You have carried out an investigation into the pigments that make up chlorophyll. Use the chromatogram you obtained when carrying out this investigation. Clearly explain the separation technique of paper chromatography in terms of partition. Calculate the Rf value of one of the constituent pigments and compare it to the book value. A paper chromatography was used to separate the pigments that make up chlorophyll to analyse, identify, and quantify the components, using paper (stationary phase – the part of the apparatus that does not move with the sample) and chromatography solvent that was 90% petroleum ether and 10% propanol (mobile phase – liquid that carries the components). A light pencil line is drawn across the strip of paper (about 2cm from one end) and the other is attached to the splint, making sure that the paper doesn’t touch the bottom of the beaker. The leaf is placed into boiling water for a minute and then mortared with a pinch of sand and a few drops of propanone to allow the extraction of the chlorophyll solution with a capillary tube. Two small dots of chlorophyll solution are placed on the pencil line on the chromatography paper. After the dots were dry a second dot was placed on top of the first couple, this process was repeated five times. A small amount of solvent was then placed into the bottom of the beaker, the paper suspended in the container, the solvent level below the pencil line; therefore the compound was placed on the stationary phase. The beaker was covered to the solvent wouldn’t evaporate. The solvent was left to pass through the paper for some time and then removed from the beaker and the distance travelled was marked and recorded (6.6cm). That point is the solvent front. The mobile phase dissolved the components and carried the individual pigments though certain distances, that distance was different to each pigment and the mixture was separated into different coloured spots. Some components in the mixture travel almost as far as the solvent did, staying really close to the solvent front, some barely moved away from the pencil line. The pigments were identified by their colour and by their Rf values, which is the distance travelled by the pigments relative to the distance travelled by the solvent: Rf = Distance moved by the substance form original position
Distance moved by solvent from the same position
There were four colours noticed and their Rf values were taken. It was observed that depending on the attraction of the individual components of chlorophyll the individual components in the chlorophyll were carried though different distances through the stationary phase. The structure of the mobile, stationary and the pigments can explain the separation: The solvent is a non-polar substance and the paper, is made of cellulose fibres that have OH groups, it is polar and can form hydrogen bonds and Van-der-walls forces. So the cellulose fibres attract water vapour from the surroundings and that interaction of the paper with the water is very important during our paper chromatography, as there is a tendency called partition, which is the tendency for a compound to divide its time between two solvents that won’t mix like water and the petroleum ether, becoming partitioned, divided. This chromatography is therefore a type of partition chromatography. In chlorophyll the differential affinities of the components for the solvent and for the water attached to the paper as they pass allowed the separation of the colours. This difference in affinity comes from the intermolecular forces acting between the chlorophyll and the stationary and mobile phases, as when one substance dissolves another, the attractive forces in both must be overcome: the dissolving solute has to be able to separate the molecules in the solvent. Therefore the highest the affinity for the stationary phase there is, the more soluble in water the pigment will be and as a result the less movement...
Please join StudyMode to read the full document