Nordic Society Oikos

Chlorophyll a Determination: Improvements in Methodology

Author(s): Osmund Holm-Hansen and Bo Riemann
Source: Oikos, Vol. 30, Fasc. 3 (1978), pp. 438-447
Published by: Wiley on behalf of Nordic Society Oikos
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OIKOS 30: 438-447. Copenhagen 1978

Chlorophylla determination:improvements
in methodology

Osmund Holm-Hansen

ScrippsInstitution
of Oceanography,Universityof California

Bo Riemann

Botanical Institute,Universityof Arhus

Holm-Hansen, 0. and Riemann, B. 1978. Chlorophylla determination:improve-

mentsin methodology.- Oikos 30: 438-447.

Chlorophylland phaeopigmentdeterminations on freshwaterand marinesamplesof

phytoplankton have shownthatthe followingconsiderationsare importantin routine
pigmentanalyses. (a) Methanolshould be used as extractionsolventinsteadof ace-
tone due to betterextractionefficiency,shorterextractiontime,and eliminationof
the need to boil or homogenizethe samples. (b) For spectrophotometric determina-
tionsof phaeopigments,the methanolextractsshouldbe acidifiedwithHCI to a final
concentrationof 3 x 10' M. (c) When a spectrophotometer is used forphaeopig-
mentdetermination, subsequentneutralizationof the acidifiedmethanolextractsto
the originalpH is required;fluorometricdeterminations do not requirethisneutrali-
zation. (d) The additionof MgCO3 to filtersor to extractsis not necessary.(e) There
is no detectableloss of chlorophyllwhen eithersample extractsor wet filtersare
storedat -20'C forthreeweeks and thenextractedwitheitheracetone or methanol.
(f) Glass fiberfiltersare effectivein retainingall phytoplanktonand have some
advantagesover membranefilters.

0. Holm-Hansen,ScrippsInstitution
of Oceanography,University of California,La
Jolla,CA 92093, USA. B. Riemann,BotanicalInstitute,
68, Nordlandsvej,DK-8240
Risskov,Denmark.

