3.16 chromatography

• Why are two solvents used?

  To ensure all of the amino acid has dissolved
• How can you suggest which compounds move more quickly through the column?

  If moving phase is non-polar e.g hexane: non-polar compounds pass through more quickly

  If moving phase is polar e.g ethanol: polar compounds pass through quickly

  It's all about solubility and IMF.
• TLC: Why do we dry in a fume cupboard?

  solvent may be toxic
• TLC: Why do we care about the depth of solvent?

  if solvent is too deep, it dissolves the sample spots from the plate
• TLC: Why do we use a lid?

  prevents evaporation of toxic solvent
• TLC: Why do we wear gloves?

  prevents contamination from hands to the plate
• TLC: Why do we use a pencil line?

  will not dissolve in the solvent
• Recall how to carry out TLC.

  Draw pencil line

  Use capillary tube to add a tiny drop of each

  Add solvent to a chamber

  Place TLC into chamber; ensuring solvent is below pencil line

  Leave for time; then remove and mark solvent front with pencil

  Dry in a fume cupboard
• What two properties can be measured to identify substances in chromatography?

  Retention times: how long each component takes from being injected to recorded

  Rf values: Distance moved by sample / distance moved by solvent

  In both instances, they are compared with standards from a data book
• Recall Gas Chromatography (GC).

  Used to separate volatile liquids

  SP: Column packed with a solid or viscious liquid

  MP: Inert gas e.g N2 passed through under pressure at high temperature

  Retention time measured
• Recall Thin Layer Chromatography (TLC).

  Used to separate and identify components in a mixture

  Stationary Phase: Plate coated with a solid

  Mobile Phase: Solvent moving up the plate
• Recall Column Chromatography (CC).

  Used to separate an organic productfrom impurities.

  Column is packed with a solid (Al2O3)and a solvent moves down the column

  More soluble components move down column faster.
• What does the movement of a substance in chromatography depend on?

  A balance between:

  Solubility in solvent

  Retention in the stationary phase
• What are the two phases in a chromatography set up? What do they normally consist of?

  Stationary phase - solid/liquid held in a solid

  Mobile phase - liquid or gas
• What is the use of chromatography?

  To separate and identify the components in a mixture
• State in general terms what determines the distance travelled by a spot in TLC.

  Balance between solubility in moving phase and retention by stationary phase
• Why do amino acids have different RF values

  each amino acid has different affinities to the stationary and mobile phase, as the movement is a balance between solubility in the moving phase with retention of the stationary phase.

  This is determined by the polarity of the amino acid.
• what is the effect of having a more polar molecule in a chromatography?

  more polar means more attraction to the plate in the stationary phase, this means the rf value will be greater
• what is the effect of having a polar solvent in chromatography

  the amino acid will be more attracted to the solvent so will move further and rf value will be bigger
• how does gas chromatography separate amino acids

  they will have a difference in their balance between solubility in the mobile phase and retention by the stationary phase causes different rf values
• how do we measure RF

  use uv

  measure difference between initial line top spots (x)

  measure distance from initial pencil line to solvent (y)

  rf value = x/y