Marine Biology t2, 184--188 (1972)
9 by Springer-Verlag 1972
Preparative and analytical extraction of pigments from brown algae
with dimethyl sulfoxide
G. R. SEELY1, M. J. DU~CCAN2 and W. E.
C. F. Kettering Research Laboratory; Yellow Springs, Ohio, USA
and
Department of Biological Sciences, Simon Fraser University; Burnaby, Canada
Abstract
Dimethyl sulfoxide (D1KSO)rapidly extracts much of the
chlorophyll v and fucoxanthin from the intact thalli of brown
algae. Subsequent extraction by acetone rapidly removes most of
the chlorophyll a and fl-carotene. An analytical procedure for
these pigments has been developed, based upon this extrac-
tion sequence, followed by partition of the acetone extract be-
tween hexane and aqueous acetone. Fueoxanthin and chloro-
phyll c can be recovered from the DMSO extract and the
aqueous acetone phase by extraction into ethyl acetate and
subsequent evaporation. The DMSO-acetone extraction se-
quence is also effective with green algae and a few red algae,
although the full scope of the method has not been investigated.
Introduction
The brown algae of the intertidal and subtidal
zones of the shorelines of the cooler oceans are an
abundant source of two photosynthetic pigments,
fucoxanthin and chlorophyll c. Conventional methods
of extracting these pigments, which require grinding
the tissue with acetone or methanol (SMIT~ and BE-
~ITEZ, 1955; BONNETT et al., t969; JEFFREY, t969),
may be lengthy and difficult because of the bulky and
rubbery consistency of many of the brown algae, and
the large amounts of mucilaginous polysaccharides
that they contain.
Desiring to obtain these pigments more quickly
and easily, and without risk of degrading them, we
explored the use of other solvents and conditions for
extraction. Among the solvents tried was dimethyl
sulfoxide (DMSO), which has acquired a considerable
reputation for its ability to penetrate membranes,
and $o denature proteins reversibly by displacing or
replacing the water around them (R~_~MLEg and
ZAFF~O~I, 1967). We found that DMSO indeed had
the ability to penetrate the tissues of brown algae
rapidly, and to extract from them the more hydro-
philie pigments such as fucoxanthin and chlorophyll c.
We furthermore found that if DMSO extraction was
followed by acetone, a large part of the chlorophyll a
and fl-earotene washed out of the tissue. The rapidity
and extent of pigment extraction was such as to
VIDIVER ~
prompt the development of analytical and preparative
procedures, based on successive extraction of algal
tissue with DMSO and acetone.
We, therefore, present procedures for the quantita-
tive extraction, analysis and recovery of photosyn-
thetic pigments from brown algae. These procedures
are especially intended for the larger brown algae,
which form the major component by weight of the
littoral and sublittoral flora of temperate zone shores.
Preliminary experiments have indicated that these
procedures can be adapted for unicellular algae, green
algae, and some red algae. They are presented with the
expectation that they may have useful applications in
marine science.
Material and methods
Algae were collected locally, in particular at
Brockton Pt., Vancouver, B. C., Departure Bay,
~qanaimo, B. C., and Pt. Roberts, Washington.
Reagent grade solvents were used for the extractions.
Acetone and hexane for chromatography were distilled
to remove material of low volatility. Commercial
icing sugar for chromatographic columns was dried
at 90 ~ overnight. Spectra were determined with a
Cary i4 Recording Spectrophotometer.
Quantitative extraction and analysis
(i) The fresh blade or other algal material is rinsed
with tap water to remove adhering salt water, blotted
dry, and weighed (adhering salt water may produce
turbidity in the DMSO extract).
(2) The blade is treated with a volume (V, ml) of
DMSO, which is 4 times the weight (g) of the blade.
Extraction is carried out in a beaker or tray and, after
about 5 rain, the yellow-brown extract (Xi) is drained
off. Extraction with less than V ml of DMSO, is
mechanically difficult and the extract is low in pig-
ment content. Much more than V ml is wasteful, and
the amount of chlorophyll a extracted could interfere
with the direct spectroscopic determination of chloro.
phyll c.
