separation of plant pigments

John Choinski johnc at CC1.UCA.EDU
Thu Nov 7 11:42:10 EST 1996

Fellow Netters

Regarding the partitioning of water soluble and lipo-soluble pigments 
from plants, I have found the following method works well:

Homogenize the tissue in 1% HCl in methanol and then add chloroform 
and water in the ratio  5ml methanol extract: 4ml chloroform: 3 ml 
water. Shake well and centrifuge, the upper layer will be 
anthocyanins and other flavonoids, the lower layer chlorophyll, etc. 
This method is cited in Tree Physiology 13: 17-27 (1993).
To:            plant-ed at
From:          MARDER at (Jonathan B. Marder)
Subject:       Re: separation of plant pigments
Date:          Thu, 07 Nov 96 07:19:38 GMT

In article <961106114200.4408045c at>, RADLICK at SIENA.EDU wrote:
>I am preparing an undergraduate lab on the separation of plant pigments from
>rapid cycling Brassica rapa (RCBr) seedlings of the following stocks - highly
>expressed anthocyanin (ANL/ANL), anthocyaninless (anl/anl), and F1 (ANL/anl)
>and have done some preliminary work extracting and separating the pigments. 
>I've extracted pigments from 3 day old seedlings by homogenization in 1% HCl 
>in 95% ethanol and run them on silica gel TLC using 10% acetic acid in water. 
>With the ANL/ANL seedlings, I get two bands at the origin and at least three 
>near the solvent front.  The ones at the origin appear orange and red under 
>I think the red one (looks green with visible light) is chlorophyl.  Could 
>other be pheophytin or carotene?  

Definitely not carotene - it wouldn't fluoresce. Also, pheophytin A 
fluorescence is very close to chlorophyll A (should be the same colour), 
whereas chlorophyll B fluorescence mught be a bit more orange.

>I would like to use a solvent system to better resolve these pigments.  The
>hydrophobic ones don't  move at all;  the hydrophilic ones all stack up at 
>top.  I would appreciate any suggestions.

You could try and partition the hydrophobic ones into hexane (or petroleum 
ether) and run them in a different system. We usually take the pigments in 
~66% acetone and shake with equal volume of hexane. This takes chlorophyll 
(but not chlorophyllide) and the carotenoids into the upper hexane phase.
The latter can be easily concentrated by evaporation and is easy to spot on 
TLC.  You could probably get a similar result with 60-70% ethanol and hexane, 
though you may have to experiment a bit.

>I would also like to identify these pigments, if possible.  Also, I would
>appreciate suggestions for standards to use along with the test samples.
By far the best way is to scrape the bands from the TLC, dissolve in solvent 
(80% acetone for chl. and carotenoids is fine) and run spectra in a 

Jonathan B. Marder             ,      Department of Agricultural Botany
E-mail: MARDER at |      The Hebrew University of Jerusalem
Phone: (08 or +9728) 9481918   | /\/  Faculty of Agriculture
Fax:   (08 or +9728) 9467763   |/  \  P.O.Box 12, Rehovot 76100, ISRAEL

Dr. John S. Choinski, Jr.
Professor of Biology
Dept. of Biology
University of Central Arkansas
Conway, AR 72035

johnc at

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