RPQ 7 chromatography

• chromatography is used to separate stuff in a mixture - once it is separated out you can often identify the components:

  • paper chromatography
  • thin layer chromatography
• how does it work?

  all types have the same basic set up:

  • a mobile phase - where the molecules can move - in both paper and TLC the mobile phase is a liquid solvent
  • stationary phase - where the molecules can't move - in paper chromatography the stationary phase is a piece of chromatography paper
  • TLC the stationary phase is a thin 0.1-0.1 mm layer of solid e.g. silica gel on a glass or plastic plate - TLC plate
• all types of chromatography work using the same basic principle:

  • the mobile phase moves through or over the stationary phase
  • the components in the mixture spend different amounts of time in the mobile phase and stationary phase
  • the components that spend longer in the mobile phase travel further or faster. the time spent in different stages separates out the components of the mixture
• investigating pigments in leaves:

  • all plants contain several different photosynthetic pigments in their leaves
  • each pigment absorbs a different wavelength of light so having more than one pigment increases the range of wavelengths of light the plant can absorb
  • in addition to photosynthetic pigments, some plants have other pigments in their leaves which play other essential roles e.g. protecting the leaves from excessive UV radiation
  • different species of plant contain different proportions and mixtures of pigments
• • a sample of pigments can be extracted from the leaves of a plant and separated using paper or TLC
  • can then identify the pigments present in the sample by calculating the Rf value
• • an Rf value is the distance a substance has moved through the stationary phase relative to the solvent
  • each pigment has a specific Rf value under specific conditions
  • can be looked up in a database
• the pattern of pigments you end up with is called a chromatagram
• e.g. use TLC to compare the pigments present in shade-tolerant plants (e.g. hostas) and shade-intolerant plants (e.g. chrysanthemums)
• make sure wearing lab coat and gloves for start
• 1. grind up several leaves from the shade-tolerant plant you are investigating with some anhydrous sodium sulfate, then add a few drops of propanone
  2. transfer the liquid to a test tube, add some petroleum ether and gently shake the tube. two distinct layers will form in the liquid - the top layer is the pigments mixed in with the petroleum ether
  3. transfer some of the liquid from the told layer into a second test tube with some anhydrous sodium sulfate
• 4. draw a horizontal pencil line near the bottom of the TLC plate - build up a concentrated spot of liquid from step three on the line by adding several drops, ensuring each one is dry before the next is added - this is the point of origin
• 5. once the plate is completely dry, put the plate into a small glass container with some prepared solvent (e.g. a mixture of propanone, cyclohexane and petroleum ether) - just enough so that the point of origin is a little bit above the solvent. put a lid on the container and leave the plate to develop. as the solvent spreads up the plate the different pigments move with it, but at different rates - so they separate
• 6. when the solvent has nearly reached the top, take the plate out and mark the solvent front (the furthest point the solvent has reached) with a pencil and leave the plate to dry in a well-ventilated place
• 7. there should be several new coloured spots on the chromatography plate between the point of origin and the solvent front. these are the separated pigments.

  you can calculate their Rf values and look them up in a database to identify what the pigments are
• calculate the Rf value using this formula:

  Rf value = distance travelled by spot

  distance travelled by solvent
• 8. repeat the process for the shade-intolerant plant and compare the pigments present in the leaves
• may find that the mixture of pigments in the shade-tolerant plant is quite different compared to the shade-intolerant plant
• • one way that shade-tolerant plants can adapt to the light conditions in their environment is by processing a different proportion of photosynthetic pigments which allows the plants to make the best use of the light available to it
• • the mixture of non-photosynthetic pigments is also likely to be different
  • e.g. the chloroplasts of shade-tolerant plants are adapted for photosynthesis in low light conditions, but really sensitive to higher levels of light
  • these plants sometimes produce dark red and purple pigments called anthocyanins which are thought to protect their chloroplasts from breif exposure to higher light levels
• propanone, petroleum ether and chromatography solvent is highly flammable and toxic
• best to do steps 2 to 5 in a fume cupboard as the chemicals used are volatile (evaporate easily) and the vapours are hazardous
• some plants can cause allergies e.g. chrysanthemums
• could use the same technique to investigate the pigments in different coloured leaves - follow same protocol just change the leaves
• Rf values are always between 0 and 1
• the stationary phase and solvent you use will affect the Rf value - if looking up Rf values need to make sure they were recorded under same conditions as experiment