MadSci Network: Botany

Re: How do you identify pigments as they show up on paper chromatography?

Date: Fri Dec 14 19:45:46 2001
Posted By: David Hershey, Faculty, Botany, NA
Area of science: Botany
ID: 1008306138.Bt

Where did you get the idea to use acetone and petroleum ether (1:9) solvent? If 
you have a reference recommending that solvent, it might have some Rf values. 
Rf values will be unique for each solvent. However, the general order of the Rf 
values should be the same because the more nonpolar pigments move farther in 
nonpolar organic solvents.

A recent plant physiology manual (Reiss 1994) identifies six pigments from 
spinach leaves extracted with hexane and chromatographed with petroleum ether-
acetone-chloroform (3:1:1) on silica-gl chromatography. The pigments and their 
Rf's were:

carotene - 0.98
chlorophyll a - 0.59
chlorophyll b - 0.42
pheophytin - 0.81
xanthophyll 1 - 0.28
xanthophyll 2 - 0.15

The color of the bands can be a general guide to identify the pigments. 
Carotene is orange. Chorophylls are green. Chlorophyll a is a blue-green. 
Chlorophyll b is a yellow-green. Xanthophylls are yellow. Phaeophytin is 
chlorophyll lacking the central magnesium ion. Pheophytin is an olive-green. 
Chlorophyll exposed to acid will be converted to pheophytin. Calcium carbonate 
is usually added during the leaf extraction process to prevent chlorophyll from 
degrading to pheophytin. You can differentiate phaeophytin from chlorophyll 
because it has weaker fluorescence in a fluorimeter. The geranium leaf might 
contain an additional carotenoid or xanthophyll.
The two bands visible under UV light would technically not be pigments because 
pigments reflect some visible light and appear colored to the human eye, hence 
the term "chroma" (color) in chromatography. Geranium leaves often contain oils 
so maybe they represent the two nonvisible bands.


Reiss, Carol 1994. Experiments in Plant Physiology. Englewood Cliffs, NJ: 
Prentice Hall.

Measuring Phaeophytin and Chlorophyll by Fluorescence

New Crops Potential of Queensland-grown Geranium (Pelargonium hybrid) for 
Essential Oil

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