• Chromatography?is an experimental technique that is used to?separate mixtures:
  • The mixture is?dissolved?in a fluid/solvent?(called the mobile phase) and the dissolved mixture then passes through a static material (called the stationary phase)
  • Different components?within the mixture travel through the material at?different speeds
  • This causes the different components to?separate
  • A retardation factor (Rf) can be calculated for each component of the mixture

Rf?value = distance travelled by component ÷ distance travelled by solvent

• Two of the most common techniques for separating these photosynthetic pigments are:
  • Paper chromatography?– the mixture of pigments is passed through paper (cellulose)
  • Thin-layer chromatography?– the mixture of pigments is passed through a thin layer of adsorbent (eg. silica gel), through which the mixture travels faster and separates more distinctly
• 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
• The Rf?value demonstrates how far a dissolved pigment travels through the stationary phase
  • A?smaller Rf?value?indicates the pigment is?less soluble?and?larger?in size
• 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.