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Accepted 17 October 1977

? 01KOS

438 OIKOS 30:3 (1978)

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 to us that distillationof the acetone and the use of
1. Introduction
MgCO3 are notnecessaryifhighqualityacetone is used.
For determinationof chlorophyllin sea water or fresh Methanol extractions:After filtrationthe filterwas
water samples most investigatorsuse the procedures foldedand eithertreatedas above (except thatabsolute
outlined by Stricklandand Parsons (1968) or in the methanolwas used insteadof acetone), or thefilterwas
UNESCO report(1966). These proceduresinvolvethe placed in 3 ml of absolutemethanol.For the samplesin
use of MgCO3 on membranefilters, grindingof samples 3 ml methanol,afterone hour of extractionthe filter
in distilledacetone, and measurementof the extracted was wrungagainstthe sides of the glass vessel usinga
chlorophyllaftershortstorageperiods. We used these glass rollerand thenrinsedtwicein methanol;the final
same procedures originally,but duringthe course of solutionwas thenfilteredthrougha Whatmanglass fi-
some years we have modified and improved many ber filterto obtain a clear solutionfreefromfilterresi-
aspectsof theabove methodology.The changeswe have dues and cell debris.To avoid oxidationand allomeri-
incorportatedinto our proceduresinvolve (a) type of zation of the pigmentsduringthe period of extraction,
filterused, (b) nature of the extractionmedium, (c) the methanol was saturated with hydrogensulphide
eliminationof MgCO3 (d) eliminationof homogeniza- beforeuse (Jensenand Sakshaug 1973).
tionof samples,(e) treatmentof thepigmentextractby The concentration of chlorophylla and phaeopigment
eitherfluorometric or spectrophotometric methods,and a in the extractswas determinedeitherfluorometrically
(f) storagetimeof frozenfilters.Our presentprocedure, in a Turnermodel No. 111 (Holm-Hansen et al. 1965)
as describedin this paper, representsa more efficient or spectrophotometrically with a Beckman DB-GT
and reliable methodologyfordeterminationof chloro- spectrophotometer. The fluorometer was equipped with
phylla and phaeopigmenta in naturalwatersamples. a CorningCS2-64 filterfor the excitationlightand a
CorningC52-64 filterfor the emittedlight.Calcula-
tions of amount of chlorophylla and phaeopigmenta
followedLorenzen (1967) and Moss (1967a, b). Ab-
2. Methodsand materials
sorptioncoefficients forpigmentsin methanolare cited
The sea water samples were obtained with alco- in the text.
hol-scrubbed8.0-1 PVC Van Dorn bottles, and the The emissionspectra of pigmentfluorescencewere
freshwater samples with2-1 polythenebottles.Repli- measured in a Turner spectrofluorometer(model
cate aliquots of waterwere filteredat a vacuum of 25 430-025) withan activationwavelengthof 440 nm. It
cm Hg througheither a 2.5 cm microfineglass fiber should be noted thatthisinstrument is an "uncorrect-
filter(Reeve Angel 984H), a 2.5 cm GF/C glass fiber ed" spectrofluorometer, and hence thespectraobtained
filter(Whatman),or an HA membranefilter(Millipo- with it are not correctedfor non-linearityassociated
re) with a pore size of 0.45 lim. When MgCO3 was withthe photodetectoras well as forotherinstrumental
added to the sample, 1.0 ml of a 1% suspensionwas artifacts.The spectrashownin thispaper (Figs 2 and 4)
added eitherto the last fewhundredml of the sample, may thusshow minordifferences withotherdata in the
or added to the filterbeforeadditionof the watersam- literature,but theyare sufficiently reliableand valid to
ple. Filterswere eitherextractedimmediatelyas out- supportour conclusions.
lined below, or were placed in 15 ml glass-stoppered Paper chromatographic separationsof chlorophyllsa
testtubes and storedat -20'C untiltimeof extraction. and b followedJensenand Liaaen-Jensen(1959) and
Acetoneextractions:Immediatelyupon completionof Jensenand Sakshaug (1973).
filtration,the filterwas folded in half and either (a)
placed in a glass-stopperedcentrifugetube, to which 3. Results and discussion
was added 10 ml of 90% acetone, or (b) groundwith
3.1. Choiceofextraction
solvent
90% acetone in a Potter-Elvehjemhomogenizeror in a
Braun Melsungen (Type 853202) watercooled homo- Most researchersin oceanographyand limnology,as
genizer, after which the slurry was transferredto well as terrestrialecology (Linder 1974), still use the
glass-stopperedcentrifugetubes and taken to a final traditionalacetone forpigmentextraction.It has been
volume of 10 ml with 90% acetone. Samples were well known,however,thatverylow acetone extraction
storedin completedarknessat room temperaturedur- efficiencyis obtainedwhencommonalgae belongingto
ing the extractionperiod,whichvaried from10 min to the Chlorophyceaeor Cyanophyceaeare extractedand
manyhours (details in text). At the end of the extrac- compared to methanol extraction(Steemann Nielsen
tionperiod,the tubeswere shakenby hand,centrifuged 1961, UNESCO 1966, Golterman 1969, Rai 1973,
at 1000 x g for5 min at 40C, and thentaken to room Riemann 1976, Sand-Jensen1976). Althoughthe use
temperatureby placingin a waterbath for10 min.The of methanolis commonin laboratoriesstudyingthebio-
acetone used in theseextractionswas reagentgrade,but chemistry and physiologyof algae, therehas been great
it was neitherdistillednor storedwithMgCO3 as sug- apathyby aquatic ecologiststo changingtheirextraction
gested by Stricklandand Parsons (1968). Years of ex- methodology.The data we present below show that
perience and manytestextractionshave demonstrated methanolextractspigmentsfromphytoplankton faster

