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The pigments of the algae with special reference to certain orders of the Chlorophyta Palmer, Mildred Ruth 1955

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THE PIGMENTS OF THE ALGAE WITH SPECIAL REFERENCE TO CERTAIN ORDERS OF THE CHLOROPHYTA by MILDRED RUTH.PALMER A THESIS SUBMITTED IN PARTIAL FTOJTTMKNT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in the Department of . BIOLOGY AND-BOTANY We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS. Members of the Department of THE UNIVERSITY OF BRITISH COLUMBIA October, 1955 ABSTRACT The chlorophyll and carotenoid pigments obtained from a number of algae were studied i n gross acetone extracts. A method proposed by Richards and Thompson, was--used to calculate the relative proportions of chlorophylls a, b, and c, astacin and non-astacin carotenoids present. Complete absorption spectra from 350 up. to 700 mn were obtained for seyeral algae, chiefly those belonging to the Chlorophyta. I t was found that the presence of chlorophyll b could be detected as a small irregularity i n the spectral curve at 460 mu. The xanthophyll pigments of twenty-eight different species of algae were investigated. Chromatographic columns were used to isolate the individ-ual pigments. A mixture of magnesia (Micron Brand) and Hyflo Super Cel was used as the adsorbent. Ethylene chloride was used as the solvent. Fourteen different xanthophyll pigments were found. Twenty two species of Green Algae were investigated i n an attempt to show that the presence or absence of certain pigments may indicate phylo-genetic relationships. Individual xanthophyll pigments appear to be of l i t t l e phylogenetic significance. Groups of these pigments, however, seem to be more significant. Fairly uniform and closely related groups such as the Zygnematales and the Ulvales contain certain characteristic groups of pigments. Less uniform groups such as the Volvocales and the Cladophorales appear to lack characteristic groups of pigments. The results agree f a i r l y well with modern phylogenetic relation-ships, based on the morphology and methods of reproduction* More extensive work with more genera and species i s needed before generalizations at the ordinal level can be made. TABLE OF CONTENTS ABSTRACT i i i I. INTRODUCTION 1 II. . ACKNOVBLEDGBiMTS 3 III. REVIEW OF LITERATURE 3 A. THE CHLOROPHYLLS V 3 B. THE PHYCOBILINS 6 C. THE CAROTENOIDS . . 7 (i) General Distribution of Carotenoids 9 ( i i ) Distribution of Carotenoids i n the Algae 10 IV. GENERAL DESCRIPTION OF METHOD 11 V. PROCEDURE 13 A. GROSS PIGMENT EXTRACTS 13 B. CHROMATOGRAPHIC WORK 14 VI. RESULTS 16 VII. DISCUSSION 22 A. GROSS PIGMENT EXTRACTS 22 B. CHROMATOGRAPHIC WORK 25 VIII. SUMMARY AND CONCLUSIONS 29 IX. BIBLIOGRAPHY 45 i i THE PIGMENTS' OF THE ALGAE WITH SPECIAL REFERENCE TO CERTAIN ORDERS OF THE CHLOROPHTTA by MILDRED RUTH PALMER I. INTRODUCTION The pigments of the algae have been used for a long time i n the systematics of the algae. The common names of the algal groups-such as Red Algae, Brown Algae, Green Algae and Blue-green Algae are indicative of the degree of importance of pigmentation i n classification. Of the three main groups of pigments - the green chlorophylls, the orange carotenoids and the blue and red phycobilins - the predominance of at least one of. these i s responsible for the naming of the different groups. For example, the Red Algae are red because of the predominance of the red phycobilin pigment, phycoerythrin. The study of - this problem was begun i n i t i a l l y i n order to evaluate and develop a method which might be useful i n characterizing marine plankton populations on the basis of pigment analyses. As the research progressed i t was f i n a l l y decided to make a more, complete and detailed examination of pigments other than the chlorophylls i n the Green Algae (Chlorophyta) and especially i n members of the morphologically diverse order Volvocales. Since i t has been shown by some authors (Strain, i n Smith 1951, p* 251) that the chlorophylls i n the Green Algae are consistently chlorophylls a and b, i t was decided to examine c r i t i c a l l y the carotenoid pigments. The carotenoida are divided into two groups, the carotenes and the xanthophyll^. Because the sample size was usually limited, i t was impossible to obtain pigment extracts which had sufficient carotene i n them to isolate and i d -entify the individual carotene pigments. Hence only the xanthophyll pig-ments were examined i n detail. Since the xanthophyll pigments are, as a group, much more variable than either the chlorophylls or the carotenes, i t was hoped that this group might prove useful i n comparing different groups of algae from a phylogenetic standpoint. Many of the samples used i n this investigation were obtained from natural blooms, although some unialgal cultures were used. Unialgal or pure cultures are superior to the natural populations because they do not contain other algae as ; contaminants. However since the size of a l l the samples obtained from natural blooms was relatively small, i n a l l analyses performed the number of contaminants could be disregarded. Any contamination which may have resulted was so slight that i t could not have provided sufficient pigments to be separated on the chromatographic column of the size used. There was no attemptmade to determine the relative, concentrations of the different xanthophyll pigments present nor was any. attempt made to compare any of the algae quantitatively with respect to these pigments under different environmental conditions. The bibliography i s not intended to be complete* particularly with respect to the older literature on the subject* I t i s chiefly com-prised of references that are most pertinent to this study and includes the more recent and useful literature* The monograph on carotenoida by Karrer and Jucker (1950) has been aost invaluable as a reference. 