It sounds like you have many other flavonoids in addition to your anthocyanins. I suggest
that you get an acidified methanolic extract, then run TLC two ways: first in BAW (tertiary
butanol: acetic acid: water at 3:1:1, then in the acetic acid. Methods are described well
in Markham's book: Techniques of flavonoid identification (1982) Academic Press.
***************
To: plant-ed at net.bio.net
From: RADLICK at siena.edu
Subject: separation of plant pigments
Date: 6 Nov 1996 08:42:38 -0800
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 UV.
I think the red one (looks green with visible light) is chlorophyl. Could the
other be pheophytin or carotene? The ones at the solvent front stack one under
the other and are blue (under UV; invisible under fluorescent), dark violet or
magenta (under UV or vis, respectively), and light lavendar (under UV and vis).
The ones that are visible under fluorescent light are, I believe, anthocyanins.
However, I don't know what the blue one is. Any ideas?
The anl/anl pattern shows the same bands at the origin and the same blue band
at the solvent front that is present in the anthocyanin expressing stock. The
anl/anl pattern also show two additional bands at the solvent front - one
yellow and the other blue. I don't know what these represent. Any ideas?
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 the
top. I would appreciate any suggestions.
I would also like to identify these pigments, if possible. Also, I would
appreciate suggestions for standards to use along with the test samples.
Thanks, in advance, for any help!
Lynn Radlick
Biology Department
Siena College
Loudonville, NY 12211
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Kevin S.Gould
School of Biological Sciences
University of Auckland
ph: 64-9-3737599 ext 7298
fax: 64-9-3737416
email: k.gould at auckland.ac.nz
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