OIKOS 30:3 (1978) 439

280

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Tab. 1. Efficiencyof 90% acetone and absolute methanolfor lates fromthe clam extractedverypoorly in acetone.
extractionof chlorophylla fromdiatomsand blue-greenalgae Complete extractionfromthe clam dinoflagellateswas
fromLake Moss0. Samples were not homogenized.Standard obtainedwithin45 min(the shortesttimeused) in met-
erroris based on fivereplicatesamples. Pigmentlevels were
determinedspectrophotometrically accordingto Moss (1967a) hanol, eitherwith or withoutgrinding;with acetone,
and Riemann (1976). however, extraction was only about 20% without
grinding,and even withgrindingit required 2.5 h for
Dominant Extraction gigchlorophyll completeextraction.
phytoplankton time a/sample
Acetone Methanol The difficulty of extractingpigmentsfromthesezoo-
(h)
xanthellaehas been previouslydescribedbyJeffrey and
1 34.2 ? 0.4 37.0 ? 0.6 Haxo (1968), who used a combinationof freezingfol-
Diatoms
~ 6 35.1 ?0.8 - lowed by methanolextraction.The yieldoffluorescence
Diatoms 12 36.2 ? 0.7 as measured in our instrument is less per unitchloro-
20 36.8 ? 0.6 -
phyllwhen methanolis used as compared to acetone.
1 28.6 ? 0.2 34.1 ? 0.4 When methanolextractsare used, a largerfactormust
6 29.4 ?04 -
thereforebe employed in the equation to equate flu-
Blue-greenalgae 12 29.8 ? 0.8
20 30.0 ? 0.5 -
orescence units to chlorophyllconcentrations.The
slightdrop in fluorescenceseen in Fig. 1B between45
and 75 min most likelyreflectschanges in instrument
sensitivitydue to eitherincompletewarmingup or to
and more completelythan does acetone, and also that voltagefluctuations on board the researchvessel,as the
thereare no inherentproblemsassociated withthe use slightdecrease was seen in boththe acetone and metha-
of methanol for either spectrophotometric or fluoro- nol extracts.
metricdeterminations of pigmentconcentration.
The extractionefficiencesof acetone and methanol
were studied withfreshwater samples fromeutrophic 100 A
Lake Moss0, Denmark, which were dominatedeither
by diatoms or by blue-greenalgae (mostlyAphanizo- 80 _
mononflos aquae Ralfs). The results(Tab. 1) show that A- ~~~~~A
afterone hourtherewas completeextractionin all met-
60 -
hanol samples, but incomplete with the acetone ex-
/1
tracts.After20 h extraction,the diatom samples were
close to 100% extraction,but the blue-greensamples 40 -
were only 88% extractedas comparedto the methanol U)
Hz
H /
samples. D 20
Many testswithcoastal marinephytoplankton have
yieldedresultssimilarto thediatomdata in Tab. 1. That O I
U)
is, if samples are groundand then extractedfora few () 0 I 2 3 4 5 6
hours,comparableresultsare obtainedwitheitherace- Ld
tone or methanol.In all thesetests,however,therewere
no detectableblue-greenor green algae presentin the
1 200 B
watersamples. Recent data indicatethatfreelivingand
> >
symbioticblue-greenalgae are, however,foundin sig-
nificantnumbersin the oligotrophicwatersof the north 150 -
and southPacificgyres(Weare et al. 1974, Mague et al. -_
1974). The widespreaddistribution of blue-greenalgae
has been previouslyreportedin intertidalenvironments 100_
and in mostocean waterwiththe exceptionof thepolar
regions(Fogg 1973). It is likelythereforethat,even in
marinestudies,one is apt to encounterblue-greenalgae 50 A-A __ __
which show poor extractioncharacteristicswith ace-
tone. Other algal groupsmay also show poor extraction
into acetone, as is seen in the data in Fig. 1. Symbiotic
0 1 2 3 4 5 6 7
dinoflagellateswere isolatedfromthe coral Pocillopora
TIME (hours)
capitata Verril(Fig. 1A) and fromthe giantclam Tri-
dacna gigas L. (Fig. 1B) and extractedwithacetone or Fig. 1. Efficiencyof extractionwithtimeof symbioticdinofla-
fromthe coral Pocillopora capitata(A) and fromthe
methanol,both with and withoutgrindingin a Pot- gellates
giantclamTridacnagigas(B). Extraction were90%
solvents
ter-Elvehjemhomogenizer. The coral dinoflagellates acetone ( ) and absolute methanol(?----), both with
extractedquite rapidlyin acetone, but the dinoflagel- (a) and without(A) grindingof the sample.