3 I I . ACKNOWLEDGEMENTS I should l i k e to acknowledge the help and encouragement of my master* s committee and particularly that of Dr. R.F. Scagel during the preparation of this thesis. I should also l i k e to thank Dr. M. Kirsh of the Chemistry Department of the- University of B r i t i s h Columbia for the use of his Beckman DU Spectrophotometer. Otherwise the f a c i l i t i e s used were those of the Department of Biology and Botany at the University of British Columbia and those of the Friday Harbor Laboratories at Friday Harbor, Washington. III. REVIEW OF LITERATURE Although this review has resulted from the examination of a large body of literature, most of which i s cited i n the bibliography, i t i s based chiefly on a few modern, comprehensive referenced on the subject (Strain, 1945; Strain, i n Frank and Loomis, 1949J Karrer andJucker, 1950; Strain, i n Smith, 1951; Rabinowiteh, 1945 and 1955). The pigments of the algae can be divided into three main groups -the fat soluble chlorophylls, the carotenoids and the water soluble phyco-b i l i n s . The chlorophylls and carotenoids are distributed universally throughout the photosynthetic. plant world* The phycobilins, however, are restricted to two algal divisions, the Cyanophyta and the Rhodophyta, A. THE CHLOROPHYLLS There are four different chlorophylls - chlorophyll a, b, a and d - found i n algae. Chlorophylls a and b are present also i n the higher plant groups and hence are the best known chlorophylls. The structural formulae for both of these chlorophylls has been worked out although there i s s t i l l seme debate as to the position of the magnesium bonds and the semi-isolated double bond (Rabinowitch 1 9 4 5 , p. 4 4 3 ) . Figures 1 and 2 (p. 4 ) show the structural formulae for chloro-phylls a and b respectively* The asterisk (*) indicates the position of the aldehyde group (*CHO) i n chlorophyll b whereas i n chlorophyll a there i s a methyl group at the same point. This i s the only difference i n the formulae of the two chlorophylls. A A A H 3 C - C C ' C C-C2H5 C - N N - C : H C MQ V C H M , C H * A A / " C H » H I H2C=CH CH0* I l H l A A A i H3C-C C' c' C-C 2H 5! C - N N-C \ = N N N - C ^ •! Hs c.-c; A\ / C - C H 3 i \ CH2 H C -4. ' H 3 9 C 2 0 0 0 C - C H 2 C00CH 3 h' y v V C H 2 H C H 3 E C 2 0 0 0 C - C H 2 COOCH3 Figure 1 . The structural formula of chlorophyll a (after Fisher, i n Rabinowitch 1 9 4 5 , p. 4 4 2 ) Figure 2 . The structural- form-ula of chlorophyll b (after Fischer, i n Rabinowitch 1 9 4 5 , p. 4 4 2 ) . Chlorophyll a i s present i n a l l photosynthetic plants except certain of the bacteria. The latte r contain a related pigment called bacteriochlorophyll. The structure of bacteriochlorophyll which ia- be-l i e v e d to catalize photosynthesis^is given i n Figure 3 (p. 4 ) . I : • ^ H 3 ; . I C O * M ! I ..«-'< yvy«..% I X - N N - < .1 H C Mg C H C = N N - C ' / 1 1 ^. H « C - C C C C-C»m 3 H ' \ „ / V / V / 3 I 1 H ^ C H-C C « 0 H39C2oOOCCH2 COOCjHs Figure 3 . Structrual formula of bacteriochlorophyll (after Fischer, i n Rabinowitch 1 9 4 5 , p* 4 4 5 ) . 5 Chlorophyll b i s present, along with chlorophyll a, i n the higher plant groups, the Chlorophyta and the-Euglenophyta. The other two chlorophylls, chlorophylls c and d, are more recently isolated pigments. Chlorophyll c occurs- i n the-Phaeophyta, the Pyrrophyta and the Chrysophyta. One alga, Vaucheria. which has been included for a long time i n the Chloro-phyta and i s an exception to practically a l l the generalizations about the Green Algae, has been removed from that division.-and placed i n the Chryso-phyta because i t contains chlorophyll _c as the accessory chlorophyll instead of chlorophyll b. Vaucheria i s green in. colour and i s coenocytic, as are-all the Siphonales, the group to which i t was thought that Vaucheria belonged. I t does not store starch, however, as do a l l the other Chlorophyta; nor does i t contain cellulose i n the c e l l walls, as do most of the other Green Algae* In most respects i t f i t s very well i n the Chrysophyta. Chlorophyll c was isolated and shown to be present i n the Brown, Algae and the Diatoms by Strain and Manning (1942,, pp. 625-636). I t had been isolated before by other workers (Willst&tter and S t o l l , 1913) but was thought by them to be an art i f a c t . Later Strain and Manning-(1943, pp. 1-19) described another chlorophyllchlorophyll d, which they had found i n the Red Algae. The Cyahophyta or Blue-green Algae contain only chlorophyll a (Strain, i n Smith 1951, p. 253). The absorption spectrum of each of these chlorophylls i s distinc-tive (see Figures 4, 5 and 6, p. 6). I t i s one of the most helpful characteristics used i n the indentification of the different pigments. 6 Chlor- fl. Chlor- b Figure 4 . The absorption spectra of chlorophylls a and b i n ethyl ether (after Zscheil and Comar 1 9 4 1 , p. 4 6 8 ) . Chlor. c • • • • 1 * • * • * » * •* 600 600 400 500 600 700 Figure 5 « The absorption spectrum of chlorophyll c (after Strain and Manning 1 9 4 2 , p 7 6 3 3 ) Figure 6 . The absorption spectrum of chlorophyll (J (after^Manning and Strain 1 9 4 3 , p. 7 ) - . B. THE PHICOBILINS The phycobilins are water soluble red and blue prpteinapeous pigments found i n the Red Algae (Rhodophyta) and the Blue-green Algae (Cyanophyta)• Originally there were described only four different pigments, c-phycocyanin and c-phycoerythrin from, the Cyanophyta and r-phycocyanin and r-phycoerythrin from the Rhodophyta. More recent study, however, has shown 7 that these pigments are more variable than was previously supposed (Haxo, O'h Eocha and Norris, 1 9 5 5 ) . Haxo et a l found at least-four different pigments related to the conventional r - and c-phycobilins. Chemically the chromophoric group of these pigments i s related to the b i l e pigments. This chromophoric group i s attached very strongly to a protein* The pigments appear to be-derived from a hypothetical pigment called bilan (Lemberg, i n Rabinowitch 1 9 4 5 , p» 4 7 7 ) . The structure of bilan i s shown i n Figure 7 (p. 7 ) . N H 2 N HZ ft H 2 N _ _ H Figure 7 . Structural formula of bilan (modified after Lemberg i n Rabinowitch, 1 9 4 5 , P* 4 7 7 ) . C» THE CAROTMQIDS Carotenoids are fat soluble yellow, orange and red pigments. At least eighty different carotenoids are-known. They are distributed gener-a l l y throughout the plant kingdom and are quite widespread throughout the animal, kingdom. These pigments are composed basically of eight iaoprene units (CH25CH-C(CH3)=CH2). I t i s characteristic of these compounds also that they are reversed at the centre so that the methyl groups occupy the 1 : 6 position at the centre rather than the 1 : 5 position. Lycopene, although not common i n algae, i s a good example of the construction of these caro-tenoid pigments (see Figure 8, p. 7 ) « Of f i f t y carotenoid pigments whose empirical formulae are known, forty-five contain forty carbon atoms (Karrer and Jucker 1 9 5 0 , p. 2 9 ) . C H S C H 3 C H ^ C H 3 CH CMCHiCH-d=CHCHlcHC=CHC#t^CHCH=CCH4CHCH=CCHJCHCH f H C H 2 > C H 3 ' H 3 C C X C H 2 > ( 4 C H 2 Figure 81 Structural formula of Lycopene. 