Consistent Sets of Spectrophotometric Chlorophyll Equations for Acetone, Methanol, And Ethanol Solvents

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  Abstract  A set of equations for determining chloro-phyll  a  (Chl  a ) and accessory chlorophylls  b ,  c 2  , c 1  + c 2 andthespecialcaseof   Acaryochlorismarina ,whichusesChl d asitsprimaryphotosyntheticpigmentandalsohasChl  a , have been developed for 90% acetone, methanoland ethanol solvents. These equations for differentsolvents give chlorophyll assays that are consistent witheach other. No algorithms for Chl  c  compounds ( c 2  ,c 1  + c 2 ) in the presence of Chl  a  have previously beenpublished for methanol or ethanol. The limits of detection (and inherent error, ± 95% confidence limit),for chlorophylls in all organisms tested, was generallylessthan0.1  l g/ml.TheChl a and b algorithmsforgreenalgae and land plants have very small inherent errors(< 0.01  l g/ml). Chl  a and d  algorithms for  Acaryochl-oris marina  are consistent with each other, giving esti-mates of Chl  d/a  ratios which are consistent withpreviouslypublishedestimatesusingHPLCandararelyused algorithm srcinally published for diethyl ether in1955. The statistical error structure of chlorophyllalgorithms is discussed. The relative error of measure-ments of chlorophylls increases hyperbolically in di-lutedchlorophyllextractsbecausetheinherenterrorsof thechlorophyllalgorithmsareconstantsindependentof the magnitude of absorbance readings. For safety rea-sons, efficient extraction of chlorophylls and the con-venience of being able to use polystyrene cuvettes, thealgorithms for ethanol are recommended for routineassays of chlorophylls. The methanol algorithms wouldbe convenient for assays associated with HPLC work. Keywords  Chlorophyll  Æ  Acetone  Æ  Methanol  Æ Ethanol  Æ  Algorithms  Æ  Chlorophyll  a  Æ  Chlorophyll  b  Æ Chlorophyll  c 2  Æ  Chlorophyll  c 1  + c 2  Æ  Chlorophyll  d  Æ Error structure Introduction Measurements of chlorophyll content of oxygenic pho-tosynthetic organisms are a fundamental measurementin many branches of plant biology and ecology. Theamountofchlorophyllinphotosyntheticorganismsisanimportant measurement in itself, particularly for chlo-rophyll  a  (Chl  a ), which is the primary photosyntheticpigment in nearly all known oxygenic photosyntheticorganisms. The presence or absence of other chloro-phylls( b, c 1  ,c 2  ,c 3 and  d )isoftaxonomic importance.Inaddition, the relative amounts of secondary chloro-phylls, such as Chl  b  in chlorophyte algae and vascularplantscomparedtoChl a varieswithbothlightintensityand the spectral quality of light (Atwell et al. 1999).Chlorophyll  a  (Chl  a ) or sometimes total chlorophyll isused as the standard basis on which to calculate photo-syntheticandrespiratoryrates(molO 2 .mgChl  a –1 .hr –1 ),themetabolicallyactivebiomassandtheproductivityof terrestrialandaquaticecosystems(gC.mgChl  a –1 .d –1 ).The amount of chlorophyll per unit of protein iscrucial in studies of chlorophyll-protein complexesbecause knowing the number and type of chlorophyllsin each type of chlorophyll complex is important inworking out their structure and function (Porra et al.1989; Porra 1991, 2002). For example, Hu et al. (1998) showed in the novel organism  Acaryochloris marina that the photosynthetic reaction centre of PS I usedChl  d  rather than Chl  a  as in other oxygenic photo-synthetic organisms. R. J. Ritchie ( & )School of Biological Sciences, The University of Sydney,Sydney, NSW 2006, Australiae-mail: Res (2006) 89:27–41DOI 10.1007/s11120-006-9065-9  1 3 REGULAR PAPER Consistent sets of spectrophotometric chlorophyll equationsfor acetone, methanol and ethanol solvents Raymond J. Ritchie Received: 1 August 2005/Accepted: 8 December 2005/Published online: 9 June 2006   Springer Science+Business Media B.V. 2006  This paper presents a consistent set of algorithms forthe routine assays of