CHROMATOGRAPHY OF PLANT PIGMENTS
Separating impure mixtures into pure substances is among the most important and fundamental of skills which must be learned by a chemist. The study of pure substances requires that the chemist learn many different techniques of separation so as to pursue the study of properties of pure substances and their reactions.
It was Alexander Tswett, a Russian botanist, who first found that he could extract pigments from plants. This extraction was done by grinding the leaves in an organic solvent. He then poured the solvent extract containing the mixture of dissolved pigments through a tube full of powdered chalk (CaCO3, or calcium carbonate). The various pigments separated into colored bands as the extract penetrated down through the powdered chalk. He carefully removed the column of chalk from the tube and separated the colored bands. Extraction of the different colored bands with the solvent produced solutions of separated pigments for study.
Many common materials around us are made up of mixtures of compounds. Examples are: gasoline, ink, cosmetics, dyes, and pigments. It is often very difficult to separate these mixtures, especially if the components are chemically very similar. One way to do this takes advantage of the slight differences in the components' solubilities, which are due to slight differences in their chemical structure. This separation method is called thin layer, or paper, chromatography.
Paper chromatography is a method used to separate very small quantities of substances from each other. The identity of these separated substances can also be determined. In paper chromatography, a small sample of a mixture is placed on porous paper which is in contact with the solvent. The solvent moves through the paper due to capillary action and dissolves the mixture spot. The components of the sample start to move along the paper at the same rate as the solvent. Components of the mixture with a stronger attraction to the paper than to the solvent will move more slowly that the components with a strong attraction to the solvent. The differences in the rates with which the components travel along the paper lead to their separation. Particular mixtures will have chromatographic patterns that are consistent and reproducible as long as the paper, solvent, and time are constant. This makes paper chromatography a qualitative method for identifying some of the components in a mixture. Different solvents will change the patterns of component separation. Colored spots on the finished and dried chromatogram can be cut out of the paper with scissors and redissolved to obtain a pure sample of one component of the mixture.
Quantitative analysis of chromatographic data includes the calculation of retention factors, or retardation factors (Rf). This gives the quantitative measure of a component’s properties in a mixture. The Rf value is calculated as
Rf = Distance traveled by solute/Distance traveled by solvent
This applies when chromatograms are made with the same solvent and developed for the same amount of time, Rf values are identical
1. To separate pigments from the leaves of spinach;
2. To identify the different pigments present in spinach leaves; 3. To compute the approximate rates of flow (Rf values) of each of the component pigments of the leaves.
MATERIALS AND APPARATUS
Whatman #1 filter paper
(pre-cut into a cylindrical shape to fit a 400 ml beaker without touching the sides of the glassware)
ordinary filter paper
stapler or scotch tape
spinach leaves (or watercress or any leafy vegetable that is in season) mortar & pestle
A. Preparing the crude chlorophyll extract
1. Obtain about 15 mature leaves of...