Chromatography of Biomolecules

As a simple demonstration of the chromatographic separation of plant pigments I followed this procedure:

  1. a leaf sample (borrowed from one of my jalapeno plants) was ground up with a small volume of acetone to create the crude extract.IMG-20150822-00090IMG-20150822-00089
  2. As much of the liquid as possible was transferred to an eppendorf tube and then spun down at high speed (13k rpm) in a microfuge to sediment out any particulates.
  3. The supernatant was then transferred to a plastic test tube and placed on a heating block at approximately 70degC to evaporate the acetone. (this step took several hours to complete)IMG-20150816-00077Havering-20150822-00095
  4. The solids remaining in the tube were then resuspended in hexane.
  5. Meanwhile, a separation column was created using a 10ml BD syringe, a small plug of cotton wool and a couple of grams of aluminium oxide. (This is the stationary phase of the column.)IMG-20150712-00067
  6. The column is first wetted with a few mLs of hexane. The column should be saturated in hexane before application of the sample.
  7. The sample from step 4 was then added to the top of the column with a pasteur pipette and a small amount of pressure applied using the plunger of the syringe.
  8. Initially clear hexane flows through the column followed by a distinctive yellow fraction (most probably carotenoid pigments), which was caught in another test tube. [While this occurs the green chlorophyll pigments have a higher affinity for the aluminium oxide solid phase than the hexane mobile phase and remain bound to the matrix of the column.]Havering-20150829-00104
  9. Once all of the yellow phase has been removed a few mLs of acetone were then added to the top of the column. After a small amount of clear mobile phase left the column it was followed by a distinctive green phase. [this constitutes the chlorophylls which have a higher affinity to the acetone mobile phase than the stationary aluminium oxide phase and so are eluted from the column]Havering-20150829-00107Havering-20150829-00110

The end result is the separation of these pigments from a single sample, and to demonstrate some basic principles of chromatography.

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Much nicer photo of the separated pigments taken by tolland. Right hand red one is an acetone extract from red pepper.

What’s next? : Analysis of other pigmented things. Chromatographic separation of other biomolecules. How about these things? :IMG-20150428-00036

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