RP 7 - Chromatography of Photosynthetic Pigments

• Chromatography is used to separate out different components in a sample.
• In this experiment, the photosynthetic pigments of a plant sample are separated into bands of colour by paper chromatography.
• The rates of migration of individual pigments will depend on their solubility, mass and affinity to the paper.
• Equipment for the experiment includes filter paper, leaf sample, distilled water, pestle and mortar, pencil, ruler, capillary tube, chromatography solvent, acetone.
• To set up the experiment, draw a straight line in pencil approximately 1cm above the bottom of the filter paper being used.
• Cut a section of leaf and place it in a mortar, add 20 drops of acetone and use the pestle to grind up the leaf sample and release the pigments.
• Use a capillary tube to extract some of the pigment and blot it onto the centre of the pencil line you have drawn.
• Suspend the paper in the solvent so that the level of the liquid does not lie above the pencil line and leave the paper until the solvent has run up the paper to near the top.
• Remove the paper from the solvent and draw a pencil line marking where the solvent moved up to.
• Calculate the Rf value for each spot (distance travelled by solute/distance travelled by solvent).
• Risk Assessment: Biohazard, Allergies; soil bacteria; contamination.
• Wash hands after use.
• Seek assistance in emergency.
• Low risk for chromatography solvent, flammable; causes irritation to eyes and skin; harmful by inhalation.
• Avoid contact with solvent; wear eye protection; keep solvent in fume cupboard; make sure room is well ventilated; keep away from naked flame.
• Wash from skin and eyes using cold water; put out fires; seek medical assistance in emergency.
• Risk for acetone, flammable; causes irritation to eyes; inhalation may lead to dizziness and drowsiness.
• Avoid contact; wear eye protection; make sure room is well ventilated; keep away from naked flame.
• Rf values should be compared to the Rf known values in a database to identify pigment.
• Affinity and solubility are factors that affect the rate of mobility.
• Pigments with lower affinities will travel further up the paper and have a higher Rf value.
• Pigments that are more soluble travel faster up the paper and will end up closer to the top at the solvent front.