chlorophylls  a, b, c 1  ( c 1  + c 2 ) and d  in acetone, methanol and ethanol solvents which giveclosely similar estimates of the chlorophylls. Chloro-phyll estimates can now be made using either acetone,methanol or ethanol solvent systems appropriate to theparticular experimental situation. Previously publishedalgorithms for estimation of chlorophylls using differ-ent solvents such as acetone and methanol were onlyavailable for Chl  a + b  and in any case were not alwaysconsistent with each other (Lichtenthaler 1987).Although acetone solvent gives very sharpchlorophyll absorption peaks and so is the solvent of choice for chlorophyll assays (see Arnon 19490;Jeffrey and Humphrey 1975; Jeffrey et al. 1997;Humphrey and Jeffrey 1997; Porra et al. 1989, Porra1991, 2002; Wright et al. 1997), acetone is sometimes apoor extractant of chlorophyll from many vascularplants and some algae, particularly green algae such as Scenedesmus ,  Chlorella  and  Nannochloris  (Sartoryand Grobbelaar 1984; Porra et al. 1989; Porra 1991,2002; Jeffrey et al. 1997; Wright et al. 1997). Methanoland ethanol are often more efficient extractants(Lichtenthaler and Wellburn 1983; Sartory andGrobbelaar 1984; Wright et al. 1997; Lichtenthaler1987). Neutralised methanol and ethanol were used inthe present study to avoid formation of allomerisationproducts of chlorophylls, which are spectrally differentto chlorophylls. Porra (1990) found that 1.5 mMsodium dithionite improved extraction of chlorophyllsfrom recalcitrant algae and discouraged the formationof breakdown products in methanol solvent (use of sodium dithionite would not affect the equationsdeveloped in the present study). The equationsdeveloped for spectrophotometric assay of Chl  a  and  b in these solvents (methanol: Porra et al. 1989; Porra1990, 1991, 2002; ethanol: Lichtenthaler 1987; Rowan 1989) are not widely used. The chlorophyll red peaks(Q y ) for Chl  c 1  + c 2  and Chl  c 2  are much lower andbroader in methanol and ethanol and there do notappear to be any published algorithms for determi-nations of Chl  a  and Chl  c 1  + c 2  or  c 2  in methanol orethanol.There is a real need for a consistent set of algorithmsfor routinely calculating chlorophyll contents of envi-ronmental samples, cultured algae and for biochemicalstudies of chlorophyll–protein complexes with somechoice of solvents to use. There is also a trade-off between choosing the best solvent for efficient quan-titative extraction of chlorophylls and use of a solventbest suited for spectrophotometric assay. Acetone isnot the ideal solvent for extraction, although it hasgreat merit as the solvent for assay of chlorophylls. Inmany practical situations, any gains in accuracy fromusing acetone as the assay solvent are lost because of its inefficient extraction of chlorophylls from cells.Replacing one solvent with another by driving off theextracting solvent using a stream of N 2  and thenreplacing it with another solvent is inefficient whenlarge numbers of samples need to be assayed and leadsto inevitable losses and oxidation.There are two other reasons why acetone is not adesirable solvent for chlorophyll assays. The first issafety. Acetone is very volatile, highly flammable,causes headache, is narcotic in high concentrations andis a skin irritant (erythema). Plastic or latex glovesprovide little protection or actually worsen the situa-tion by being attacked by the acetone. It is not adesirable solvent to use in a teaching laboratory and itsflammability, security concerns and volatility make itproblematic to transport by air for fieldwork. Thewidespread use of plastic laboratory-ware also leads todifficulties because acetone attacks polystyrene andpolymethylacrylates (PMMA) and, therefore, plasticspectrophotometer cuvettes cannot be used for acetonebased chlorophyll assays.Methanol is a very good extractant for chlorophylls,particularly from recalcitrant vascular plants and algae(Porra