In this article we will discuss about the isolation of pigments and their characterization.
Theory of Plant Pigments:
Plant pigments are mostly the colouring substances found either in cell sap (as water-soluble forms, or in the plastids (water-insoluble forms). Normally, the plastidial pigments are responsible for photosynthesis while cell sap pigments are responsible for coloration of floral parts and various plant organs.
Isolation of cell sap pigments are very simple, which can be done by boiling water or by alcohol. On the other hand, plastidial pigments are isolated by solvent extraction method using acetone, petroleum ether, methanol, chloroform, alcohol etc.
Depending on the variation of solubility of different plastidial pigments in different solvent systems, they can easily be separated by separating funnel or by chromatographic techniques.
(A) Extractions, Separation and Characterisation of Plastidial Pigments:
Materials and Equipment’s Required:
1. Leaves of Cucurbita sp. or spinach or Eichhornia sp.
2. Mortar and pestle, separating funnel (Fig. 3.31), beaker, linen cloth, cotton
3. Acetone, alcohol methanol, petroleum ether, KOH, chloroform, etc.
4. Stand, test tubes, filter papers, chalk, petridishes, scissors, forceps etc.
5. Spectroscope, solutions of Cu acetate, Zn acetate, etc.
Procedure:
1. Chop the leaves into small pieces and crush them in mortar in presence of acetone and 95% alcohol mixture (15: 85 ml).
2. Strain the extract of pigment through a linen to remove the leaf debris.
3. Use this acetone extract of pigment for separation of pigments by solvent extract method or by chromatography.
Method I: Solvent Extraction Method:
1. Take about 100 ml acetone extract of plastidial pigments in a separating funnel (500 ml capacity) and add petroleum ether (about 80-100 ml).
2. Add a few ml of dist. water in the mixture and then shake thoroughly and wait till the acetone water layer settles down.
3. Wash pigments layer twice by the addition of dist. water and discard the aqueous layers.
4. Add a few ml of methanolic KOH soln. and then shake thoroughly and wait for the separation of the layers — upper ethereal layer containing chlorophyll-a and traces of carotene and lower methanol layer containing chlorophyll-b and traces of xanthophyll.
5. Draw the two layers separately for further characterization.
Method II: Solvent Extraction Method:
1. Take about 100 ml of acetone extract of pigment and then add about 100 ml of solvent ether and a few ml of distilled water. Mix the content and shake in a separating funnel.
2. Discard the lower aqueous acetone layer and then add KOH soln. in excess to esterify the chlorophylls.
3. Discard the esterified pigments and collect only yellow pigments from the ethereal layer, which is further separated by the addition of methanol: Upper layer carotene and lower layer xanthophyll.
4. Collect both the layers separately for further characterization.
Method III: Separation by Chromatography:
1. Take a small quantity of the crude extract of pigments in a petridish.
2. Place a chalk (white) on it and allow it to soak the pigment for 30-40 minutes. Dry the chalk and record the vertical movement of pigments due to solvent movement.
3. Crude pigments extract, on the contrary, may be spotted on chromatography paper or white filter paper and then the solvents are allowed to pass through it. Pigments will separate on the paper depending on the differential rate of solvent mediated movement of pigments.
Characterization of Chlorophyll:
(a) Fluorescence:
It is an important optical property of chlorophylls.
Procedure:
Place a few drops/ml of chlorophyll extract in a test tube and expose to sunlight for a few minutes.
Observation:
1. Extract of pigments showing reddish fluorescence indicating the presence of chlorophylls.
(b) Phaeophytin Formation:
Procedure:
Treatment of chlorophyll extract with dilute acids results in the formation of a brown-coloured solution called phaeophytin in which magnesium is absent.
Observation:
However, the position of magnesium may be substituted by addition of a few drops of Ca-acetate, Zn-acetate or Mg-acetate solution, as indicated by appearance of partial greenish coloration of the treated extract.
(c) Spectroscopic Properties:
Chlorophyll can absorb a range of light as revealed by spectroscopic analysis.
Procedure:
Take a small quantity of chlorophyll extract and place it in between a light source and spectroscope.
Observation:
On visual examination, dark bands are observed in the red and blue violet regions of the spectrum indicating that chlorophyll can absorb light in between ± 400 nm to ± 600 nm.
However, detailed steps in the separation of plastidial pigments are tabulated:
(B) Extraction of Water-Soluble Anthocyanin Pigments and its Characterization:
Materials and Equipment’s Required:
1. Flower petals of different colours
2. Test tubes, ethyl alcohol, amyl alcohol, water, HCl (dilute), NaOH soln., etc.
3. Beaker, clamp etc.
Procedure:
1. Prepare an extract of coloured petals of flower either in boiled water or in alcohol and then filter the extract.
2. Perform the following tests for characterization of extract:
Test I:
To a small quantity of the extract add a few drops of dil. acid (HCl or H2SO4), the solution turns red, indicating the pH sensitivity of extract.
Test II:
Similarly, to a small quantity of extract, add a few drops of NaOH soln, the solution turns bluish indicating the pH sensitivity of extract.
Test III:
To a small quantity of watery extract add a few ml of lead acetate. The solution turns bluish-green or green ppt. appears.