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Thin layer chromatography for plant pigments
(from Osmosis 8, Autumn 1995)
 

Read these instructions carefully before you start. Work quickly to get the best extracts.

WARNING: The solvents used in this investigation are flammable.

Instructions

1. Cut a TLC plate into small stips e.g.1.25 x 6.7 cm, so that they fit your tubes. Do not touch the surface of the plates.

2. Place 2 - 3 wheat or grass leaves on a slide. Use a second slide to scrape the juice out.

3. Add 6 drops of propanone to the green mush and mix. Transfer the dark green liquid to a small watch glass.


4. Repeat steps 2 and 3 until you have about 20 drops of extract. Use a hair dryer to remove all the water from the extract.

5. When the extract is completely dry, add 3 - 4 drops of propanone and mix the extract with a fine paint brush.

6. Use the brush to transfer tiny drops of extract to the TLC strip. Keep the spot diameter to less than 2 mm. Dry the spot thoroughly between each addition. Repeat until the spot is a very dark green.


7. Slot the TLC strip into a slit in the cork and put it into an empty tube. Mark the tube below the level of the spot. Remove the TLC strip.

8. Add running solvent to the mark. Put the TLC strip back in the tube. Make sure that it does not touch the sides of the tube. Watch the chromatogram develop.

9. After about 4 minutes remove the strip and immediately use a pencil to mark the solvent front.


10. Measure the distance run by the solvent front and by each of the pigments. All measurements should be made from the centre of the original spot to the front of each pigment spot.

11. Calculate how far the pigment has gone relative to the solvent front. This is the Rf value. (Rf = the distance run by the pigment divided by the distance run by the solvent.)

12. Draw a suitable results table. For each pigment record: the distance run, its colour, Rf value and possible identity.

Questions
  • Why is it important not to let your fingers touch the TLC plate (e.g. in Step 1)?

  • Why will you probably get a better extract if you work fast (Steps 2 and 3)?

  • Why is it important to get rid of all the water in Step 3?

  • Study the absorption spectra for various plant pigments (in most biology texts). Which pigments absorb most light from the red end of the spectrum? What colour are they?

  • If chlorophyll is the most important photosynthetic pigment, make a hypothesis to suggest which colours of light are most useful to a plant for photosynthesis?

  • Light at the blue end of the spectrum penetrates most easily underwater. Why are seaweeds often yellow-brown?

  • Name some plants whose leaves are not green. How could you find out what pigments they contained?

Further work
  • For a given plant, compare thin layer chromatograms with paper chromatograms of the same pigments. What are the advantages and the disadvantages of each method?

  • Compare leaf extracts from different plants.

  • You may isolate the different pigments by scraping each of them off the chromatogram, with a razor blade. into a different small tube. Re-dissolve the pigments in a very small volume of running solvent. These pure pigment solutions may be viewed with a spectroscope to confirm the absorption of the different light wavelengths shown in absorption spectra graphs.

Acknowledgements Stephen Tomkins, Homerton College, Cambridge, and Barry Miller, Douglas Ewart High School, Newton Stewart.

Notes for teachers

Apparatus:

  • 2 or 3 young wheat plants (grown for 2 weeks under a light bank) or grass.
  • 2 glass microscope slides
  • 1 glass teat pipette
  • 10 cm3 propanone
  • 1 small watch glass or translucent Fuji film can lid
  • 1 electric hair drier
  • 1 very fine paintbrush
  • 1 or more TLC chromatography strips*, (1.25 cm x 6.7 cm)
  • 1 glass specimen tube, (2 cm x 7.5 cm)
  • 1 cork to fit the tube, with a horizontal V-slit
  • 7 cm3 chromatography running solvent per tube**
  • 1 marker pen

*TLC chromatography strips available from: Camlab Limited, Nuffield Road, Cambridge, CB4 1TH England. Tel: 01223 424222 Fax: 01223 420856

Order: Precoated plastic sheets Polygram SIL G 5 x 20 cm MN/805 012

**Running solvent
  • 5 parts of cyclohexane
  • 3 parts of propanone
  • 2 parts of petroleum ether (40 - 60°C)

Different running solvents often produce different Rf values.

For this running solvent typical Rf values are as follows:

  • Chlorophyll a 0.60
  • Chlorophyll b 0.50
  • Carotene 0.95
  • Xanthophyll 0.35
  • Phaeophytin 0.80 (a breakdown product)

Reference

Tomkins, S. P. and Miller, M. B. (1994), A rapid extraction and fast separation of leaf pigments using thin layer chromatography. School Science review75 (273), 69 - 72.

A SAPS SCOTLAND VERSION OF THIS PROTOCOL IS AVAILABLE FROM THE SAPS SCOTLAND OFFICE IF REQUIRED.

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