Chromatography

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• Series of analytical techniques that can be used to separate mixtures of compounds for further use or for analysis
• In all forms of chromatography, a mobile phase moves through or across a stationary phase
• Stationary phase: This phase does not move.
  • Compounds in the mixture are attracted to it (adsorbed) and slowed down.
  • Either a solid or a liquid
• Mobile Phase: This phase moves.
  • The more soluble compounds in the mixture are carried faster as the mobile phase moves.
  • Either a liquid or a gas
• Paper chromatography
• Thin Layer chromatography
• Column chromatograph
• Gas chromatography
• High-performance liquid chromatography

Paper Chromatography

• Used to separate mixtures, especially dyes or pigments
• Dots of single dyes are placed alongside a dot of the unknown mixture
• The solvent is drawn up the paper by capillary action
• As the solvent moves up the paper, the pattern of the single dyes can be compared to that of the unknown mixture
• The distance the solvent travels relies upon its polarity
• The more polar molecules will be more adsorbed to the paper, and thus will travel less

Thin Layer Chromatography (TLC)

• All chromatography involves a stationary phase and a mobile phase
• Stationary phase is a layer of silica gel (polar) fixed on a rigid glass plate
• The mobile phase is a solvent which travels up the plate, carrying the substances
• Uses same principles as paper chromatography
  • Capillary action still draws the solvent up the matrix
  • However, while the molecules in paper chromatography are separated based on mace, in TLC, separation often depends on solubility or charge, due to the interaction of solute and matrix
  • A dry sample is placed in the silica gel matrix. As the solvent front moves up the gel, it dissolves the sample and carries it up the matrix with it
  • Some of the particles in the sample stick more strongly to the silica gel than others so they lag behind the solvent
    • The more polar molecules have a stronger IMF between itself and the gel, thus it sticks more strongly, and lags
  • Eventually the different substances in the sample separate out, with similar molecules travelling a similar distance
• The solvent (i.e. the mobile phase) is drawn up the stationary phase by capillary action
  • Adhesive forces are between molecules of different substances
    • Adhesion enables solvent molecules to bond to stationary phase
  • Cohesive forces are between molecules of the same substance
    • Cohesion enables solvent molecules to bond to each other and move each other through stationary phase
  • Capillary action causes liquids to form a meniscus in a narrow glass tube.

TLC or Paper Chromatography?

• TLC has a lot of advantages
  • Glass plate is rigid, not flexible like paper, so it is easy to control
  • After separation, the substances in the mixture can be recovered
    • The silica gel holding the separated substances is scraped of the glass plate and added to a solvent
    • The substance will dissolve and the silica gel can easily be removed by filtration
    • The glass plates can be re-coated with silica gel and be used over and over again

Retention Factor (Rf)

• Ratio of the distance traveled of the solute to the solvent
• Different pigments have different Rf values $Rf=\frac{Solut{e}_{distance}}{Solven{t}_{distance}}$
• Greater Rf means it is more non-polar

UV and Locating Agents

• Many substances are white or colourless, and so aren’t visible on a TLC plate
• One way of making colourless substances show up is to use UV light.
• This usually works well for organic compounds
• An alternative method is to use a chemical locating agent – a chemical that reacts with the substance to form a coloured compound.
  • For example, when ninhydrin is exposed to an organic compound it stains it purple-brown.

Gas Chromatography

• Used widely in many analytical laboratories, including forensic police labs, synthetic chemical labs, and drugs testing labs.
• Suitable for low molecular weight samples
• In paper and thin layer chromatography, the mobile phase is a liquid
• However, as the name implies, the mobile phase in GC is an inert gas.
• The stationary phase is usually a long thin tube of silica gel.
• Like all forms of chromatography, GC uses a stationary phase to impede the movement of a mobile test substance.
• Different substances are attracted to the matrix by different amounts, and therefore journey along it at different speeds.
• In GC, the sample is injected into the machine, where it is vaporised. It is then washed over the matrix by an inert gas.
• Some substances will be more attracted to the matrix than others. These will take much longer to reach the detector.
  • Adhesion forces are stronger than cohesive forces
• The detector measures the abundance of a substance at a given time, and this data is plotted on a graph.

High Performance Liquid Chromatography (HPLC)

• High performance liquid chromatography (HPLC) is a development of column chromatography in which the eluent is pumped through the column at high pressure.
  • Eluent: In chromatography, a solvent used in order to effect separation by elution.
• This results in better and faster separation than can be achieved in standard column chromatography.

GC	HPLC
Mobile phase changes	Mobile phase is constant
Constant temperature	Increasing temperature
Compounds partition from the mobile phase based on solubility	Compounds partition from the mobile phase based on volatility
Elution is generally time or volume dependent	Elution is generally temperature dependent

Column Chromatography (Extension)

• Mobile phase is passed through (under pressure) the stationary phase
• Interaction between the MP and SP with the compounds in the mixture enable separation