et al. 1989; Porra 1990, 1991, 2002). It is less volatile and flammable than acetone but is notoriouslytoxic. It is an insidious poison because it is readilyadsorbed by inhalation and through the skin and soshould not be used in a teaching laboratory if it can bereplaced by ethanol. Methanol slowly fogs polystyrenespectrophotometer cuvettes leading to false readingsand cannot be used at all with PMMA cuvettes.Methanol is the usual solvent for HPLC systems toassay chlorophylls: a set of spectrophotometric equa-tions for a methanol solvent system would therefore bea very useful adjunct to HPLC work on chlorophylls(Wright and Jeffrey 1997; Jeffrey and Wright 1997;Mantoura et al. 1997).Ethanol is a much safer solvent than either acetoneor methanol (Wright et al. 1997) but is not used veryoften for the assay of chlorophylls although equationsfor  Chl a  and  b  are available (Lichtenthaler 1987;Rowan 1989). Although flammable it is not very toxicand is suitable for use in a teaching laboratory. Ethanoldoes not attack polystyrene and so polystyrene plasticspectrophotometer cuvettes can be used. There areconsiderable practical, safety and economic advantagesin using ethanol as the solvent for chlorophyll extractand assay. Algorithms developed for ethanol in thepresent study now allow routine assays of chlorophylls a, a + b, c 1  + c 2  , c 2  and  d  in a very safe and convenientsolvent. 28 Photosynth Res (2006) 89:27–41  1 3  Diethyl ether is a very popular solvent for chloro-phylls for research purposes, particularly for preparingpure pigments (see Porra et al. 1989, Porra 1991;Scheer 1991; Jeffrey et al. 1997). Many of the diag-nostic spectra of chlorophyll pigments are for diethylether as solvent (Jeffrey et al. 1997). Except for freeze-dried material, it cannot be directly used as a chloro-phyll extractant because it is not miscible in water. It isnot a solvent of choice for routine and class laboratorywork because it is extremely volatile, flammable,explosive and narcotic. The explosion hazard in par-ticular restricts it use. Ether also attacks plastic cu-vettes and most plastic laboratory ware. Diethyl etherhad to be used in part of the present study because theonly published formulae for determining Chl  a  and  d  inmixtures of these chlorophylls were for diethyl ethersolvent (Smith and Benitez 1955; French 1960).Porra et al. (1989) and Wright et al. (1997) discussthe merits of other solvents used for chlorophyllextraction and assay such as chloroform, dimethylsulphoxide (DMSO) and dimethyl formamide (DMF).These solvents are more dangerous and even lessdesirable than acetone and methanol for routineresearch purposes and in the teaching laboratory butchlorophyll algorithms for a DMSO solvent system arecurrently under development in our laboratory.The present study compares the use of the solventsacetone, methanol and ethanol in determining con-centrations of chlorophylls  a, b, c 1  + c 2  , c 2  and  d . Newalgorithms for determining concentrations of chloro-phyll mixtures in the three solvents are presented alongwith error estimates. Materials and methods Synechococcus R-2  (PCC 7942) srcinating from thePasteur Culture Collection was used as an example of acyanobacterium with only Chl  a . It was grown in BG-11medium (Allen 1973). English spinach ( Spinacia olera-cea  L., Chenopodiaceae) was used as an example of avascular plant with Chl  a  and  b . Hydroponically grownspinachwasusuallyusedfreshfromalocalsupermarketand had a Chl  b/a  ratio of about 0.35–0.25, consistentwith being grown in bright light. Where shade adaptedplants were required, spinach plants were kept in asouth-facing shaded sunroom for several days and thenewshade-adaptedleaveswereusedasasourceofChl a and  b . The marine diatom,  Phaeodactylum  sp. (SydneyUniversity Teaching Collection)was usedasasource of Chl  a  and  c 1  + c 2 .  