 Vol. 12, ~u 2, 1972 G.R. Sl~r~ye~ at.: Brown algae pigment extract.ion
(3) The blade is treated with V mt of acetone; the
dark green extract (X2) is drained off after 5 rain or
less.
(4) Because of the dry and shrunken condition of
the blade a~ this point, immediate fresher extraction
with acetone is unprofitable, t[owever, if the blade is
treated with water for a few minutes, vh'tuMly all of
the pigments can be extracted from the swelled blade
by 2 or 3 treatments with acetone. Particularly tough
tissue may have to be treated with water and ex.
~raeted with acetone again. The blade, now mostly
colorless or light brown, is reduced to about 6 % of its
original wet weight.
(5) The acetone extraet~ contain fucoxanthin and
chlorophyll c as wetl as chlorophyll a and fl-earotene.
The former two pigments are separated fl'om the latter
two by partition and ex~action. To a volume V' of
the acetone extracts, which may be treated sepaxately
or combined, are added (~1/3)V' ml hexane and (114)V'
ml water. The phases are separated, and the upper
(hexane) phase is extracted twice with (1/9)V' ml 75 %
methanol. W h e n these extracts are combined with the
lower (aqueous acetone) phase, a small hexane-rieh
phase appears which is separated and combined with
the main hexa~e phase. The hexane phase is now
extracted t~ice wi%h (I/9)V' ml 80% methanol. The
first extract is usually added to the combined lower
phases, but the seeomt is usually discarded because it
is colorless or very" pale yellow.
(6) The hexane phase (X2A) is analyzed spectro-
scopically for chlorophyl] a and fi-caxotene after dilu-
tion with acetone (usually 10 to 20 volumes), as
described below. The DMSO extract (Xt) and the
combined aqueous acetone-methanol phase (X2B) are
analyzed speetroseoNcally for fucoxanthin, chloro-
phyll c and chlorophyll a.
Preparative extraction
The following procedures have been found saris.
factory for recovering the pigments from the extracts
in a form suitable for chromatography.
(t) The hexane phase (X2A) is evaporated to
dr3~ness at. or below room temperature and at reduced
pressure in a rotary evaporator. The residue of chloro-
phyll a, fl-carotene and other lipids may be somewhat
oily but is suitable for chromatography on sugar.
(2) To Vi ml of DI~ISO extract XI in a separatory
funnel are added (1/2) V1 ml ethyl acetate and V1 ml
0.5 5I (NHd)2SO4 solution. Phase separation is accel-
erated by refrigeration for ca. ~[h. The upper phase is
extracted at least 4 times with at least (1/5) V~ mt
0.55I (NHa)~SO4 solution, and dried with small
amounts of saturated (NHa)~SO4 solution and anJay-
drous NaeSO4. I t is necessary to e~x~.rac~ the ethyl
acetate phase with (t/I0) saturated (NHa)~SO~I solu-
tion (0.5 ND to ensure rapid phase sepaxation. (A
white precipitate may appeax in the lower phase during
24.
t85
the initial partition into ethyl acetate, especially with
extracts from older specimens. This precipitate re-
teoins chlorophyll c. The recovery of chlorophyll c
may be improved by diIuting the separated lower phase
with an equal volume of water, and adding about
[i/10] volume of ethyl acetate).
(3) To V2 ml of X2B in a sepaxatory funnel are
added (2/5) V2 ml ethyl acetate and (3/4) V~ ml 0.5 M
(NHd)2S04 solution. The lower phase is colorless. The
upper phase is extracted at least 4 times with 0.5 iV[
(4) The ethyl acetate exr are combined and
evaporated to dr~mess at or below room temperature
in a rotary evaporator.
The steps (2) and (8) above are optimized for
paxt~tioning into ethyl acetate. I t should be simple to
adapt the procedures for partitioning into ethyl ether,
a solvent more often used for the purpose.