8 The nomenclature of these pigments i s rather confused, especially with regard to the term xanthophyll. In 1837 B era alius coined the term xanthophyll for what he believed to be a single yellow pigment found i n autumn leaves* Part of the pigment turned out to be the result, of the . presence of the-same pigment as that found i n carrot roots. This fraction was f i r s t called carotene but, i s now known to be a mixture of at least two pigments, * and {3 carotene* The term carotene i s f a i r l y well established as the generic name for the- unsaturated hydrocarbons such as lycopene* Willstatter and S t o l l (1913) f i r s t isolated l u t e i n , later found (GrillAm and Heilbron 1935, p. 1064) to be a mixture of lutein and zeaxanthin, from egg yolk* This pigment, lutein, i s the same as one of the pigments called xanthophyll by Berzelius (Rabinowitch 1945, p* 470). I t i s an alcohol and has many isomers. The term carotenol has been suggested to cover these pigments (Hogertj i n Rabinowitch 1945» p. 471) with the main pigment of the class called luteal. Karrer and Jucker (1950) give the generic name-as phytoxanthins and c a l l the main pigment xanthophyll. Strain (in Smith 1951, p. 245) suggests xanthophyll as the generic name of the oxygen derivatives of the carotenes, and c a l l s the chief pigment of t h i s class lutein. None of this nomenclature i s perfect. Carotenol leaves out a l l the oxygen derivatives which are not alcohols* The term phytoxanthins suggests that these are plant pigments only. Xanthophyll implies that the pigments occur only i n leaves. The last two mentioned do include a l l the oxygen derivatives of the carotenes, and i t i s just a matter of preference which i s used. The terminology suggested by Strain has been followed i n this thesis* The carotenoid pigments owe their colour properties to the large number of conjugated double bonds i n the molecule* There are, as. a.rulej eleven or more double bonds of which at least nine are conjugated. The individual pigments have characteristic absorption spectra* As a whole they have absorption spectra with three peaks between 400 mu and 500 mu, the centre one being the highest. As more double bonds enter the conjugat-ed system, a l l of these peaks are displaced towards the red end of the spectrum. The addition of one double bond to the conjugated system dis-places the maxima 20-22 mu towards the red. The addition of an isolated ethylenic bond displaces the maxima 9-11 mu toward the.red. I f a carbonyl group enters the conjugated system the effect i s very pronounced, result-ing i n a shifting of the maxima about 40 mu towards the red* These changes occur only, i n the terminal rings. I f one ring i s closed the are displaced 4-5 mu toward the blue. Introduction of a hydroxy! group has very l i t t l e effect (1-2 mu). Replacing a conjugated double bond with an epoxide group shifts the maxima 6-9 mu towards the blue* Most of these pigments are present as the trans-isomer but there are a few with one cis double bond* A change from the trans-isomer to the els form shifts the maxima 3-4 mu towards the shorter wave lengths. Cis-trans-isomerization i s readily induced by treating a solution of the pig-ment with strong mineral acids or heat (Karrer and Jucker 1950, p. 28)* ( i ) General Distribution of Carotenoids The carotenoid pigments are distributed throughout the plant kingdom. They are present i n coloured bacteria, coloured fungi, etiolated seedlings and a l l plants containing chlorophyll* How the plants form these pigments and what use they have for them i s obscure although numerous invest-igations have been carried out to find the answer to these problems. In animals these carotenoids are present sometimes i n the form of what might.be called provitamins (Karrer and Jucker 1950, pp. 11-15). One 10 molecule of y9 carotene- plus two molecules of water w i l l give two molecules of vitamin A (I.e. pp. 11-15). The pigments of the retina also appear to be carotenoid i n character (Karrer and Jucker 1950, pp. 16-17). Carotenoids are widely distributed throughout the animal world as colouring matter. Arthropods, molluscs, echinoderms, nemertians, nlychaetes, bryozoans, brachiopods., coelenterates, sponges, tunicates, f i s h , amphibians, reptiles, birds and mammals a l l contain carotenoids of one sort or another (Karrer and Jucker 1950, pp. 66-99). ( i i ) Distribution of Carotenoids i n the Algae The algae as a whole contain an interesting assemblage of caroten-oids. The Chlorophyta contain «. and (9 carotene i n varying amounts and up to half a dozen or more different xanthophylls. The xanthophylls are i n general the same as those found i n the higher plants. In addition to being found i n higher plants the following are also found i n the algae: lutein (usually about half the t o t a l xanthophylls present), violaxanthin, flavoxanthin, ne©<* xanthin, zeaxanthin, and possibly some cryptoxanthin* (Strain, i n Smith 1951, P. 251). The Siphonales, a coenocytic order of the Chlorophyta, contain- two carotenoids, namely siphonein and siphonaxanthin (Strain, i n Smith 1951, p. 251) which have not been found i n any other order of Green Algae. This order also contains a carotene as the chief carotene, i n contrast to most other plants where |3 carotene i s the most common. The Phaeophyta and the Bacillariophyceae i n the Chrysophyta contain f3 carotene and a number of xanthophylls found nowhere elso i n the plant king-dom. These pigments are fucoxanthin, which i s very abundant, neofucoxanthin A and B. The Brown Algae also contain violaxanthin, flavoxanthin and neo-#Cryptoxanthin was never found during the course of this research. 