1.3.2 Chromatography: Amino Acids

• Chromatography is a technique that can be used to separate a mixture into its individual components
• Chromatography relies on differences in the solubility of the different chemicals (called ‘solutes’) within a mixture
• All chromatography techniques use two phases:
  • The mobile phase
  • The stationary phase

• The components in the mixture separate as the mobile phase travels over the stationary phase
• Differences in the solubility of each component in the mobile phase which affects how far each component can travel
• Those components with higher solubility will travel further than the others
• This is because they spend more time in the mobile phase and are thus carried further up the paper than the less soluble components

Paper Chromatography

• Paper chromatography is one specific form of chromatography
• In paper chromatography:
  • The mobile phase is the solvent in which the sample molecules can move, which in paper chromatography is a liquid e.g. water or ethanol
  • The stationary phase in paper chromatography is the chromatography paper

Paper chromatography method

• A spot of the mixture (that you want to separate) is placed on chromatography paper and left to dry
• The chromatography paper is then suspended in a solvent
• As the solvent travels up through the chromatography paper, the different components within the mixture begin to move up the paper at different speeds
  • Larger molecules move slower than smaller ones
  • This causes the original mixture to separate out into different spots or bands on the chromatography paper

• This produces what is known as a chromatogram

An example of a chromatogram that has been produced by using paper chromatography to separate a spot of ink

Using chromatography to separate a mixture of Amino Acids

• Paper chromatography can be used to separate a mixture of amino acids
• A spot of the unknown amino acid sample mixture is placed on a line at the bottom of the chromatography paper
• Spots of known standard solutions of different amino acids are then placed on the line beside the unknown sample spot
• The chromatography paper is then suspended in a solvent
• Each amino acid will be more or less soluble in the mobile phase than others and will therefore separate out of the mixture travelling with the solvent at different times/distances from the line, depending on their:
  • Charge
  • Size

• The unknown amino acid(s) can then be identified by comparing and matching them with the chromatograms of the known standard solutions of different amino acids
  • If a spot from the amino acid sample mixture is at the same distance from the line as a spot from one the known standard solutions, then the mixture must contain this amino acid

• • In order to view the spots from the different amino acids, it may be necessary to first dry the chromatography paper and then spray it with ninhydrin solution (this chemical reacts with amino acids, producing an easily visible blue-violet colour)

How chromatography can be used to separate a mixture of amino acids and identify the individual components