Chromatography
For purfficat~on of major pigments and detection
of minor pigments it is necessary to resort to chroma-
tography, and suitable procedures are in the literature
(JEFFREY, 1968a; G~lCSIDE and RIL1~y, 1969). XSre
employed successive chromatography of the extracts
on a cotumn of dried icing sugar.
The X2A residue is dissolved in hexane, and
developed with hexane-aeetone mixtures, fi-carotene
and other lipids axe eluted with 0 to 5 % (v/v) acetone
in hexane, and chlorophyll a with 5 to t0% acetone.
This solvent system is not intended to separate the
Iipids of the carotene fraction. Since neither chloro-
phyll b nor lutein is present in brown algae, the
chlorophyll a thus obtained is spectroscopically pure.
With fresh algal material, the bands of pheophytin a
and chlorophyll a' are very faint, proving that chtoro-
p~711 a is not appreciably degraded during the extrac-
tion.
The residues of X t and X2B are dissolved in h e y
ane with 0 to t0 % acetone, and after passage of the
small chlorophyll a band, the xanthophy]t fraction is
developed and eluted with 7.5 to 20% acetone in
hexane. Most of the xanthophytt fraction is native
fueoxanthin. However, there are always a number of
minor bands, including violaxanthin, isomers of fu-
coxanthin, and ehlorins. Red crystals of fucexanthin
separate out of the eluate on refl'igeration overnight.
Chlorophyll c remains fixed to the top of the column
during elation of fbeoxanthin. Upon development
~4th 6:4:1 hexane:ethyl acetate:acetone, the chloro-
phyll c fraction is resolved into 2 or 3 green bands,
which are partially separated on elution from the
column. I t is known that there are at least two com-
pounds with similar spectra in the chlorophyll v
fraction (Safrr~ and B~rxT~z, t955; D o u a ~ R ~ u et al.,
t966, t970; JEFF~u t968b, 1969; VY~SL~u et al.,
1970). Preced&ng and overlapping the first chlorophyI1
186 G.R. SEELIr et al.: Bro~m algae pigment extraction Max, Biol.

o band is the band of a chlorin (absorption bands at
657, 440 n m in the eluate). This substance always
appears to accompany chlorophyll c. The same or a
similar substance appears in spectTa of chlorophyll
reported b y PAI~SO~qS (i96I).
~ e have not a t t e m p t e d to identify the numerous
minor pigments that appear on chromatography of
the fractions. Because of the rapiditffand the gentle
conditions of the extraction, and because f~he major
pigments are recovered in good yield, we believe t h a t
the minor pigments are natural degradation products,
the quantity of which m ~ v be iudieative of the age or
condition of the alga.
Spectroscz)py
I t was necessary to determine specific absorp-
r ~ ~~m ~ of the pigunents in solvents having the
same composition as the extracts. This was done b y
comparing spectra of the pigments in these solvents
with spectra in reference solvents for which absorptivi-
absorptivities of the 630 and 580 nm bands was more
nealqy independent of solvent and species t h a n either
absorptivity alone. Our use of the sum of the ab-
sorbanees near these 2 wavelengths partly overcomes
the difficulty of estima~ng chlorophyll c accurately
without separafAug the 2 @ecies.
