Methodology for Pigments


Carotenoids, the tetraterpenoid (C40) compounds, are ubiquitous in plants. These terpenoids existing as hydrocarbons (carotenes) or oxygenated derivatives, are accessory pigments in photosynthetic systems and give characteristic color to plant parts, particularly flowers and fruits. Carotenes occurring in different chemical forms have characteristic features and functions. Their levels are altered during physiological and pathological conditions. A method to extract total carotenoids from fresh plant materials, and to separate most of the components from each other is given below.


The total carotenoids are extracted and partitioned in organic solvents on the basis of their solubility. The separation of individual components is effected by chromatography on activated magnesia.

» Acetone-dry, alcohol-free. To dry treat with anhydrous sodium sulphate and distil over granular zinc (10 mesh).
» Hexane-distilled over KOH
» Activated Magnesia
» Diatomaceous Earth - Hyflo Supercel
» MgCO3

Finely cut material with scissors/knife or grind in food chopper to assure representative sample. (If analysis cannot be performed immediately, blanch in boiling water for 5-10 min and store frozen.)
Place 2-5g weighed sample in a high-speed blender. Add 40mL acetone, 60mL hexane and 0.1g MgCO3. Blend for 5 min.
Filter with suction or let residue settle and decant into a separator.
Wash the residue twice with 25mL portions acetone, then with 25mL hexane and combine the extracts.
Wash acetone from extract with five 100mL portions H2O, transfer upper layer to 100mL volumetric flask containing 9mL acetone, and dilute to volume with hexane.
Pack activated magnesia-diatomaceous earth (1:1) mixture in a chromatographic tube (17.5 X 2.2cm OD). To prepare the column, place small glass wool or cotton plug inside the tube, add loose adsorbent to 15cm depth, attach tube to suction flask and apply full vacuum of water pump. The packed column should be about 10cm
high. Place lcm layer of anhydrous sodium sulphate above adsorbent.
Pour the extract into the column with vacuum continuously applied to the flask.
Use 50mL acetone-hexane (1: 9) mixture to develop the chromatogram and wash visible carotenes through adsorbent. Keep the top of the column covered with solvent during entire operation.
Collect the entire eluate. (Carotenes pass rapidly through the column; bands of xanthophylls, carotene oxidation products and chlorophylls are retained.)
Transfer the eluate, which has been reduced in volume by the loss of vapor through water-pump, to a 100mL volumetric flask, dilute to volume with the acetone-hexane mixture.
Determine the absorbance of the solution as soon as possible with a spectrophotometer at 436nm. Calibrate the instrument first with solutions of different concentrations of high purity b-carotene.
Prepare the calibration curve and calculate the carotene content (mg/100g) in the sample material using the curve.


Alcohol may be used instead of acetone for extraction. Use 80mL alcohol and 60mL hexane in blender; other volumes are same as for acetone extraction.
The extract volume will get reduced due to evaporation at room temperature. Maintain the volume at appropriate stages.
Carotenoids that are bound as esters are not estimated in this procedure. A portion of the extract may be hydrolyzed using methanolic KOH in a water bath for 5-10 min and then subjected to chromatography.