440 OIKOS 30:3 (1978)

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 Previous studies have reportedsimilarlow acetone A ore
extractionefficiency usingcommonalgae like Chlorella ~~~~~~~Bef
vulgaris (Cambridge Culture Collection 211/1lh) acid
(Steemann Nielsen 1961), Scenedesmus quadricauda (664)
Brebisson (Rai 1973), or the filamentousCladophora w 0.12M
and Vaucheria,whichcan onlybe extractedcompletely z
C (663)
in methanol(Marker 1972). When methanolis used to CD M~~~~~~~~I2
extractsuchcells it is not necessaryto homogenizeor to (656)
boil the filters,which resultsfrequentlyin significant Cn ~~~~~4M
losses of chlorophylla and increasedamountsof phae- CD (655)
opigmenta (Riemann 1976).
Specific absorptioncoefficientsfor chlorophylla in
methanolare givenas 74.5 1g-' cm-' (Mackinney1941,
Seely and Jensen1965) or as 75 1 g` cm-1(Lenz and
Zeitzschel 1968). Using an acid factorof 1.5 forchlo- 600 650 700
rophylla in methanol,Marker (1972) gives a specific
absorptioncoefficientfor phaeophytina in methanol WAVELENGTH
(nm)
between 49.6 to 50.0 1 g71 cm-', depending on the
choice of absorptioncoefficientforchlorophylla.
On the basis of the above data, it is suggestedthat
absolute methanolshould be used ratherthan acetone
B
because of 1) shorterextractiontimes,and 2) elimina-
tion of the requirementforhomogenizationor boiling.
Furtherreductionof the likelihoodof oxidationof the
pigmentsduringextractionmay be affordedby using
methanolsaturatedwithhydrogensulphide(Jensenand --Before acid
Sakshaug 1973). We are not aware of any problemsor
drawbacksassociated withthe use of methanolas com-
pared to acetone forpigmentdeterminations. z
0
Cl)
3.2. Photometric
andfluorometric
measurements
of Cl)

pigments inmethanol
extracted
w 4M
3.2.1. Acidification procedure
The spectrophotometric (Lorenzen 1967, Moss 1967a, 1.2M
b) and the fluorometric(Holm-Hansen et al. 1965,
Loftus and Carpenter1971) determinations of chloro- 0.12 M
phylla and phaeopigmenta involve acid treatmentof
the extractedpigments.A numberof chemicalchanges
occur when chlorophyllextractsare treatedwith acid.
These changesvaryin different extractionsolventsand
depend upon the natureof thepigments,the amountof
acid added, and the period of timethe acid reactswith
650 700 750
the extract.
The acidificationstep was studiedin a cultureofDu- WAVELENGTH
(nm)
naliella tertiolecta
Butcher(containingboth chlorophyll Fig.2. Absorption(A) and emission(B) spectraofpigments
a and b) extractedin methanol.Fig. 2 shows the ab- extractedin methanolfroma cultureofDunaliella tertiolecta.
sorptionand emissionspectrabeforeand afteraddition To acidify 0.1 mlofvariousHCL solutions
extracts, (0.12,1.2,
of HCl to different finalmolarities.The red absorption and4.0 M ) wereaddedto the4 mlofmethanol extract.
maximumof the phaeopigmentsresultingfromacidifi-
cation depends upon the concentrationof HCl in the
finalacidifiedsolution(Fig. 2A). Highermolarityof the spectively.These changeswere probablydue to further
HCl reduces the readingsat 665 nm due to changesin degradationofchlorophyll b forming phaeophytinb and
the absorptionspectrum. resultingin increasedemission(see below and Fig. 4D).
The wavelengthof the maximumemissionpeak did Acidificationto 3 x 10-3 M degradated only chloro-
not change significantly with differentacid concentra- phyll a to phaeophytina. This is in accordance with
tions(Fig. 2B); increasedemissionwas observed,how- Joslynand Mackinney (1938) and Schanderl et al.
ever, when acidifiedto 3 x 10-2 and 1 x 10-1 M re- (1962) who reportedthatdegradationof chlorophyllb

OIKOS 30:3 (1978) 441

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to phaeophytinb was 5.5 to 9 timesslowerthandegra- 8
dationof chlorophylla to phaeophytina, and therateof ._._._..--*-@1. XI -3 M

degradationof chlorophyllb depended on the molarity 7 -1.

