3.3.16 Chromatography

Cards (17)

  • Thin layer chromatography
    > The stationary phase is a coating of powdered silica or alumina on a sheet of glass or plastic
    > The mobile phase is a solvent such as water or ethanol
    > The method for TLC is the same as paper chromatography and Rf values can be calculated
  • Stationary phase does not move
    Mobile phase (solvent) - moves through the stationary phase carrying the dissolved substance with it
    > The different substance are attracted to the two phases to different extents, so they become separated as the mobile phase moves.
  • TLC chromatography method:
    > A horizontal line in pencil is drawn at the bottom of the plate (stationary phase)
    > Drops of the same and known compounds if available are spotted on the line on the plate and dried
    > The plate is placed in a container containing a small amount of solvent (mobile phase) and a lid is placed to prevent loss of solvent
  • TLC chromatography method pt 2:
    > As the solvent rises the different substances separate out.
    > The solubility of the substances in the solvent is important
    > The substance will rise further up the plate if a more effective solvent is used (a greater Rf value)
  • TLC for amino acids:
    > Mixtures of amino acids are separated using thin layer chromatography (TLC)known amino acids at the bottom of the plate
    > Apply a small spot of the mixture at the bottom of the chromatography plate (plate is covered with silica)
    > Add spots of known amino acids at the bottom of the plate
    > Place the plate in a solvent such as ethanol
    > The solvent will carry the amino acids up the plate at different rates and the amino acids will separate out
    > You can identify the amino acids by comparing the separated spots from the mixture with the known amino acids spots
  • The plate is stained using ninhydrin so the amino acids become visible
    TLC is also used to monitor organic reactions by mounting the disappearance of staring material or appearance of product.
  • Column Chromatography:
    > The stationary phase is powdered alumina or silica packed into a glass column
    > The mobile phase (called eluent) is an organic solvent
    > The mixture is added to the top of the column and allowed to run into the stationary phase
    > Solvent is added to the top to wash the different components down the column
    > This process is called elution
    > The different components are collected in test tubes or flasks and then analysed purifying compounds from mixtures
    > Column chromatography is a useful way of separating and purifying compounds from mixtures
  • The retention time of a substance is the time taken for it to leave the column
  • The retention time for a solute depends on:
    > Its attraction to the stationary phase
    > Its solubility in the mobile phase (solvent)
  • The retention time is low (fast) if the substance is attracted weakly to the stationary phase but strongly attracted to the solvent (mobile phase).
    The substance has a high solubility in the mobile phase.
  • If the substance is polar it will be attracted more strongly to the stationary phase than the solvent
    This means it will have a high retention time (slow)
  • Gas-liquid chromatography:
    > GLC is used to separate mixtures of volatile liquids.
    >The stationary phase is a liquid with a high boiling point coated onto a powder.
    > This coated powder is packed into a long stainless steel or glass coiled tube (called the column).
    >The mobile phase is an inert gas such as nitrogen, also called the carrier gas
    > The sample vaporises when it is injected into the hot column and it is pushed through the column by the carrier gas
  • Different compounds exit the column at different times depending on:
    > The boiling point of the compound compared to the temperature of the column.
    > The solubility of the compound in the liquid of the stationary phase
  • If the compound is highly soluble in the liquid of the stationary phase it will take longer to exit the column.
    This means it will have a high retention time (slow).
    If the compound has a low solubility in the liquid of the stationary phase it will have a low retention time (fast)
  • Gas-liquid chromatography:
    > A detector is used to monitor the outlet stream from the column.
    > Each compound appears as a peak on a chart and has it own retention time.
    > Retention times can be used to identify the compound
    > The area under each peak is proportional to the amount of each compound
  • Chromatography can be used to separate and identify the components in a mixture.
    Types of chromatography include:
    • thin-layer chromatography (TLC) - a plate is coated with a solid and a solvent moves up the plate
    • column chromatography (CC) - a column is packed with a solid and a solvent moves down the column
    • gas chromatography (GC) - a column is packed with a solid or with a solid coated by a liquid, and a gas is passed through the column under pressure at high temperature.
  • Separation depends on the balance between solubility in the moving phase and retention by the stationary phase.