Absorptivities were determined in 4 : i DI~ISO:
water, the approximate composition of X t , in acetone
for X 2 A (hexane diluted with J0 or more volumes o f
acet~)ne), and in a 3: J: l acetone: m e t h a n o l : w a t e r
mixture representing the approximate composition
of X2B. The resatt~ are collected in Table L
Based on these values, pigment concentrations in
the extracts axe calculated from the following formulae
Table i. S p ~ f l v s ~/ficient~ /.or pigmen~ a~ wase-
lengths ar~d in solvva~s used/or a.nalysis,
Chl: J~lorophyll; car: ca~'otene
Solvent Wavelength Abs(~rption coefficient, ~0.1%
1 cm (l/g)

ties have been reported. Our absorp~ivities are based (nm) Chl a C~t ~ fx ear
upon the following set of published absorptivities,
Fueoxanthin: ~1 ~0.~%
em = t65 l/g a t 449 n m in petro- 4:I 665 72,8~ 0 0
leum ether (J~.~sn~, i96J). D~ISO: 63t 12.7 34A 0
water 582 9.0 27,7 0
"'ft.carotene: ,~~ -- 250.51/g a t 451 n m in petro- 480 3.6 44.7 ~30
leum ether (D~vx~s, t965). Acetone 661 83.3 0 0 0
w2 " i %--
- 96.6 t/g a~ 660.5 nm in 628 Ji.3 32.9~ 0 0
Chlorophyll a: ~1r 580 8.9 29.1 0 0
ethyl ether (ST~AI~ et al., t963). 480 2,7 10~6 1t8 ~93
Chlorophyll c: The esb~ml_a~ion of fkis substance 470 2.6 47,9 142 J92
presenf~ some problems. The 2 compounds under this 3:'1:~ 664 73,6 0 0
acetone: 631 I3.0 36,9 0
name (chlorophyUides ca a n d c~) differ in their degree of methanol: 58f 9A 25.3 0
unsaturation and in the ratio of their absorption bands water 470 2.0 77,0 Jdl
near 630 and 589 rim. T h e y occur in varying propor-
tions in seaweed (g~t~t~Exr, t968b, 1969), and because In 100 % D~ISO; pigment not very soluble in 80 % D~ISO.
of the similarity of their spectra it appeaxs impossible b Insoluble.
to determine t h e m individually with m u c h accuracy, I t is assumed ~ha~ E ~ + E ~ = 62.0 I~.
without chromatographic sepax~ion. Before the pffbli-
c~tion of ~u et al. (1970) values of the absorp-
ti~dties of the chromatographicMly separated methyl eontMning the absorbances ~ a~ appropriate wave-
chlorophyllides, the most reliablevalues were those of lengths i . C~)ncentrations are expressed in g/t and these
STR-.AIN and Sv~c (1966) for a mixture of u n k n o w n abbreviations are used: c h l = chlorophyll, f x = fuco-
proportions of ca and 89 Our absorp~vities were ori- xanthin, ear = fl-carotene,
ginally based on these, viz. ~,~0,1%
~ ~m _- 29,6 i/g at 627 n m X l : ca. 4 : t D ~ S O : w a t e r
and 27.4 l/g a t 578 n m in ether, and determined with [chl a] = D~sj72.8
a chromatographic fraction which had nearly the same [chl o] = (eG31 + e ~ s ~ - 0.297 essD/6i.8
ratio of absorp~ivities as theirs. According to the data [fx] = (~ds0-- 0.722 (~6~1+ ~s2 -- 0.297 ~s65)
of WASLEr et al. (1970), a mixture of the methyl - - 0.049 (%~5)/130
ehlorophyllides e~ and c~ having the same ratio
X2A: ca. i 0 : l aeetone:hexane
~ era = 32.9 I/g at 6-~7 n m and 30.5 I/g a~
would have ~o.~%
579 a m in ether, or 33.6 and 31.2 1/g. respectively, for [car] = (eds0 -- 0.033 ~6~1)/193
the unesterified "chlorophyltides. 0 ~ ' values m the
different solvent mixtures have been adjusted so thab X2B : ca. 3 : i : l acetone: methanol: water
they are now based on these. [cht a] = e~64/73,6
Estimation of chlorophyll c is assisted b y our ob- [chl c] = (q~l + e ~ - 0.300 ~ ) / 0 2 . 2
servation, subsequently borne out b y the published [fx] = (~o~0 -- 's (~at + ~ s l -- 0.300 ~6~a)
values of ]VAsL~r et al. (i970), ~hat ~he sum of the - - 0.0275 ~664)/14L
Vol. 12, No. 2, 1972 G.R. S ~ Y e~ al.: Brown algae pigment extxaction 187

Rcsult$
I t will suffice to illustrate the results of the extrae.
tion me~hod with 3 species, JL~t.minaria saccharina,
Sargassum muticum, and tZucus distichvz (Table 2).
