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Chlorophyll using DMSO - equations

daemon at net.bio.net daemon at net.bio.net
Thu Mar 12 19:58:00 EST 1998

It has been requested that I post answers once I received them.  Here are
the answers, from those that had no reservations about posting them.

Also, since the experiment and question had to do with mosses, some
comment about mosses:

Before going through and doing extractions on all 160 moss tips, I did a
time series on some of the moss tips that weren't in the experiment...
Some fresh, locally collected Fontinalis duriaei, and some from  the
species in the experiment from British Columbia (some in the refridgerator
for about two weeks, others left in a sorting aquarium at room temp).
Readings taken at 40 min to about 20 h; no significant decrease in PQ
occurred, and 12 h appears to be the optimal extraction time, as Geert
Raeymaekers suggested.

Also, for the 160 stem tips done for the experiment itself (after the 12
h), very few of the individual PQs were low enough to indicate any
significant degradation.

It has been suggested to me that

1)  Mosses have cell walls that necissitate greater extraction times than
higher vascular plants, simple algae, and lichens.

2)  Bryophytes have proteins that protect the chlorophyll - apparently
goes into solution with the chlorophyll, keeping it from degrading during

The second comment... there has actually been quite some research (I
recall reading it a few years ago when I was thinking of what to do for
thesis research before going on hiatus) on protective proteins in mosses -
necessary, or at least advantageous for a plant has to extend through long
dry periods without the benefit of a vascular system, heavy cuticle, or
other typical water-loss prevention traits.  The research for this has
mostly been done with desert species, but I'm guessing it extends to other
mosses as well... there is anecdotal evidence in that mosses kept as
herbarium specimens for years can be rewet, still green, and begin
photosynthesizing in a few hours.


=46ollowing are the responses.


X-Sender: tgo2 at postoffice.mail.cornell.edu
Date: Wed, 04 Mar 1998 15:42:33 -0500
To: wjboelem at mtu.edu (Bill Boelema)
=46rom: "Thomas G. Owens" <tgo2 at cornell.edu>
Subject: Re: Chlorophyll using DMSO - equations

I believe that Porra and coworkers (mid-late 80's) did a systematic
evaluation of chlorophyll extraction and measurements in several solvents
including DMSO.  If you don't know the extinction coefficient for
pheophytin in your particular solvent system, you can determine it
empriically - take soem pure chlorophyll a (scrape if off a TLC plate
N2 and measure its absorption spectrum, add a small amount of acid (<1%
volume) and remeasure the spectrum.  The acid will convert the chl to pheo
and you can calculate the extinction at any wavelength.  If you wish to
quantify both chl and pheo in samples, it is best to use whole spectra (or
at least multiple wavelengths), or better yet use an HPLC.

Tom Owens

At 10:43 AM 3/4/98 -0500, you wrote:
[my original post snipped]
Thomas G. Owens
Section of Plant Biology
Cornell University
Ithaca, NY  14853-5908

607 255-8516 phone
607 255-5407 fax


Date: Thu, 05 Mar 1998 09:55:13 +0100
=46rom: Anders R=CAbild <Anders.Raebild at botge.kvl.dk>
To: wjboelem at mtu.edu
Subject: Chlorophyll extraction with DMSO
Content-Disposition: inline

A recent publication giving equations for DMSO and other solvents using
different spectrophotometers is:

 Wellburn AR (1994) The spectral determination of chlorophylls a and b, as
well as total carotenoids, using various solvents with spectrophotometers of
different resolution. J Plant Physiol 144: 307-313

Hope you find it useful

Best regards

Anders Raebild
Arboretum, KVL
Kirkegaardsvej 3A
Dk-2970 Hoersholm

Phone: +45 35 28 36 35
=46ax: +45 35 28 36 29
E-mail: anr at kvl.dk


Date: Thu, 05 Mar 1998 14:33:48 -0500
=46rom: David Kirschtel <dkirscht at zoo.uvm.edu>
To: Bill Boelema <wjboelem at MTU.EDU>
Subject: Re: Chlorophyll using DMSO - equations

Bill -

When I've done chl extraction from algae with DMSO, we've mixed with
acetone and then used the acetone fraction for analysis (if my memory
serves me correctly). As far as I remember this allowed us to use the
acetone equation for quantifying chla. The neat thing about using
DMSO for us was that it allowed us to coextract ATP. (see refs below)

As for degradation of chl to phaeo. Again, with algae, the bulk of the
degradation occurs after extraction . If the extract is dense enough
you can sit there and watch it happen, changing from a limey-green to

Most of the people I know think I'm excessively obsessive about
protecting chl but here are my rules of thumb:

	1 always extract the samples in the dark
		use a light proof box or container during extract.
	2 when doing the analysis keep the room as dark as possible
 	   -generally I use a 15W yellow "bug" light at the
	   opposite corner of the room from the spectrophotometer
	   -if this isnt possible absolutely no flourescent lights
	3 keep things as cool as possible but be careful DMSO
	  freezes at a relatively high temp. If you wind up going
	  over to acetone then keep the samples on ice

Also during the extraction process I'll add MgCO(3) as a saturated
sol'n to force the equlibrium back to chl.(APHA ref)

- - - - - - - - -


Palumbo, Mulholland and Elwood. 1987. Extraction with DMSO to
simultaeously measure photosynthesis, chl and ATP. Limnol &
Oceanogr. 32:464-471

Shoaf and Lium. 1976. Improved extraction of chl a and b from algae
using DMSO. Limnol and Oceanogr. 21:926-928

APHA. Standard Methods for the analysis of water and wastewater.