1 1 xanthin. The Diatoms (Bacillariophyceae) have diatoxanthin and diadinoxan-thin. The Dinophyceae i n the Pyrrophyta contain peridinin, which i s the most abundant xanthophyll present, diadinoxanthin, dinoxanthin, and neodinoxanthin (Strain, i£ Smith 1 9 5 1 , p . 2 5 3 ) , (see Table 1 , p. 1 1 ) . Pigment Phaeophyceae Bacillariophyceae Dinophyceae fucoxanthin neofucoxanthin A neofucoxanthin B violaxanthin flavoxanthin neoxanthin diatoxanthin diadinoxanthin peridinin dinoxanthin neodinoxanthin Key * present - not present Table 1 . The distribution of xanthophyll pigments i n the Phaeophyceae, the Bacillariophyceae and the Dinophyceae (modified after Strain, i n Smith 1 9 5 1 , p. 2 5 3 ) . The Red Algae as a rule have only one or two xanthophylls, one of which i s always lutein. The Cyanophyta (Myxophyta of some authors) contain myxoxanthin and myxoxanthophyll. Fucjoxanthin has been reported i n Poly-slphonia nigrescens and: Callithamnion -pikeanum (Carter, Heilbron and Lythgoe 1 9 3 9 , p. 1 9 3 ) * Both these Red Algae are much branched, filamentous forms and are commonly covered with diatoms. Unti l these algae can be obtained i n a diatom free state, the presence of fucoxanthin i n Red Algae must be regard-ed as questionable. 17. GENERAL DESCRIPTION OF METHOD Two general methods were used i n the work. Fir s t was the method of Richards and Thompson ( 1 9 5 2 ) for characterising plankton populations using 12 gross pigment extracts. This w i l l be discussed more f u l l y under the section dealing with procedure. Second was the method chosen for the separation of the xanthophyll pigments. This was the chromatographic column. Because these xanthophyll pigments are so similar chemically the standard purification methods of sep-aration using immiscible solvents followed by recrystalization are very unsatisfactory unless they are used i n conjunction with a highly sensitive method such as, chromatography. So far chromatography i s the only method known which, w i l l permit- separation of.these pigments i n a pure enough state to identify them. The*^chromatographic column i s made up of a finely divided material which i s packed into a tube such as the one shown i n Figure 9 (p. 12). The — adsorbing properties of the material used must be considered i n selecting a suitable adsorbent* The materials, to be selected as adsorbents vary greatly i n their adsorption properties. Sub-stances such as powdered sucrose or starch have weak adsorption properties whereas those such as magnesia (Micron Brand) or charcoal are the most active adsorbents. The adsorption capacity of a l l adsor-bents, however, depends on the solvent used. Adsorption i s greatest from saturated hydrocar-Figure 9* bons such as petroleum ether, less-from cyclic hydrocarbons and 'chlorinated hydrocarbons such as benzene and ethylene chloride, s t i l l less from alcohols and least of a l l from water* The combination of adsorbent and solvent used i s of great importance 13 and the choice i s made through references to the literature and through the t r i a l and error method. In this research a combination of magnesia and ethylene chloride was found most satisfactory. Powdered sugar (C & H Brand), petroleum ether and benzene were t r i e d but did not y i e l d good results. Some-times a l i t t l e methanol (1$ to 5/6) was added to the ethylene chloride to speed up the developing process. V. PROCEDURE The samples used for this work were collected from freshwater lakes, streams, ponds, ditches and puddles, from various marine substrata at low tide, from floats, and from unialgal cultures. The organisms were identified to genus and wherever possible to species. A. GROSS PIGMENT EXTRACTS As a rule a sample of not more than 50 ml. of material was requir-. ed for a complete pigment analysis using the method proposed by Richards and Thompson (1952). The organisms were concentrated by f i l t e r i n g the sample through a millipore aersol f i l t e r which was then dried i n a vacuum dessicator. The sample was then extracted for twelve hours i n the refrigerator with 5 a l . of acetone. The extract was decanted after centrifuging to remove the cells and undissolved f i l t e r . From this extract a complete spectral analysis between wave lengths 350 mu to 700 mu was recorded, reading optical densities at every 10 mu. A Beckman DU Spectrophotometer was used for these readings. Where samples con-taining a mixture of pigments were analysed the calculations were made according to formulae given by Richards and Thompson (1952> p. 158), to determine the relative amounts of chlorophylls a, b, £, astacin and non-astacin carotenoids present. 14 For those samples used only to calculate these five pigments or pigment groups, optical density readings were taken at 480 mu, 510 mu, 630 mu, 645 mu and 665 mu. These wavelengths represent the points of least inter-ference i n the curves for chlorophylls a, b, and c, astacin and non-astacin carotenoids respectively. B. CHROMATOGRAPHIC WORK In preparing^ the sample for extraction, several different methods were used to remove a l l the excess water from the plants. Unicellular forms were centrifuged with a Foerst continuous flow,centrifuge. This method yielded samples-having a volume of from one to two m i l l i l i t r e s of c e l l s . Filamentous forms were washed i n tap water to remove as many contaminants, both plant and animal, as possible, then squeezed dry and extracted. Samples weighed from five to ten grams when treated i n this way. The samples were extracted for two to ten hours (depending on the ease of extraction) i n the dark using absolute methanol as the extractant. Methanol i s a useful solvent because i t i s water soluble and w i l l thus pen-etrate the cells of the fresh organisms and extract the soluble carotenoids and chlorophylls. After extraction the solution was f i l t e r e d and then saponified with alcoholic KOH (15 gm. KOH to 150 ml. methanol solution). Saponifaction makes the chlorophylls water soluble and releases any of the carotenoid pigments which are present as esters. The extract was diluted with about 400 ml. of concentrated sodium chloride solution and the carotenoid pigments removed with ethyl ether. The ether solution was washed with d i s t i l l e d water (to remove the methanol) and then dried with calcium chloride. The