The results with Laminaria are typical of the k d p s ,
including Nereocystis, Alaria, and Costaria. Two-
thirds of the chlorophyll c is extracted b y DMSO,
and 3/4 of the chlorophyll a is removed in the first
acetone exVratt. The pigments obtained in this ex-
traction were purified and used to determine the ab-
sorptivities in Table L
Discussion
The DMS0-acetone extraction procedure permits
recovery of pigments from large brown algae, including
kelps, qnlekty and with a minimum nm~ber of
operations. Only t r e a t m e n t with solvent a t room
temperature and decantation are required to get clear
extracts of pigments.
Although 3 treatments with acetone are usually
necessary to extract the pigments quantitatively for
analytical purposes, it is usually unprofitable to per-
form more t h a n i acetone extraction ff the objective

Table 2. Analysis o/ pigment content in brown algal blades. Total pigment weight
(g/kg wet blade) and distribution by percent into the various extracts
Species and sample Extrac~ Chl a Chl c fx car
weight

Lam inaria saccharina Wt., g[kg 0.538 0.075 0.243 0.013

120 g Xi 7A % 65.5 % 56.5 % 0%
X2A~ 67.0 0 0 87.5
X2B 8.6 23.5 28.9 0
X3A I6.0 0 0 12.5
X3:B 1.4 11.0 ~4.6 0
Sargassum muticum W~., g~kg 0.631 0.080 0.293 0.027
5g X1 10.7 77.7 78.4 0
X2Ab 81.7 0 0 100
X2B 7.6 22.3 21.6 0
Fucus distichus Wt., g,~g 0.383 0.038 0A56 0.018
~9A g XI 2A 42.0 46.0 0
X2Aa 20.6 0 0 40A
X2B t.7 t3.i 22.9 0
X3A 6I .8 0 0 59.9
X3B 1.8 37.8 20.2 0
X4~ IL9 7.2 11.0

Acetone extracts X2 and X3 p~rtitioned separately.

b Acetone extracts X2, X3, and X4 combined and partitioned together into
X2A and X2B.
o Chlorophyll (Chl) and fucoxanthin (fx) were estimated directly in acetone
with the following formulae:
[chtc] = (~s2s+ ~ss0- 0.239 g~1)/62.0.
[fx] = (~470- 0.774 (es2s + ~ss0- 0.239 ~ssl) - 0.031 ~s61)/t42.
Carotene (car) was not determined.

The pigments are very readily extracted from
Sargassum; it m a y be worth noting t h a t J~FF~Er
(1963, t969) isolated her chlorophyl] ~ from S. flavi-
ca~s, and P ~ s o ~ s and ST~CKLAND (i963) obtained
their fucoxanthin from S. muticum.
As might be expected from its habitat, Nucus is
more difficult to extract. The last 2 acetone extractions
were lengthy, ca. t h. The wiry fronds of Desmareztla
intermedia (?) required 4 treatments with acetone for
q u a n t i t a t i v e extraction. Application of the procedure
to a species of Dezmarestia (probably viridis) with
acid-containing vacuoles gave a r~pid and quantita-
tive yield of pheophytin a and acid rearrangement
products of fucoxanthin.
is pigment preparation without ~he necessity of quan-
titative recovery. Extracts containing about 80% of
the pigment content of a brown alga can, thus, be
prepared within 10 to t5 min of receipt of the sample.
The mildness of the methods used and the rapidity of
extraction afford the highest assurance t h a t the pig-
ments extracted are actually native to the alga. The
method appears particularly suited to the reliable
detection and extraction of minor pigments in these
a~ae.
When the met~hod was applied to samples of green
algae of the genera Ulva and E~ero~wrpha, DMSO
extracted mainly violaxanthin, and acetone extracted
chlorophylls a and b and lutein along with the rest of
i88 G. 1:~.SEELY et al.: Brown algae pigment extraction
t h e v i o l a x a n t h i n . T h e e x t r a c t i o n s are r a p i d , a n d appli-
cations to pigment recovery and analysis appear
promising. IVitella is also r a p i d l y e x t r a c t e d b y t h i s
method.
