Practical: chromatography

• What do chloroplasts contain that absorbs light?

  Photosynthetic pigments
• What is the purpose of chromatography in this context?

  To separate and identify chloroplast pigments
• What are the main pigment groups found in chloroplasts?

  • Chlorophyll a
  • Chlorophyll b
  • β-Carotene
  • Xanthophyll
• What are the colors of the main chloroplast pigments?

  • Chlorophyll a: Blue-green
  • Chlorophyll b: Yellow-green
  • β-Carotene: Orange
  • Xanthophyll: Yellow
• What is chromatography?

  An experimental technique to separate mixtures
• Why do different components in chromatography separate?

  They travel at different speeds due to size and charge
• What is the retardation factor (Rf)?

  A value calculated for each component
• What are the two common techniques for separating photosynthetic pigments?

  Paper chromatography and thin-layer chromatography
• What is used as the stationary phase in paper chromatography?

  Paper (cellulose)
• What is used as the stationary phase in thin-layer chromatography?

  Thin layer of absorbent (e.g., silica gel)
• What should you avoid using to draw the line on filter paper?

  A pen
• Why is it important to use a healthy leaf in the experiment?

  It ensures many active photosystems are present
• What does acetone do in the pigment extraction process?

  Dissolves fats and breaks down membranes
• What is the mobile phase in chromatography?

  The solvent that carries the dissolved mixture
• How do you calculate the Rf value?

  $Rf =$$\frac{\text{distance travelled by component}}{\text{distance travelled by solvent}}$
• What does a higher Rf value indicate about a pigment?

  It is more soluble in the mobile phase
• What does a lower Rf value indicate about a pigment?

  It is less soluble and/or larger in size
• What are the general Rf values for chloroplast pigments?

  • Carotenoids: Highest Rf values (close to 1)
  • Chlorophyll b: Much lower Rf value
  • Chlorophyll a: Between carotenoids and chlorophyll b
• What is a limitation of paper chromatography?

  It is not as specific as other techniques
• What does chromatography not provide information about?

  The amount of each pigment present
• What technique can be used to calculate the amount of each pigment?

  Colorimetry
• What is the difference between the color of pigments and the colors of light they absorb?

  Pigments have color; absorption is different
• Why should you learn the approximate Rf values for chloroplast pigments?

  To identify pigments based on Rf values