resulting ethereal solution was then evaporated under vacuum u n t i l a l l of the ether was gone. The pigments 15 obtained were then redissolved i n a small amount of ethylene chloride (CH2CICH2CI) and this solution was poured through a column containing a mix-ture of magnesia (Micron Brand) and Hyflo Super Cel i n equal amounts. The column was*then washedwith fresh ethylene chloride. I f the pigments were strongly adsorbed i t was usually found better to develop the column with ethylene chloride to which 1 to % methanol was added. I f the pigments were less strongly adsorbed, ethylene chloride alone was found satisfactory* When the pigments separated into distinct bands the column was sucked almost to dryness. Best results were obtained when the chromatographs were run under suction provided by a high vacuum pump. At thi s point the column was removed.from i t s container and the pigment bands were separated manually. The pigments were eluted with 95$ ethanol and the magnesia and Hyflo Super Cel were removed by Centrifuging fdr five minutes at about 4000 rpm. Packing the column, according: to the literature (strain 1945$ p. 41; Karrer and Jucker 1950, p. 27) Is a c r i t i c a l feature i n making a satisfactory chromatographic column. According to these authorities i t must be done by pouring i n about one centimeter of adsorbent at a time and tamping i t down with a rod of some sort. This procedure was t r i e d but gave unsat-isfactory results* When the columns were prepared i n this manner, the f i l t r a t i o n rate was very slow, the pigments would not separate and the column could not be removed from the container* The method f i n a l l y used was as follows. Half and inch of adsorbent cotton was placed at the bottom of the container so that the material would not run right through the tube* About one centimeter of adsorbent at a time was placed i n the tube and the suction flask under the column was struck on the table a few times following each addition so that the adsorbent settled 16 into a f a i r l y compact column. Suction was applied to help pack the column. The f i n a l portion of adsorbent at the top of the column was tamped down with a cork on a rod. This gave a f a i r l y firm surface on which the pigment mix-ture and developer could be poured. The columns were 2.5 centimeters i n diameter and from 10 to> 30 cm. long. The length varied directly with the number and type of pigments to be separated. The columns packed i n this manner gave good results and required only about five minutes to prepare. The columns required from f i v e to twenty minutes to develop. Having obtained the ethanolic solution of the pigments with the procedure outlined above, a complete absorption spectrum was run on each pigment on the.Beckman DU Spectrophotometer. Optical density readings were taken at 350 mu through to about 500 mu. When approaching the maxima, read-ings of optical density were taken at every 2 mu on the scale, this being the readable limit of the Beckman Spectrophotometer between 400 mu and 500 mu. Between the maxima, readings were taken at every 4 mu and near the extremities of the curves at every 10 mu. These readings were graphed with the wave lengths as the abscissa and the optical density as the ordinate. Identification of the pigments was based primarily on the absorp-tion curve and the position of the maxima. The following features were also used i n identifying the pigmentsj the shape of the absorption curve, the position and colour of the pigment oflthe column and the reaction of a diethyl ether solution of the pigment with 37$ HC1. With some pigments the acid layer turns blue (Karrer and Jucker 1950, p. 7). VI. RESULTS The following table shows the results obtained using the method outlined by Richards and Thompson (1952, pp. 156-172). The numbers indicate the relative concentrations of chlorophylls a, b and c, astacin and non-17 aatacin carotenoids. Alga Chloro- Chloro- Chloro- Astacin Non-astacin phyll a phyll b phyll c CHLOROPHYTA Order VOLVOCALES Dunalieila 5.291 .730 .593 — 3.146 Chlamydomonas 6.391 2.703 3.042 — 2.903 Haematococcus 4.784 1.991 2.120 3.602 1.497 Volvox 4.089 1.874 2.293 2.347 Order CHLOROCOCCALES Chlorella 1.796 ,328 .790 — 1.626 Order ULOTRICHALES Chaetophora 1,913 .613 .116 — .745 Order ULVALES Ulva 5.314 3.103 4.264 — 1.115 CHRYSOPHYTA Class BACILLARIOPHYGEAE F r a ^ l a r i a 4.332 .956 2.943 .106 1.915 Table 2. Relative concentrations of Chlorophyll a, b, c and astacin and non-astacin carotenoids as calculated from formulae given by Richards and Thompson (1952, p. 158). Figures 10 to 16 show the curves obtained from spectrophot©metric analyses of gross pigment extracts i n 90% acetone. Some of the data i n Table 1 have been obtained from these same extracts. The following table, Table 3 , contains the names (where known), the structural formulae (where known), the absorption maxima, the absorption spectra and the reactions with HC1 of a l l the pigments studied and discussed i n this thesis. The maxima, unless otherwise indicated, are given for ethanolic solutions of the pigments and those ma-rjma tram, the literature^ unless otherwise indicated, are from Karrer and Jucker (1950). Table 3. The oharaoterization of the different xanthoph; (oontinued on page 19) PIGMENT MAXIMA (in mu) Literature Found Lutein (17, 23>* 476, 446.5, 420 476, 446, indis-tinot Violaxanthin (18, 24) 471.5, 442.5, 417.5 472, 442, 418 Taraxanthin (25) 472, 443, 418 472, 442, 418 Neoxanthin (26) 467,437,415 467,437,414 Flavoxanthin (19, 27 448, 421, not given 452, 424, 402 a and b) 452, 422, not given (Strain, 1938) Zeaxanthin (20, 28) 483, 451, 423.5 480, 451 482, 452 (Strain, 1945) 479, 452 (Zsoheile et a l , 1942) Astaoene (21, 29) 510 (in CSg) 478 500 (in pyridine) 480 (approximately in ethanol) pigments found during the work for this thesis. REACTION WITH COMMENTS  57% HOI no reaction Forms a diffuse"yellow orange band on the adsorption column whioh moves down the oolumo. f a i r l y rapidly* stable blue Forms a f a i r l y sharp band above colouration lutein and is orange yellow i n oolour. no reaction Forms a sharp yellow band above violaxanthin. stable blue Forms a sharp, bright yellow band colouration near the top of the oolumn« stable blue Forms a pale yellow