Chromatography

What is chromatography?

• Separation of a mixture into all its constituents

• There are two key components:

  • Stationary phase

  • Mobile phase

Stationary phase

• This is the chromatography paper or the think layer chromatography plate (TLC)

• The TLC is a thin sheet of paper with a thin layer of silica gel or aluminium hydroxide

• There are free OH- groups pointing outwards in contact with the mobile phase

Mobile phase

• This is the solvent for the biological molecules

• Water can be used to polar molecules

• Ethanol for non polar molecules

• Mobile phase flows through and across the stationary phase carrying the biological molecules with it

How does this occur?

• Solvent travels up the paper or plate with components of the solution mixture with it

• Different components travel at different speeds dependent on their solubility in the mobile phase; this is also dependent on the polarity of the molecule as well

• Exposed OH- groups make the surface of the plate or paper very polar, it forms hydrogen bonds with the molecules

• A highly polar solute will stick to the surface and move more slowly

• A non polar solute will travel quickly

How to identify pigments

• Measure distance from the pencil line to the centre spot of the pigment and the distance from the pencil line to solvent front

• Rf value= spot distance/ front distance

• If the experiment is repeated under the same conditions each pigment will always have the same Rf value

• If you know the Rf values of particular pigments it allows you to identify them

Chromatography with colourless molecules

Using TLC there are ways to see colourless molecules:

• Ultraviolet light

  • Under the UV light the plate will glow and the spots ink travelled will not

• Ninhydrin

  • To see amino acids

  • Allow plate to dry and spray with ninhydrin

  • Binds to amino acids to make visible brown/ purple spots

• Iodine

  • Allow plate to dry and place in a container with iodine crystals

  • Iodine forms a gas, which binds to the molecules of each spot

How is chromatography used?

• Monitor progress of reactions

• Urine testing athletes for drugs

• Analysing drugs for purity

• Analysis of food for contaminants