M o s t r e d algae t e s t e d were r e s i s t a n t to e x t r a c t i o n ,
b u t two, species o f Callophyllis a n d Rhodymenia, were
susceptible. T h e c h l o r o p h y l l a a n d c a r o t e n o i d s were
e x t r a c t e d , leaving t h e p i n k p h y c o b i l i n s inside t h e blade.
P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e a p p l i c a t i o n to dia-
t o m s a n d o t h e r m i c r o o r g a n i s m s , b u t these h a v e n o t
yet been adequately investigated.
T h e success o f t h e p r e s e n t e x t r a c t i o n t e c h n i q u e is
e v i d e n t l y d e p e n d e n t on t h e a b i l i t y o f D M S O t o pene-
t r a t e m e m b r a n e s a n d disorganize t h e i r c o n s t i t u e n t s .
I n t h i s connection, we h a v e n o t e d t h a t D M S O a t a
c o n c e n t r a t i o n o f o n l y 20 % in w a t e r evokes a d i s t i n c t
c h a n g e ' i n t h e s p e c t r u m o f a n i n t a c t b l a d e of Nereo-
cystis, especially in t h e r e g i o n of f u c o x a n t h i n absorp-
tion. Also, t h e o r d e r of a p p l i c a t i o n o f solvents is
i m p o r t a n t . E x t r a c t i o n b y DMSO followed b y a c e t o n e
is m o r e effective t h a n e x t r a c t i o n b y a c e t o n e followed
b y DMSO, or b y t w o e x t r a c t i o n s w i t h i : i D M S O :
acetone. I t a p p e a r s t h a t DMSO, in a d d i t i o n to ex-
t r a c t i n g t h e m o r e h y d r o p h f l i c p i g m e n t s , affects t h e
m e m b r a n e s so t h a t t h e m o r e lipophilie p i g m e n t s are
r a p i d l y e x t r a c t e d on t r e a t m e n t w i t h acetone. I t w o u l d
a p p e a r w o r t h e x p l o r i n g t h e effect o f D M S O on m e m -
b r a n e s t r u c t u r e a n d p h y s i o l o g y in t h e s e algae.
Summsl'y
I. D i m e t h y l sulfoxide (DMSO) e x t r a c t s m u c h o f t h e
c h l o r o p h y l l c a n d f u c o x a n t h i n f r o m b r o w n algae,
a n d also p r e p a r e s t h e algae for r a p i d e x t r a c t i o n of
chlorophyll a and carotene by acetone.
2. l~elativeIy simple a n d r a p i d p r o c e d u r e s for
a n a l y s i s of t h e s e p i g m e n t s a n d for t h e i r p r e p a r a t i v e
r e c o v e r y a r e p r e s e n t e d , which do n o t r e q u i r e h e a t i n g ,
freezing, grinding, or centrifuging t h e algal tissue.
3. Because o f t h e h e t e r o g e n e i t y in t h e c o m p o s i t i o n
o f c h l o r o p h y l l c, i t is r e c o m m e n d e d t h a t its e s t i m a t i o n
be b a s e d on t h e s u m o f a b s o r b a n c e s of its b a n d s n e a r
580 a n d 630 n m .
Acknowledgements. We are grateful to Mr. C. VAN
NET~E~ for his observations concerning the extractability of
Nitella, and to Dr. L. D. DRUERL for assistance in the identifi-
cation of algae. This work was performed at Simon Fraser
University while G.R.S. was on leave from the C. F. Kettering
Research Laboratory, and was supported in part by National
Date of final manuscript acceptance: September 6, 1971. Communicated by T. R. PARSONS, Vancouver
:Responsible for presentation of text: Professor Dr. O. KINNE, Biologische knstalt Helgoland, Palmaille 9, 2 Hamburg 50, Germany.