band just below colouration neoxanthin on the column. no reaotion This pigment moves f a i r l y rapidly down the oolumn separating just above the lutein band. It is red orange in oolour and quite distinc-t i v e . no reaotion This pigment is usually found in animals (Kuhn and Lederer, 1933, p. 488), although i t i s also found in Buglena and Haematooooous (Tisoher, 1944). It moves rapidly through the oolumn and the band is a bright pink Table 3. The characterization of the different xanthoph; (concluded on page 20). PIGMENT MAXIMA (in mu). Literature Pound Auroxanthin (22, 30 430, 402, 382 430, 404, 383 a and b) IM known Xan- 472, 451 thophyll 1 (31) Unknown Xanthophyll (423j) 472, 446 2 (32) Unknown Xanthophyll 3 (33) Unknown Xanthophyll 4 (34) 472, 444, 420 472, 445, 424 pigments found during the work for this thesis REACTION WITH 87% BC1 COMMENTS light blue oolour This pigment is strongly adsorbed and forms a light yellow band at the top of the column. The adsorp-tion spectrum is very similar to that reoorded for auroxanthin. However, aooording to Karrer and Juoker (1950, p. 197) auroxanthin characteristically forms a blue oolour with 15$ HC1. This pigment does not. In spite of this difference i t was tentatively deoided to refer to this pigment ob-tained from StigeooIonium farotum and Spangomorpha ooalita as auroxanthin. This pigment ooours as a diffuse red orange band above the lutein band. It separates slowly from lutein and has only been found in red algae of the Bangioideae. This pigment separates slowly from . neoxanthin when 2$ methanol is added to the ethylene ohloride when devel-oping. It forms a f a i r l y sharp orange band and never ooours in large amounts* blue Forms a yellow orange band near the oolouration middle of the ooluim« no reaction no reaotion no reaotion This pigment forms an orange band below lutein on the ooluma. It was found only in Spongomorpha ooalita. PIGMENT MAXIMA (in mu) REACTION WITH Literature Found 37% HC1 COMMENTS Unknown Xanthophyll 479, 450 5 (35) Unknown Xanthophyll 6 (36) no blue oolour- This pigment forms a diffuse ation but aoid yellow orange band, very much layer yellow. like that of lutein, below zeaxanthin on the column* 477, 447, 424 — This pigment was only found once i n this surveys Table 3* The oharaoterization of the different xanthophyll pigments found during the work for this thesis *The Figure numbers of the structural formulae and the absorption spectra respectively are given in brackets after the name of the pigment* Where only one Figure number is given i t i s for the absorption speotrum. 21 CH 3 /CH 3 CH3/CH5 C CH3 CH3 CH3 CM3 / C N i CH2 CCH=CHC=CHGH=CHC = CHCH=CHCH=C-CH=CHCH=CCH=CH-CH CHj < HOCH .CCH3 H3C CJ ^HOH \ H Z CH Figure 17* Structural formula of lutein. / C \ ? H 3 C H 3 CH3 CH3 C I CH2 C-CH=CHC»CHCH=CHC=CHCH=CHCH=CCH=CHCH=CCH=CHC CH2 1 HOCH c£ C H O H ! CH2 CH3 CH3 NCH 2 i Figure 18. Structural formula of violaxanthin* C H , / C H 5 ; CH2 C=CH CH3 CH3 CH3 CH3 .C ' HOCH /C CH C=CHCH=CH-C=CHCH=CHCH=C CH=CHCH=C CH=CH CH CH» CHjl 0 H3CC CHOH c h 3 \ H ; Figure 19. Structural formula of flavoxanthin. CH3 /Hj, . CH3 / C H 3 ~ X ? H 3 C H * C H 3 CH3 > CH2 CCH=CHC=CHCH=CHC=CHCH=CHCH=CCH=CHCH=CCH=CHC CH2 t H I HOCH VCCH S HjC'C .CHOH ^CH2 _ N c ^ Figure 20. Structural formula of zeaxanthin. C ^ 3 /CH3 ' ~ ~ <U*3 /CH3 > l C H 3 C H 3 <?H3 C H » CH» X-CH-CHC-CHCH=CHC«CHCH=CHCH=C CH=CHCH=CCH=CH-C CH, i H II I , CO C C H i H.C-C .CO ! _ _ • \< Figure 21. Structural formula of aetacene. CHj / C H 3 C H , C H 3 C / \ / \ C H 2 C = C H C H 3 C H 3 C H 3 C H 3 C H = C CHZ , H O C H C C H C = C H C H = C H C = C H C H = C H C H = C C H = C H C H = C • C H C £ H O H ! CH2 0 0 ' CH2 i C H 3 C H 3 Figure 22. Structural formula of auroxanthin. 22 For the distribution i n the algae of the fourteen pigments studied i n this research and summarized i n Table 3 (pp. 18-20), see Table 4 (pp. 23 and 24). VII. DISCUSSION A. GROSS PIGMENT EXTRACTS The method proposed by Richards and Thompson (1952) was i n i t i a l -l y devised for the characterization of plankton populations i n the-ocean. Its advantage l i e s i n the fact that only small samples (1-2 l i t r e s ) of seawater are-required. Diatoms (Bacillariophyceae) and Dinoflagellates (Pyrrophyta) apparently make up the major part of the phytoplankton of the sea. Since these contain only chlorophyll c as an accessory chlorophyll i t can be assumed, that whenever any chlorophyll b i s indicated by this method there are probably some green flagellates, i n the nannoplankton. Since these flagellates usually disintegrate with the customary methods used at sea for preservation of. plankton, i t would be useful i f this method could be adopted to give an indication of their presence. I t was hoped that this method would be of some use i n this research and thus eliminate the tedious procedure of separating the pigments individually. Unfortunately the data obtained from the few samples analysed i n this way show that i t i s d i f f i c u l t to interpret the results obtained. Whenever chlorophyll b was present, as undoubtedly i t i s i n Ulva, very high read-ings were obtained for chlorophyll c. I t i s impossible that a species of Ulva could be so contaminated with diatoms that the chlorophyll c content would be almost as high as the chlorophyll a, especially since the diatoms also contain chlorophyll a as their major chlorophyll. Hence, i t appears that the presence of any chlorophyll b interferes with the chlorophyll c Table 4. Distribution of xanthophyll pigments i n the different algae examined ALGA CHLOROPHYTA Order VOLVOCALES Phacotus sp, Lobomonas pyriformas  Yolvox sp. Order CHLOROCOCCALE3 Chlorella sp, Pediastrum boryanum Order ULOTRICHALES Stigeoclonlum faretum  Draparnaldia sp. Order 0ED0GONIALES Qedogonium sp. Order ZYGNEttATALES -Spirdgyra varians^  Spirogyra sp, Zygagoniam ericetoreaa  Sirogonium sp, Mougeotia sp. Mougeotia sp. Order GLADOPHORALES Spongomorpha cdalita  Urospora penicilliformis Order SCHIZOGONIALES Prasiola merldionalis E < • ™ o p> a » N *' I i l l " «*• c+- C+ f+ o' • c«-a a a a a a 1 £ § 1 _ _ # * * - * - - * -- - * 1 H to \J» p- \n 0-* * tt - # - - * # * - • - . * - - - - - . - - * ; - . - • • -* -c B B E E K « * " c+ 5 P 5 5 5 $u a> £t £ £t £ St ° tr cr pr jr cr « ALGA "^ is" S b fcJ fcJ a> b : H 10 u> 4sr Nja ON Order ULVALES Snteromorpha intestinalis * * * Enteromorpha linza *• * Ulva expansa •a- * * Ulva sp. •* * Ulva stenophylla * EUGLSNOPHYTA Euglena sp. CHRYSOPHYTA Class XANTHOPHYCEAE Vaucheria sp. RHODOPHYTA Subclass BANGIOIDEAE Bangia fucospurpurea < < - _ _ _ _ _ _ _ - & _ _ _ _ Porphyra perforata Subclass FL0R3DEAE Antithamnion pacifica  Irid&ea heterocarpum Table 4 (concluded). Distribution of xanthophyll pigments i n the different algae examined. Key * present - absent 25 reading to such an extent that the values obtained from the readings are very incorrect, even after the corrections introduced by Richards and Thomp-son (1952, p. 156) have been applied. And conversely, although not to such an extent, any chlorophyll c present w i l l result i n excess chlorophyll b values. I t i s therefore necessary to be extremely cautious i n drawing con-clusions from data obtained using the method proposed by Richards and Thomp-son. In spite of the limitations apparent i n the method, certain d i f -ferences are suggested by comparing the complete absorption spectra of mixed pigment extracts. There i s a distinct irregularity i n the curve between 46O mu and 470 mu i n some instances. At this point chlorophyll b has a max-imum. In most of the Green Algae that were analyzed (Figures 10-13) this point showed up more or less clearly. In analyses of. samples of the genus Dunaliella, however, there i s no chlorophyll b maximum (Figure 14). Even from the calculation for chlorophyll b using Richards and Thompson's formulae very l i t t l e chlorophyll b or c was indicated i n this alga. Dunaliella i s generally classified i n an order, the Volvocales, of the Chlorophyta. Since the Chlorophyta generally contain chlorophyll b as the accessory chlorophyll, one would expect to find chlorophyll b i n this alga. I t i s possible that chlorophyll b is. present i n very small, quantities i n the flagellate, but since no chromatographic study of i t s pigments has been made, i t i s impos-sible to say how valid this assumption may be. B. CHROMATOGRAPHIC WORK The.Volvocales, as a group, seem, to be quite variable i n their pigmentation, both with respect-to. their- chlorophylls and xanthophylls. Three genera, Phacotus. Lobomonas, and Vplyox, i n this group were studied with respect to their xanthophyll pigments and i t was found that they were 26 rather diversified. Of the 1 three*genera examined each had three different xanthophylls* Phaftotus and Lobomonas had two pigments i n common, violaxanthin and zeaxanthin. Volvox had one pigment>taraxanthin, i n common with Lobomonas, and none the same as those of Phacotus. The Volvocales are i n many respects a rather diverse group. Veg-etatively they vary from unicellular be- or quadra-flagellate forms through colonies of many shapes. The colonies may be f l a t - plates such as Gonium -or spherical colonies such as Pandorina, or hollow spheres* such as Volvox. The reproductive cells vary from isogamy i n Dunaliella, through heterogamy i n many of the Chlamydomonas species, to oogamy i n Volvox. I t i s perhaps reasonable to find that their pigments also are rather variable. Genera which appear to be morphologically uniform and f a i r l y closely related, such as the members of the Zygnematales, contain quite similar pigments.. The Zygnematales i s an order of Green Algae, which, a l -though f a i r l y diverse morphologically, possesses a unique method of sexual reproduction called conjugation. The order consists of two groups, the> filamentous forms such as Spirogyra and Zygnema and the unicellular and colonial Desmids. In this order four different genera, Spirogyra, Zygagonium, Siro-gonium and Mougeotia, and six different species were examined. I t was found that zeaxanthin was always present, and. unknown, xanthophyll, xanthophyll 5, was present i n five out of s i x species and lutein was absent i n the same five out of s i x entities, Xanthophyll 5 apparently can take the place of lutein. The absence of lutein i n these organisms i s of interest because, according to Strain (in Smith, 1951, pp. 251 and 253) lutein i s always present i n the Chlorophyta and usually as the major-xanthophyll. I t was lacking i n Phacotus (Volvocales), Lobemonas-(Volvocales}.-. Stigeoclonium farctum (Ulotrichales) and Pediastrum boryanum (Chlorococcales). 27 The closely related marine genera Ulva and Enteromorpha were found to contain very similar pigments, although Ulva.-usually had one-or two more carotenoids - than the more- primitive Enteromorpha* Prasiola, which i s an alga having a parenchymatous organization, i n appearance very much l i k e a dimin-utive Ulva, grows along the seashore i n the splash zone on rocks where there i s a plentiful supply of seagul droppings. There has been some debate (Fritsch 1935, p. 220) concerning the order i n which Prasiola should be placed. Some phycologists have placed i t i n the Ulotrlcales (Fritsch 1935, p. 217) and others have removed i t to an order of i t s own, the. Schizogoniales (Smith 1950, p. 195). A study of the pigments i n Prasiola reveals that this plant con-tains the same three pigments (lutein, violaxanthin and neoxanthin) found i n Enteromorpha, but i t possesses two additional pigments (zeaxanthin and an unknown xanthophyll, xanthophyll 3) not found i n any of the members of the Ulvales studied; This suggests a common origin, at least of the Ulvales and the Schizogoniales. Although these three pigments - lutein, violaxanthin and neoxanthin - are distributed quite commonly throughout the Green Algae> they occur together-only i n three other algae examined! namely, Vovox sp.