Chromatography is a separation process that relies upon the differential distribtuions of a mixture between a mobile liquid phase (the chromatography solvent) and a stationary solid phase (in this case, paper)
Aim
Aim
- To separate and identify the photosynthetic pigments from leaf chloroplasts obtained from intertidal marine seaweed
- We are using chemicals such as anhydrous sodium sulfate (VI), cyclohexane, ethylethanoate and propanone. We should therefore eye protection and do not inhale solvents
- We should also work in a well ventilated room with no naked flames as we are working with flammable chemicals
- Propanone can cause eye irritation so wear necessary eye protection (safety glasses)
- Grind the seaweed with two stones, this breaks the seaweed up and exposes the pigment originally held within the cell
- Add a small spatula measure of anhydrous sodium sulfate (VI) to the bottom of a plastic Eppendorf tube. This is to remove any moisture that may be present
- Place small pieces of a soft green leaf in the bootom of the Eppendorf tube. Add 5 drops of Solvent A. Solvent A consists of 2 parts ethyl ethanoate and 3 parts propanone
- Press and stir the leaf with a metal probe so as to extract as much colour as possible
- Now use a fine brush to place a small dot of coloured solvent about 5mm from the bottom edge of the TLC plate. Mark this position on the side of the plate
- Blow on the dot to dry it before putting another dot of coloured solution on top of it
- Use a dropper pipette to place ~0.5cm3 of solvent B (the developing solvent) into a clean vial so that the depth is about 5mm. The level of the solvent must be below the level of the dot on the chromatography plate
- After a few minutes, when the ‘solvent front’ has moved between 7/8ths of the way up the plate, remove the plate from the vial (use forceps / tweezers) and let it dry. Mark the position of the solvent front.
- Record the results on the results sheet – RF value can be found by doing the Distance travelled by pigment in (mm) / Distance travelled by solvent front (mm)