2.2.17 Chromatography

• 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 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 monosaccharides

• Paper chromatography can be used to separate a mixture of monosaccharides
• Mixtures containing coloured molecules, such as ink or chlorophyll, do not have to be stained as they are already coloured
• Mixtures of colourless molecules, such as a mixture of monosaccharides, have to be stained first
• A spot of the stained monosaccharide sample mixture is placed on a line at the bottom of the chromatography paper
• Spots of known standard solutions of different monosaccharides are then placed on the line beside the sample spot
• The chromatography paper is then suspended in a solvent
• As the solvent travels up through the chromatography paper, the different monosaccharides within the mixture separate out at different distances from the line
• The unknown monosaccharides can then be identified by comparing and matching them with the chromatograms of the known standard solutions of different monosaccharides
  • If a spot from the monosaccharide sample mixture is at the same distance from the line as a spot from one of the known standard solutions, then the mixture must contain this monosaccharide

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

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

Calculating the Rf value

• After a chromatogram has been obtained the molecules present in the sample mixture can be identified by calculating their retardation factor (Rf)
• In order to calculate Rf values, a line must be drawn across the chromatogram to show how far the solvent travelled
  • This line is known as the solvent front
  • The distance between the origin line and the solvent front is the distance moved by the solvent
  • The origin line is the line at the bottom of the paper on which the samples were placed at the beginning of the experiment

• The R_f value demonstrates how far a dissolved molecule travels during the mobile phase
  • A smaller R_f value indicates the molecule is less soluble and larger in size

• The Rf value of each solute (each spot on the chromatogram) is calculated and then compared to the Rf values of known molecules/substances
• The equation is:

Rf = distance moved by solute ÷ distance moved by solvent

• The Rf value is a ratio so it is always lower than one
  • It has no units

Using Rf values to identify chloroplast pigments

• Chromatography can be used to separate and identify chloroplast pigments that have been extracted from a leaf as each pigment will have a unique Rf value
• Although specific Rf values depend on the solvent that is being used, in general:
  • Carotenoids have the highest Rf values (usually close to 1)
  • Chlorophyll B has a much lower Rf value
  • Chlorophyll A has an Rf value somewhere between those of carotenoids and chlorophyll B
  • Small Rf values indicate the pigment is less soluble and larger in size

Paper chromatography can be used to separate the photosynthetic pigments found within chloroplasts. Rf values can then be calculated for each pigment and compared to known Rf values for the different pigments.