required practical 7
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what is the aim of the practical?
use of chromatography to investigate the pigments isolated from leaves of different plants
what is the method for the practical?
• draw a straight pencil line approx 1cm from the bottom of the filter paper being used • cut section of leaf and place in mortar • add 20 drops of acetone and use pestle to grind up leaf and release pigments • extract some of pigment using capillary tube and blot onto centre of pencil line • suspend paper in solvent so level of solvent is below pencil line, and leave paper until pigments have separated and solvent is near but not at the top • remove paper from solvent and draw pencil line to mark solvent front and • calculate Rf value for each spot, measuring from the centre (distance travelled by pigment/distance travelled by solvent)
why should the line be drawn in pencil and not pen?
with pen the ink will separate into pigments and obscure the results
what type of leaf should be used?
one which is healthy and has been exposed to direct sunlight so it contains many active photosystems
why is acetone used?
it is an organic solvent so is dissolves fats eg. those in cell membranes - combined with the mechanical pressure of the pestle, it breaks down the cell, chloroplast and thylakoid membranes so the pigments are released
what does the calculated Rf value measure?
the distance moved by each pigment in the stationary phase in relation to the distance moved by the solvent
what does it mean if a pigment has a higher Rf value?
it has a higher affinity in the liquid mobile phase eg. because it is non-polar, highly soluble in the solvent and small
what does it mean if a pigment has a lower Rf value?
it has a higher affinity with the solid stationary phase eg. because it is polar, less soluble in the solvent and large
how can Rf values be used to identify pigments?
compare them to known Rf values in a database for paper chromatography and that specific solvent