Extraction and Chromatography of Plant Pigments

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Extraction and Chromatography of Plant Pigments

Aim: To extract the following plant pigments carotenoids and chlorophyll from tomato and spinach samples.

Introduction

Chlorophyll is one of the pigment that can be found in fruits and vegetables which gives plant such as spinach its green colour, carotenoid on the other hand gives the orange / yellow colour of fruit such as tomato. The presence of these two pigments apart from the colour they give plants, fruits and vegetables, they are also very useful for photosynthesis. There are different other types of pigments that adds to the colours of many other plants but chlorophyll a, chlorophyll b and β-Carotene will be mentioned.

 

Experimental Procedure

Spinach

  • Spinach (10g) was weighed and crushed in methanol (25cm3) using a pestle and mortar.
  • The methanolic solution was decanted carefully by pressing the spinach to free the solvent as much as possible; the methanolic solution obtained was discarded.
  • The spinach residue was grinded with a mixture of methanol (10cm3) and petroleum ether (40-60) (15cm3).
  • The mixture was filtered through a filter funnel, which was plugged with a little cotton wool into the separating funnel.
  • The spinach was regrinded with the extract and methanol (19cm3) and petroleum ether (40am3) was also added.
  • The second filtrate was filtered and collected in the separating funnel.
  • The lower methanol phase was collected and discarded.
  • Water (20cm3) was added to the petroleum ether solution, stopper was added on the funnel and was shaken gently.
  • The layer was allowed to separate; the lower aqueous phase was collected and discarded.
  • The washing was repeated using petroleum ether solution in order to remove the entire dissolved methanol.
  • The petroleum ether solution was poured in a conical flask and was then dried by adding anhydrous sodium sulphate and was swirled for five minutes.
  • The solution was filtered into a round-bottomed flask, the residue was washed with several portions of petroleum ether and the solution was concentrated with approximately 5cm3 by careful distillation of the solvent at atmospheric pressure on a steam bath using a condenser.
  • The crude extract was examined by thin layer chromatography.
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Tomato Paste

Tomato paste (10g) was weighed and the experimental procedure carried out was the same procedure as spinach above.

Thin-Layer chromatography

The spinach and tomato extracts was examined on the same cellulose thin-layer plates. A mixture of methanol:acetone:water, 15:15:1 v/v was used to develop the spots. Alumina plates was also used, which was developed by using acetone:petroleum ether, 5:95 v/v. the spots was studied under both the visible and ultraviolet light.


Results

From the two different TLC plates used, the spots observed are as follows:

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