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1. Thin Layer Chromatography

1. Thin Layer Chromatography - Thin Layer Chromatography...

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Thin Layer Chromatography Experiment 1 Introduction The aim of this experiment was to examine the components of an extract of spinach and to observe how they behaved in three different solvents. Method A small handful of spinach leaves was taken (4-5 leaves) and placed in a 100cm 3 beaker. A mixture of chloroform (4cm 3 ) (TOXIC) and methanol (12cm 3 ) (TOXIC, FLAMMABLE) was then thoroughly mixed in a fume cupboard and added to the spinach leaves. The suspension was then stirred with a glass rod for 8 minutes whilst warming GENTLY on a steam bath in a fume cupboard. To ensure the suspension did not boil it was necessary to remove it from the heat occasionally to allow the temperature to decrease a little. After 8 minutes the dark green extract was removed from the paler leaves and decanted into a 25cm 3 conical flask. A portion of this extract (approx. 1cm 3 ) was put onto a watch glass and blown upon (in a fume cupboard) in order to evaporate the organic solvent. When the evaporation was complete, the bottom of the watch glass no longer felt cold on continued blowing. The watch glass then had a green sludge suspended in an almost colourless liquid remaining. At this point chloroform (2cm 3 ) was added and the green sludge dissolved completely. A Pasteur pipette was used to remove the outer, dark green chloroform layer to a small test tube. This extract in the small test tube was used to make three thin layer chromatography plates. The first of the three solvents was prepared by thoroughly mixing ethyl acetate (3cm 3 ) (FLAMMABLE) and petroleum ether (b.p. 40-60°C) (7cm 3 ) (FLAMMABLE, TOXIC). A piece of saturation paper was inserted into a solvent bottle and pushed flat against the sides, and then the solvent mixture was poured in. On one of the chromatoplates a small pencil circle was drawn (2mm diameter) approximately 1cm from one end and equidistant from both sides. A concentrated spot of the chloroform extract was made in the circle following the method described in the script. Using tweezers so that skin oils did not appear on the plate, the chromatoplate was placed carefully into the solvent bottle with the extract spot at the bottom. The extract spot was correctly positioned above the solvent level. The plate was observed carefully so that when the solvent front had risen three-quarters of the length of the plate, the plate was removed from the bottle with tweezers. Using a pencil the solvent front was quickly marked on the plate before it evaporated. Then the plate was placed in a fume cupboard and the solvent fully evaporated off. The appearance and measurements of the coloured spots on the plate was recorded immediately. The above application and development was then repeated using exactly the same method but with two new solvent mixtures, namely, ethyl acetate (2cm3) (FLAMMABLE) and petroleum ether (b.p. 40-60°C) (8cm3) (FLAMMABLE, TOXIC) and ethyl acetate(1cm3) (FLAMMABLE) and petroleum ether (b.p. 40-60°C) (9cm3) (FLAMMABLE, TOXIC). Between each test the solvent bottles were thoroughly rinsed with acetone and distilled water and a new saturation pad was inserted. Fresh applicators
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