of acid in the acidifiedextract.
6-
The absorptionspectrain Fig. 2A includealso inter-
ferenceof phaeophytinb, whenacidifiedto 3 X 10-2 or p. x I2M
1.2
1 x 10' M HCl. The absorptionspectrumof phaeo- F-
phytinb is, however,close to the spectrumof chloro- z 54
phyllb in the red region,makingthe differencebefore
and afteracidificationinsignificant. Here the observed
changes upon acidificationare probably caused by
spectralchangesof phaeophytina due to changesin pH,
like those reportedby Livingstoneet al. (1953) and
Marker (1972), thus requiringneutralizationof the
acidifiedextractsto thepH foundin theoriginalextract.
Subsequent neutralizationof the acidifiedextractsto
the pH found in the solutionbefore acidification(pH T ME (minutes)
7.7) changed all the spectraof acidifiedextractsto one
identicalspectrumwithan absorptionmaximumclose Fig. 3. Rates of neutralizationin absolute methanolacidified
withHCI and neutralizedwith25 Mg MgCO3 per mlmethanol.
to 665 nm,thusindicatingthatthe spectralchanges of The molarityof the HO1 in the acidifiedextractis indicatedon
the phaeopigmenta was due to pH changesin the ex- the figure.
tracts.
Spectrophotometricand fluorometricdrift in the
acidifiedextractsmaybe caused by (1) incompletecon- perchromatographyandreported
thatcompletephae-
versionof chlorophylla to phaeophytina, (2) phaeo- ophytinization whentheconcentration
occurred ofHCI
phytinization of other chlorophyllsthan chlorophylla, in the acidifiedextractwas as low as 4 x 10' M. The
and (3) further chemicalreactions.Both spectrophoto- concentration
ofacidneededto affect
thischemicalre-
metricand fluorometric driftwas observedwhenchlor- actionis farless thanmanyproceduresrecommendedin
ophyllextractswere acidified.Using verydiluteHCl (3 the literature,whichrangefrom0.01 to 0.2 M (Loren-
X 10' M in the extract),driftingpracticallystopped zen 1967, Moss 1967a, b, Stricklandand Parsons 1968,
withinthree minuteswhen reading the absorptionat Marker 1972).
665 nm. In contrastto this,1 x 101 M HCl or stronger In view of the above results,the followingacidifica-
in the acidifiedextractincreaseddrifting in an anomal- tion proceduresare proposed when pigmentsare ex-
ous way, indicatingfurtherchemicalreactions.Conco- tractedin absolute methanol.
mitantwiththisdrifting, a markedincreasein absorp- Spectrophotometric determinations:the molarityof
tionat 750 nmwas also noted. Absorptionat 750 nm is the HOl should be about 3 x 10-3 M in the acidified
normallyused to correctfor absorptionof othercom- extract.Reaction timeis threeminutes.Neutralization
ponentsthanchlorophyllsand shouldnot exceed 0.005. is accomplishedby additionof 25 Mg MgC03 per ml
In the fluorometer,driftingnormallystopped within extract,and takes about 10 minwithslow stirring. Sub-
three minuteswhen acidifiedto 3 x 10-3 M; further sequent filtration througha Whatmanglass fiberfilter
additionof HCl altered the emissionas shown by the produces a clear solution.
data in Fig. 2B. Fluorometricdeterminations: the molarityof the HCI
When a spectrophotometer is used neutralizationof should be about 3 x 10-3 M in the acidifiedextract.
the acidifiedpigmentsis required to (1) convertthe Reaction time is less than three minutes,and subse-
acidifiedphaeophytina spectrumto the neutralform, quent neutralizationis not required.
(2) stop furtherdrifting at otherwavelengths.
The rate of neutralizationof acidifiedextractswas 3.2.2. Influenceof chlorophyllb on thedetermination
studied in methanolextractsby measuringthe "appa- of otherpigments
rent pH" (Fig. 3). When methanolextractsare made In many eutrophicwaters green algae, which contain
1.2 x 10-2 M with HCl, neutralizationby additionof both chlorophyllsa and b, oftenconstitutea significant
MgCO3 requiresmore thanone hour; ifthe concentra- proportionof the phytoplankton. As chlorophyllb can
tion of HCl is 1.2 mM, however,the extractcan be interferewiththe determination of phaeophytina, both
neutralizedby MgCO3 within 10 min. Consequently chlorophyllsa and b were separatedfromspecies of the
whenacidificationis made in methanol,verydiluteHCl grass Poa by paper chromatographic procedures(Jen-
is required(finalconcentrationin the extractshould be sen and Liaaen-Jensen 1959, Jensen and Sakshaug
about 1 mM) to facilitatethe subsequentneutralization 1973) and theirabsorptionand emissionspectradeter-
step and also to minimizedrifting. Riemann (1976) has minedin absolutemethanol.Afteracidificationof these
followedthephaeophytinization of chlorophylla by pa- solutions, aliquots were chrornatographedand the

