separation of plant pigments
johnc at CC1.UCA.EDU
Thu Nov 7 11:42:10 EST 1996
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 net.bio.net
From: MARDER at agri.huji.ac.il (Jonathan B. Marder)
Subject: Re: separation of plant pigments
Date: Thu, 07 Nov 96 07:19:38 GMT
In article <961106114200.4408045c at siena.edu>, 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 agri.huji.ac.il | 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 cc1.uca.edu
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