Chromatography of Chloroplast Pigments (Cambridge International A-Level Biology)

Spec point — describe and use chromatography to separate and identify chloroplast pigments

Chromatography of chloroplast pigments

• 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 (R_f) can be calculated for each component of the mixture

R_fvalue = 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 (e.g. 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 R_f value

• The R_f value demonstrates how far a dissolved pigment travels through the stationary phase

  • A smaller R_fvalue indicates the pigment is less soluble and larger

• Although specific R_f values depend on the solvent that is being used, in general:

  • Carotenoids have the highest R_fvalues (usually close to 1)
  • Chlorophyll B has a much lower R_fvalue
  • Chlorophyll A has an R_f value somewhere between those of carotenoids and chlorophyll B
  • Small R_fvalues indicate the pigment is less soluble and larger

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

Exam Hint Make sure you learn the approximate R_fvalues for the different pigments within chloroplasts (or at least their values relative to each other). This means you should be able to identify different chloroplast pigments based on their R_f values alone.