Heterocapsa pygmae  (a small marinedinoflagellate, Sydney University TeachingCollection),andzooxanthellae( Symbiodinium sp.,Dinophyta)froma zoanthid ( Zoanthus robustus  (Carlgren, Coelentera-ta)) and  Rhodomonas spN23  (Chroomonas spN23,Cryptophyta, Sydney University Teaching Collection)were tried out as sources of Chl  a  and  c 2 .  Rhodomonas spN23  could be grown easily in large amounts and wasusedasthestandardsourceofChl a and c 2 preparations.  Acaryochlorismarina wasakindgiftfromDrMinChen(Sydney University).  Acaryochloris marina  is a marineoxyphotobacterium with Chl  d  as its major photosyn-thetic pigment with some Chl  a  (Miyashita et al. 1997,2003; Akiyama et al. 2001; Kuhl et al. 2005). A free- living oxyphotobacterium with Chl  d  as its dominantchlorophyll was recently isolated from the Salton Sea intheUSA(CCMEE5410,Milleretal.2005)andanotheras epiphytes on rhodophyte algae (Murakami et al.2004).  Phaeodactylum  sp.,  Heterocapsa pygmae , Rhodomonas spN23  and  Acaryochloris marina  were allgrown in enriched f-2 seawater as described byMcLachlan (1973) but using Fe-citrate rather thanFe-EDTA as the iron source.The algae were grown on an orbital shaker(  80 rpm) fitted with overhead fluorescent lights(Sylvania Gro-Lux). The light intensity was approxi-mately 80  l E m –2 s –1 (PAR, using a Li-Cor photon fluxmeter Model LI-189). However,  Acaryochloris marina consistently grew better on the edge of the shakerwhere the light intensity was lower (    40  l E m –2 s –1 ).  Acaryochloris marina  consistently grows better at lowlight intensities.Laboratory procedures were performed in a natu-rally low-lighted laboratory with the fluorescent lightsoff. The normal lighting in the laboratory undersuch conditions was about 2  l E m –2 s –1 (400–700 nmPAR) (Li-Cor Quantum photometer Model LI-189).Exposure of chlorophyll extracts to light was avoided.Analytical grade acetone, methanol and ethanolwere from Mallinckrodt Baker BV, Deventer, Holland.Denatured dry alcohol (Ethyl alcohol 99.5%, Chem-Supply Ltd, Gillman, SA, Australia), denatured withdenatonium benzoate 0.00066%, fluorescein 0.0001%and methyl isobutyl ketone 0.25%, was found to befree of spectroscopic contaminants in the visible rangeand so could also be successfully used. Analytical die-thyl ether was from Merck Pty Ltd, Kilsyth, Victoria,Australia.Commercial acetone and methanol are often highlyacidic. A 90% acetone was made up using a saturatedsolution of magnesium carbonate hydroxide to removeany acid present. To ensure that 100% methanol, 100%ethanol and denatured 99.5% ethanol were acid-free, asmall amount of magnesium carbonate was added, andthen the suspension was clarified by filtration throughfilter paper. Excessively alkaline extractants should Photosynth Res (2006) 89:27–41 29  1 3  also be avoided because of allomerisation of chloro-phylls and in particular the formation of rhodochlorinsin alkaline solvent (Porra et al. 1989; Porra 1990).Aqueous preparations of methanol and ethanol werenot used in the present study. Papista et al. (2002)reported that chlorophylls form Chl-monosolvate andChl-disolvate mixtures in methanol/water and ethanol/water mixtures lead to misleading results. Solventswere kept at 4  C.Microalgae were collected by first centrifuging themat 3000  ·  g  for 10 min, then resuspending in deionisedwater and pelleted a second time. After decanting andresuspension of the hard pellet, the pigments wereextracted in a 1:1:1 mixture of 90% acetone, 100%methanol and 100 % (99.5%) ethanol, all neutralisedwith magnesium carbonate. The crude extract wasallowed to stand in a refrigerator in the dark at 4   C forabout 30 min before being cleared by centrifugationand the pellet discarded. To extract Chl  a  and  b  fromspinach, the leaves were cut up into small pieces andground in a glass-glass tissue grinder in the mixedacetone/methanol/ethanol extractant. The extract wasthen pelleted by centrifugation and the pellet dis-carded. All concentrated extracts were