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Isolation of Plant Pigments by Column Chromatography



To introduce the students to column chromatography and  apply it for  separation  of  compounds in  mixture.




Very few compounds exist pure in nature. They must first be extracted and then purified and if not extractable they need to be synthesized.  Separation involves the application of chemical principle, properties and techniques to separate specific compounds from mixtures. The mixtures to be extracted may be Homogeneous (having same composition all over the volume) or Heterogeneous (differ in composition) depending on the solvent and the solutes.


A plant pigment is any type of coloured compound produced by a plant. A chemical compound which absorbs visible radiation between 380 nm (Violet) and 760 nm (ruby-red) is considered as a pigment. The energy from these radiations is used by plants during photosynthesis for the production of glucose from CO2 and H2O. Different types of plant pigments exist in nature and include various classes of organic compounds (xanthophyll, carotenoids, lycopene etc). Plant pigments give colour to leaves, flowers and fruits and are also important in controlling photosynthesis, growth and development.

Chlorophyll chemically is a complex molecule that can have several modifications among species of plants as well as other photosynthetic organisms. Chlorophyll a, green pigment found in all land plants, few water plants and algae, is the major pigment that captures energy from light (380-760nm) to produce glucose. Additional pigments known as "accessory pigments" include chlorophyll b and carotenoids that capture different wavelengths of light and pass the energy to chlorophyll a for use during photosynthesis.  Accessory pigments also help function as a sunscreen to protect plant cells from sunlight damage. Theories supporting plant origin in sea have been supported by the absorbance of light by these pigments at the range of red-orange (shorter wavelength), which below 5 meters depth do not penetrate so much.  Longer wavelength penetrates more, which is an advantage to the submerged sea algae, supposed to have evolved as land plants.

Carotenoids include a major part of plant derived compounds classified under the class terpenes and are tetrapenes (eight isoprene units).  Some examples of carotenoides include Lycopene, the compound responsible for the red colour of tomatoes and watermelons as well as β-carotene the precursor compound of vitamin A, causes carrot and apricot to be orange in colour. Spinach leaves are known to be a major source of both chlorophylls and carotenoids and also have trace amounts of other pigments too.


Structure of Chlorophyll and β-carotene:


Chlorophyll- a

Chlorophyll- b


The commercial application of β-carotene include; used as natural colour additive in foods and as previously mentioned is the precursor for vitamin A. Chlorophyll isolated could be used in deodarants and leather dyes.




The word 'chromatography', formed from the Greek word 'Khroma' meaning colour and 'graphein' meaning to draw a graph or to write, was coined by the Russian botanist M.S.Tswett around 1906, to describe his process of separating mixtures of plant pigments. The general principle employed in all kind of chromatography is movement of a mixture of compounds over a mobile phase, which passes over selectively adsorbing surface, the stationary phase. Two different techniques have been employed in this experiment to separate and study the components present in spinach leaves.


Column chromatography is devised on the basis of differential adsorbance  of substances on solid adsorbent (silica or alumina) to an extent that depends on the substance polarity  and other chemical properties and structural properties. Some compounds adsorb more strongly to the stationary phase than others, as they elute (wash down) the column at a very slow rate.  Thus they can be separated on the basis of their elution rate accordingly. This technique is used to separate naturally colored components of leaves such as spinach ( Spinacia oleracea).The dark colour of the spinach is a combination of β-carotene and chlorophylls.





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