Responsible for advertisements: E. SEIDLER, D-1 Berlin 15, Kurffirstendamm 237. Springer-Verlag - Berlin 9 Heidelberg 9 New York
Printed in Germany by Carl ICitter & Co., Wiesbaden
Copyright 9 by Springer-Verlag Berlin. Heidelberg 1972
.Mar. Biol.
Research Council of Canada Grant 51o. A2908, and by Iqational
Science Foundation (USA) Grant 5Io. GB7893.
Literature cited
B0~ET% 1%, A. K. M~LA~S, A. A. S P ~ x , J. L. TEE, B. C. L.
WEEDOI~ and A. McCoa~Icx: Carotenoids and related
compounds. Part XX. Structure and reactions of fuco-
xanthin. J. chem. Soc. (C) 1969, 429---454 (1969).
DAvIEs, B. ~I.: Analysis of carotenoid pigments. In: Che-
mistry and biochemistry of plant pigments, pp 489--532.
Ed. by T. W. GOODWIN.London: Academic Press 1965.
DOVG~E~TY, R. C., It. It. STRAIN,W. A. SvEc, R. A. UFHAVS
and J. J. KATZ: Structure of chlorophyll c. J. Am.
chem. Soc. 88, 5037--5038 (4966).
The structure, properties, and distribution of
chlorophyll c. J. Am. chem. Soc. 92, 2826--2833 (4970).
G/~SIDE, C. and J. P. RILEY: A thin-layer chromatographic
method for the determination of plant pigments in sea
water and cultures. AnalyCica chim. Aeta 46, 479--191 (1969).
JEFFREY, S. W. : Purification and properties of chlorophyll o
from Sa~gassum flavicans. Biochem. J. 86, 343--3~18 (1963).
- - Quantitative thin-layer chromatography of chlorophylls
and carotenoids from marine algae. Biochim. biophys.
Acta 162, 274--285 (4968a).
- - T w o spectrally distinct components in preparations of
chlorophyll c. Nature, Lond. 220, 4032--4033 (1968b).
- - Properties of two spectrally different components in chloro-
phyll c preparations. Biochim. biophys. Aeta 177, 456---
467 (4969).
JE~SB~, A.: Algal carotenoids I. fucoxanthin monoacetate.
Acta chem. seand. 15, 4604--4605 (4961).
P ~ s o ~ s , T. R. : On the pigment composition of eleven species
of marine phytoplankters. J. Fish. Res. Bd Can. 18, I017--
4025 (4964).
- - and J. D. H. ST~ICX~AND: Discussion of spectrophoto-
metric determination of marine-plant pigments, with
revised equations for ascertaining chlorophylls and caro-
tenoids. J. mar. Res. 21, 455--163 (1963).
R ~ L E R , D. H. and A. Z A ~ o ~ r ~ : Biological implications
of DMSO based on a review on its chemical properties.
Ann. 2q. Y. Acad. Sci. 141, 13--23 (([967).
SMIT~, J. 1~. C. and A. BE~TEZ: Chlorophylls: analysis in
plant materials. B. Chlorophyll c. In: Moderne Methoden
der Pfianzenanalyse, Vol. 4, pp 162--164. Ed. by K.
PAEe~ and M. V. TRACEr. Berlin: Springer-Verlag 1955.
STBAIN, H. H. and W. A. SvEc: Extraction, separation, estima-
tion, and isolation of the chlorophylls. In: The chloro-
phylls, pp 21--66. Ed. by L. P. VERNON and G. 1%. SEELu
New York: Academic Press t[966.
- - , M. R. THOMAS and J. J. KATZ: Spectral absorption pro-
perties of ordinary and fully deuteriatcd chlorophylls a and
b. Biochim. biophys. Acta 75, 306--3ti (1963).
WASL~u J. W. F., W. T. SCOTT and A. S. HOLT: Chloro-
phyllides c. Can. J. Biochcm. 48, 376-383 (4970).
First author's address: Dr. G. R. S~ELY
C. F. Kettering Research Laboratory
Yellow Springs
Ohio
USA