made up toabout 6 ml and stored in the dark in a freezer at –20  Cfor no more than 7 days.Extraction of chlorophyll by soaking algae or vas-cular plants in solvents overnight was not employedbecause it provides an opportunity for chlorophyllaseto convert chlorophylls to chlorophyllides.Spectrophotometric readings were made using aShimadzu UV-2550 UV–visible spectrophotometerusing standard scanning settings and a 1 nm bandwidthand 1 nm sampling interval. Quartz cuvettes were usedunless otherwise stated. Polystyrene cuvettes werefrom Sarstedt International (Numbrecht, Germany).Concentrated pigment extracts were used to make updiluted samples for the spectrophotometric study. A50  l l of pigment extract was diluted with assay solventto make up to 1.0 ml of assay mixture. Where mixturesof chlorophyll extracts were being assayed it was en-sured that the diluted sample was not contaminatedwith more than 6.7% of foreign solvents. Thus a 1.0 mlmixture of Chl  a  from  Synechococcus  and Chl  a  and  b from spinach, made up in 90% acetone would containno more than 3.3% methanol and 3.3% ethanol. Allchlorophyll assays on the concentrated extracts wererun in acetone, methanol and ethanol so that directcross-comparisons of chlorophyll assays using the threesolvent systems could be made.In the present study, all error-bars are ±95% confi-dence limits (CL) with the number of replicates inbrackets. All chlorophyll algorithms have been workedout for 1 cm light path cuvettes. Absorbance readingshave dimensions A cm –1 and hence the absorbancecoefficients have dimensions mg l –1 cm A –1 . Spectrophotometry theory French (1960), Porra (1991, 2002) and Jeffrey andWelschmeyer (1997) give general outlines of thesimultaneous equation approach to estimating sepa-rately the chlorophylls in mixtures of chlorophylls. Thismethod of estimating the component chlorophylls inplants was popularised by Arnon (1949). In spectro-photometry of chlorophylls, it is customary to zerospectrophotometers at 750 nm to correct for turbidityand contaminating coloured compounds. Thus, thesimplest chlorophyll algorithms capable of resolvingtwo chlorophylls (1 and 2) in a mixture have the gen-eral form,Chl ð 1 Þð l g = ml Þ E  k 1 ; 1  A k 1 þ E  k 2 ; 1  A k 2 Chl ð 2 Þð l g = ml Þ E  k 1 ; 2  A k 1 þ E  k 2 ; 2  A k 2 ð 1a ; b Þ where, E  k 1,1 istheabsorbancecoefficient(mg l -1 cm A -1 )for the red peak ( k 1) of Chlorophyll (1),  E  k 1,2  is theabsorbance coefficient (mg l -1 cm A -1 ) for the red peak( k 1) of Chlorophyll (2),  A k 1  is the absorbance of thepigment extract at wavelength ( k 1) nm.More complex algorithms using measurements atthree or more wavelengths can be developed, however,the more complex the algorithm, the more difficult it isto fit to a data set and the larger the inherent error(Appendix 1). Hence, the least complex algorithm,consistent with a good fit to the data, should beadopted.For organisms containing two types of chlorophyll,readings at two wavelengths (the red absorption peaksQ y  of the two chlorophylls present) are usually em-ployed and so chlorophyll equations are usually of theform  z  =  ax  +  by . Jeffrey and Humphrey (1975) alsopublished a trichroic formula to determine Chl  a, b  and c 1  + c 2  in acetone extracts from mixed phytoplanktonpopulations.In most oxygenic photosynthetic organisms (  Acary-ochloris marina  is an exception), Chl  a  is the pre-dominant chlorophyll. Absorption by Chl  a  over mostof the red part of the spectrum strongly interferes withdeterminations of Chl  b, c 2  and ( c 1  + c 2 ). Chlorophyllalgorithms require measurements of absorbances attwo wavelengths: one is at the red absorption peak forChl  a  and the other is at the red peak of the otherchlorophyll. The Chl  a  equation is usually very accu-rate but the equation for the minor pigment will be less 30 Photosynth Res (2006) 89:27–41  1 3
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