Hope this is helpful

David Kirschtel               |
Dept. of Botany & Ag. Biochem.|    Nature didn't know we were coming
Marsh Life Sci Bldg           |    and, quite frankly, doesn't give
Univ. of Vermont              |         a damn that we're here.
Burlington, VT 05405-0086     |
tel:  802.656.0429            |      - Stephen J. Gould,
email: dkirscht at zoo.uvm.edu   |        Darwin's Revolution in Thought:
http://www.uvm.edu/~dkirscht  |        an an illustrated lecture


Date: Thu, 05 Mar 1998 14:15:42 -0700
=46rom: Barry Pogson <pogson at asu.edu>
Subject: Re: Chlorophyll using DMSO - equations
To: Bill Boelema <wjboelem at mtu.edu>
Reply-to: pogson at asu.edu
Organization: Arizona State University

Dear Bill,
I suggest you check out Porra et al.  This paper is cited alot and seems
to me to be a default for measuring chl.

Details are:
Porra, R. J.
Thompson, W. A.
Kriedemann, P. E. 1989
Determination of accurate extinction coefficients and simultaneous
equations for assaying chlorophylls a and b extracted with four
different solvents: verification of the concentration of chlorophyll
standards by atomic absorption spectroscopy
Biochim. Biophys. Acta 975: 384-394

The extinction coefficients for chlorophylls a and b in diethylether
(Smith, J.H.C. and Benitez, A. (1955) in Modern Methods of Plant
Analysis (Paech, K. and Tracey, M.V., eds.), Vol. 4, pp. 143-196,
Springer-Verlag, Berlin), used this paper as primary standards, were
verified, to within an error of less than 1%, by magnesium determination
using atomic absorbance spectrophotometry. We also report the
determination of accurate extinction coefficients for chlorophylls a and
b in N,N'-dimethylformamide, methanol or buffered 80% aqueous acetone.
Highly purified chlorophylls were used and methods were employed which
not only minimize errors due to evaporation of the volatile solvents
employed in their estimation but also eliminate variable
micro-contamination by chlorophyll degradation products, a potential
source of inconsistency between the extinction coefficients obtained in
each of these three solvents. Using these new coefficients, expressed as
both millimolar and specific coefficients, we have derived new
simultaneous equations to obtain chlorophyll concentrations of nmol/ml
and microgram/ml, respectively. These equations were applied to data
obtained with leaf discs from spinach and Flindersia brayleyana
extracted with the three specified solvents and to a concentrated
solution (in N,N'-dimethylformamide) of a chlorophyll a + b mixture
added to the three solvent systems. The validity of these equations is
proven by the consistency of the chlorophyll determination and of the
chlorophyll a/b ratios. New simultaneous equations, compatible with the
equations derived for the three solvents, are presented for the assay of
chlorophylls a and b converted to their cyclic hydroxylactone
derivatives by extraction with alkaline pyridine reagent (2.1 M pyridine
in 0.35 M NaOH). Most chlorophyll analyses in higher plants, including
the chlorophyll content and chlorophyll a/b ratios of plant thylakoids
and chlorophyll-protein complexes, have been obtained in 80% aqueous
acetone with the much used simultaneous equations of Arnon (Arnon, D.I.
(1949) Plant Physiol. 24, 1-15). For this reason we include conversion
factors which correct these earlier data and make it compatible with
data calculated with the simultaneos equations presented in this paper.
The importance of these corrections to the formulation of meaningful
models of the photosynthetic apparatus is demonstrated. Our results also
indicate that grinding leaf discs with N,N'-dimethylformamide is a more
reliable method for extracting all chlorophylls than shaking with this
solvent for 24 h.


Dr Barry Pogson
Photosynthesis Center, Dept. of Plant Biology, Box 871601
Arizona State University, Tempe, AZ 85287, USA.
Email: pogson at asu.edu; Fax: 1 (602) 965-6899; Ph: 1 (602) 965-2583


=46rom: KOURIL at prfnw.upol.cz
Organization:  Palacky Univ. Fac. of Science
To: wjboelem at mtu.edu
Date:          Mon, 9 Mar 1998 16:16:33 MET-1
Subject:       chlorophyll equations
X-Confirm-Reading-To: <KOURIL at prfnw.upol.cz>
X-Pmrqc:       1
Priority: normal

Literature for determining chlorophylls and phaeophytin
Lichtenthaler, H.K.: Chlorophylla and carotenoids: Pigments of
photosynthetic membranes. Meth.Enzym. 148, 350-382 (1987)
Wellburn, A.R.: The spectral determination of chlorophylls a and b,
as well as total carotenoids, using various solvents with
spectrophotometers of different resolution. J. Plant Physiol.
Vol.144, 307-313 (1994).


  Bill Boelema           |         Graduate Student, Biological Sciences
wjboelem at mtu.edu         |           Michigan Technological University

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