« Chlorella sp., and Urospora penicilliformis. Figure 37 (p. 2S) which illustrates-a generally accepted modern phylogenetic arrangement (after Papenfuss 1951, p. 6) of the-orders of the Green Algae, indicates that the Volvocales (Volvox) and the Chlorococcales (Chlorella) are i n the evolutionary lines leading to the Ulvales and the Cladophorales (Urospora). The xanthophyll pigments of these algae could be used to support the arrangement i n Figure 37. However the pigments of other, genera studied i n these orders do not support the arrangement as well. More exhaustive study of more species and genera would have to be done before any definite conclusions about the phylogenetic relationships of these orders could be 28 ULOTRICHALES ULVALES SCHIZOGON; OEDOGONIALES ZYGNEMATALES DASYCLADALES SIPHONALES SIPHONOCLADALES CLADOPHORALES CraOROCOCCALES VOLVOCALES Figure 37* Diagrammatic representation of the probable inter-relationships of the orders of Green Algae (Papenfuss 1951, p. 6).: drawn at the ordinal level using the xanthophyll pigments as a criterion* I t i s probable that the presence of any individual xanthophyll pigment has l i t t l e phylogenetic significance* The pigments are very similar chemically and perhaps somewhat interchangeable physiologically* The d i f -ferences between any two xanthophylls i s sometimes not very great chemically and could possible result from a single gene change (mutation) i n the nucleus. I f more were known about the function and mode of formation of the xanthophyll pigments, speculation about their phylogenetic significance would probably be more f r u i t f u l . While the presence of a single xanthophyll has probably l i t t l e phylogenetic importance, the consistent presence of two or more xanthophylls as i n the case of the Ulvales and Prasiola meridionalis (Schizogoniales), i s possibly significant. This i s also true of the Zygnematales where zeaxanthin and Unknown xanthophyll 5 were almost always present together* However these two. pigments were found together i n the unrelated Pediastrum boryanum 29 (Chlorococcales) and Stigeoclonium farctum (Ulotrichales). Lutein occurs commonly i n the Rhodophyta and i s frequently the only xanthophyll present (Strain, i n Smith 1951, p. 253} Carter et a l 1939, p. 102). The two members of-the Bangioideae which were examined, Bangia  fucospurpurea•and Porphyra perforata, contained lutein and one other unidentified xanthophyll, Unknown xanthophyll 1. Unfortunately there were no other members of this group available for a comparative study. VIII. SUMMARY AND CONCLUSIONS The Richards and Thompson method of characterizing plankton pop-ulations by gross pigment analyses requires more thorough investigation before results obtained by this method can be interpreted with any degree of confidence. This study shows, however, that the complete absorption spectra of gross pigment extracts w i l l frequently indicate the presence or absence of certain pigments, particularly chlorophyll b. The carotenoid pigments of-the Green Algae are surprisingly con-sistent considering the great variation i n size, form, c e l l structure and the methods of reproduction found i n this group. Orders such as the Volvo-cales, i n which there i s much morphological and reproductive diversity, appear to be quite variable i n their pigmentation. Three genera, Phacotus, Lobomonas and Vol vox, i n theVolvocales were examined. Each had three d i f -ferent xanthophyll pigments with a to t a l of six different xanthophylls i n the group. Phacotus and Lobomonas had two xanthophyll pigments (taraxanthin and zeaxanthin) i n common. Volvox had one pigment (violaxanthin) i n common with Lobomonas and none the same as those of Phacotus. The orders Zygnematales and Ulvales, i n which the method of sexual reproduction i s f a i r l y uniform, were found to contain groups of two or three xanthophyll pigments f a i r l y regularly. The Zygnematales usually contained 30 zeaxanthin and Unknown xanthophyll 5, plus a number of other pigments which appeared less consistently. The group of pigments characteristic of a l l members of the Ulvales examined consisted of lutein, violaxanthin and neo-xanthin. The presence of one xanthophyll pigment alone i n an alga does not appear to have any phylogenetic significance. The consistent presence of groups of pigments appears to be more important phylogenetically. In general the results obtained from the pigment analyses support relationships recognized on the basis of morphological and reproductive characteristics. Some phylogenetic considerations have been suggested, but these should be supported by a -more exhaustive study of more species and genera before futher conclusions can be drawn. 31 — i 1 1 1 L__: ' i -4 0 0 500 6 0 0 X i n m ^ Figure 10. Absorption spectrum of a gross pigment extract of Ulva sp. i n acetone. 32 Figure 11. Absorption spectrum, of a gross pigment extract of Chlorella sp. i n acetone. 400 500 s o o X in m/i. Figure 12. Absorption spectrum of a gross pigment extract of Chaetophora sp. i n 30% acetone. 6 0 0 Xinm/t 3 3 Figure 13* Absorption spectrum of a gross pigment extract of Chlamydomonas sp. i n 90$ acetone. 34 Figure 14* Absorption spectrum of a gross pigment extract of Dunaliella sp. i n 90% acetone. 35 Figure 15* Absorption spectrum of a gross pigment extract of Volvox sp. i n 90% acetone. 36 Figure 16. Absorption apectrum of a gross pigment extract of Fragilaria sp* i n 90% acetone. 37 Figure 23. Absorption spectrum of Lutein i n 95$ ethanol. f • • 1 * * i i I i i r i 400 450 X in m/i Figure 24. Absorption spectrum of Violaxanthin i n 95% ethanol 3a DENSI • • / \ ICAL / \ OPTI •i TARAXANTHIN ^ 4 0 0 ' 450 ' X i n m / i Figure 25. Absorption spectrum of Taraxanthin i n 95$ ethanol. Figure 26. Absorption apectrum of Neoxanthin i n 9% ethauox. 39 Figure 27a. Absorption spectrum of Flavoxanthin i n 95$ ethanol. — i 1 i 4 00 4 50 Xin^l Figure 27b. Absorption spectrum of Flavoxanthin i n 95# ethanol (from Karrer and Jucker 1950, p. 264). 40 Figure 28. Absorption spectrum of Zeaxanthin i n 95$ ethanol. Figure 29. Absorption spectrum of Astacene i n pyridine (from Karrer and Jucker 1950, p. 265). 41 AUROXANTHIN • • i • I '. i I i i 1—:—l 40 0 450 X in rryt Figure 30a. Absorption spectrum of Auroxanthin i n 95$ ethanol. Figure 30b. Absorption spectrum Of Auroxanthin i n benzene (from Karrer and Jucker 1950, p. 263). 42 UNKNOWN I i i i • i i i i i I i i—.—i i 400 450 X in m/x. Figure 31• Absorption spectrum of Unknown Xanthophyll 1 i n 95$ ethanol. UNKNOWN 2 1 i i > 1 l 1 1 i i I i • • • i | 400 450 X in m/i. I Figure 32.' Absorption spectrum of Unknown Xanthophyll 2 i n 95$ ethanol. 4 3 4 ° o 450 X in m/i Figure 3 3 . Absorption spectrum of Unknown Xanthophyll 3 i n 9% ethanol. Figure 3 4 . Absorption spectrum of Unknown Xanthophyll 4 i n 9% ethanol. 4 4 Figure 35. 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