Upper secondary · 20 min

Chromatography of leaf pigments

Separate green-leaf pigments as coloured bands and compare their migration with the solvent front.

Goal

Observe how polarity and differential adsorption/solubility separate a mixture; calculate a retention factor for a paper or thin-layer chromatogram.

Apparatus and reagents

Spinach or other green leaves, mortar, sand, acetone or ethanol for extraction, chromatography paper or silica plate, suitable non-polar mobile phase, capillary, pencil and ruler.

Procedure

Extract leaf pigments, draw a pencil origin line and place a small concentrated spot on the paper or plate.
Place the strip in a closed developing chamber with the origin above the solvent level; observe the bands separate as the solvent rises.
Mark the solvent front before it evaporates; measure the distance from the origin to each band centre and to the solvent front.
Calculate Rf = distance travelled by pigment / distance travelled by solvent; identify the fastest and slowest-moving pigments.

What to observe

The coloured mixture separates into orange, yellow and green bands. In a typical normal-phase leaf-pigment separation, non-polar β-carotene travels farthest; more polar chlorophylls are retained more strongly.
Rf is dimensionless and depends on the stationary phase, solvent composition, temperature and chamber saturation; compare values only under the same conditions.

Explanation

Partition between the stationary phase (cellulose/silica) and the moving solvent sorts pigments by polarity: non-polar β-carotene travels farthest, then xanthophylls, chlorophyll a and chlorophyll b. Rf = (distance to band centre)/(distance to solvent front) is dimensionless and is only comparable under identical conditions.

History of the experiment

In 1906 the botanist Mikhail Tswett passed a leaf extract through a column of chalk and watched green and yellow bands separate — 'chromatography', literally 'colour writing'. His method was ignored for three decades until Kuhn and Lederer revived it in the 1930s to isolate carotenes, and Martin and Synge's partition theory (1941) turned it into the analytical backbone it is today.