Thin Layer Chromatography (TLC) 3) Gas Chromatography (GC) 4) High Pressure (Performance) Liquid

    Chromatography (HPLC)

  • Column Chromatography The stationary phase is a powdered adsorbent which is held in a vertically positioned glass column.

    The separation mechanism is usually ADSORPTION.

    The mixture to be analyzed is loaded on top of this column.

    http://www.m2c3.com/chemistry/VLI/M4_Topic2/M4_Topic2_print.html

  • Column Chromatography The mobile phase is a solvent poured on top of the loaded column; it flows down under the force of gravity. The sample components set up multiple, repeating physical equilibria between the stationary and mobile phases. If analytical conditions are correct, some components elute in the early fractions; other components elute from the column only after more mobile phase has flowed.

    http://orgchem.colorado.edu/hndbksupport/colchrom/colchrom.html

  • Polarity • Polarity results from the

    uneven partial charge distribution between various atoms in a compound.

    • Atoms, such as nitrogen, oxygen, and halogens, that are more electronegative have a tendency to have partial negative charges.

    • Atoms, such as carbon and hydrogen, have a tendency to be more neutral or have partial positive charges.

  • Column Chromatography ØThe polarity of the solvent which is passed

    through the column affects the relative rates at which compounds move through the column.

    ØOften a series of increasingly polar solvent systems are used to elute a column.

    Ø If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. If a solvent is not polar enough, no compounds will elute from the column.

    ( a non-polar solvent is first used to elute a less-polar compound. A more-polar solvent is added to the column to elute the more-polar compound.)

  • Column Chromatography ØQualitative information:

    Based on color and order of elution - the progress of the separation can simply be monitored visually.

    ØQuantitaitve information: (semi-quantitative at best – approximate concentration) Based on intensity of color/hue

    ØFractions of the eluent are often collected sequentially and the composition of each fraction is analyzed by thin layer chromatography or some other method.

  • Column Chromatography • Slurry Method Preparation:

    – A mixture of the adsorbent (e.g.alumina or silica) with the solvent is prepared and this slurry is poured into the column containing glass wool or cotton.

    – Always ensure that there is a continuous flow of mobile phase. The column must not go dry.

    – The advantage of slurry methods is that they eliminate air bubbles from forming in the column as it packs.

    – Air bubbles may lead to channels

  • Column Chromatography

    • Dry – Packed Method – Easier to prepare than slurry method – A dry column containing glass wool or

    cotton is filled with solvent. Dry alumina is added to the column containing the solvent and allowed to settle.

    – Disadvantage of dry – packed method is that there is more chance for the formation of bubbles and channels in the column.

  • Thin Layer Chromatography (TLC) • A TLC plate is a sheet of glass, metal, or plastic

    which is coated with a thin layer of a solid adsorbent (usually silica or alumina) – The stationary phase.

    • A liquid, or eluent, is the mobile phase (usually an organic solvent).

    • A small amount of the mixture to be analyzed is spotted near the bottom of this plate.

    • The mobile phase slowly rises up the TLC plate by capillary action.

    • The separation mechanism is usually ADSORPTION.

  • Thin Layer Chromatography (TLC)

    • The TLC plate is placed in a reservoir of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid.

    • The components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others.

    http://www.coleparmer.co.uk/products/chromatography/Chromatography.asp

  • Thin Layer Chromatography

    http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html

  • Thin Layer Chromatography

    • The Rf (retention factor) can provide corroborative evidence as to the identity of a compound.- (Qualitative information – “what is present”)

    • If two substances have the same Rf value, they are likely (but not necessarily) the same compound.

  • Thin Layer Chromatography

    • Rf is dependent on – The solvent system – Adsorbent – Thickness of the adsorbent – Amount of material spotted – Temperature

    Factors are difficult to keep constant from experiment to experiment – considered relative (compared to standards run on same plate)

  • Thin Layer Chromatography • Some samples are colored.

    Simply mark by circling the spots before fading.

    • Others colorless samples may require UV to visualize samples or to spray with an appropriate reagent.

  • Thin Layer Chromatography • If a set of standards that cover the concentration

    range of interest are separated on the same plate as the