442 OIKOS 30:3 (1978)

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 t)
(A and
Fig.4. Absorption
B) andemission(C andD)
\ U') spectraforchlorophyllsa
A L B andb andphaeophytinsa
_ s I A -D B andb in methanol.Pigments
L) wereisolatedfromspecies
l \ _ (9 ofthegrassPoa bypaper
W chromatographictechniques.
Wn
LOi
O \ S
z O
z\ wasdoneby
Acidification
< l <C /'4 adding0.1 ml of 1.2 M HCl
M i 1 zIr / 5to 4 ml of methanolextract.
o 0

ct ll \ | CHLOROPHYLL <4
w w
> >
b
~~~~~~~~~CHLOROPHYLL
PHAEOPHYTINa -PHAEOPHYTIN b

650 700 750 650 700 750

a.)
C D

ILO

z z
0 CHLOROPHYLLa 0
Cn C PHAEOPHYTINb
w w

PHAEOPHYTIN CHLOROPHYLLb

650 700 750 650 700 750

(nm)
WAVELENGTH

phaeophytinsa and b were isolated and used foranaly- lar to those we show in Fig. 4 in methanol,in thatboth
sis of their absorption and emission spectra. Fig. 4 absorptionand emissionof phaeophytina decreased as
showstheabsorptionand emissionspectraforall fourof comparedto chlorophylla, whilethe emissionof phae-
these pigmentcomponentsin methanol.Both the ab- ophytinb increaseddramaticallyas comparedto chlor-
sorptionand emissionof phaeophytina decrease com- ophyll b. These data indicate that the calculationsof
pared withchlorophylla, but the peaks also changedto phaeophytina upon acidificationin methanolor ace-
shorterwavelengths(Figs 4A and 4C). The absorption tone extractswill be very much dependent upon the
of phaeophytinb in the red regionis nearlythe same as ratioof the phaeophytinsa and b.
the absorptionof chlorophyllb, althougha small de- The absorptionspectra in the red regionof chloro-
crease was found upon acidificationof chlorophyllb. phyllb and phaeophytinb are nearlythe same (Fig.
The fluorescenceof phaeophytinb, however,was mar- 4B). Consequentlythe determinationof chlorophylla
kedlydifferent fromthatof chlorophyllb; upon acidifi- and phaeophytina will be complicatedby the presence
cation of chlorophyllb, the emissionincreasedsignifi- ofchlorophyllb. When theconcentration ofchlorophyll
cantlyand the peak decreased from671 nm to 658 nm. b is smallrelativeto chlorophylla, the effecton chloro-
Loftus and Carpenter (1971) showed the emission phylla determinations willbe minorand can be ignored.
spectra of chlorophyllsa and b and their respective When the ratio of chlorophyllb/chlorophyll a exceeds
phaeophytinsin acetone. Their spectraare fairlysimi- -0.4, however,the opticaldensityof the acidifiedsolu-

OIKOS 30:3 (1978)

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tionwillbe highdue to the absorptionofphaeophytinb. do not have an advertised"pore size", and this has
In thisway the amountof phaeopigmenta willbe over- given rise to apprehensionsregardingthe efficiency of
estimatedwhen the spectrophotometric equations are the glass fiberfiltersto retain small cells. Glass fiber
used. filterswere suggestedby Vollenweider(1969), with a
The influenceof chlorophyllb on the determination warningthatsuch filtersmay not retainas manysmall
of chlorophylla by fluorometry or spectrophotometry cells as the membranefilters.Sheldon (1972) has pro-
are discussed by Loftus and Carpenter (1971). The vided data thatminimizesthispossible objectionto the
most directway to discernif the calculated amountof use of glass fiberfilters.By the use of an electronic
phaeopigmenta representsan increase in the ratio of particle counter,Sheldon has shown that the median
chlorophyllb/a,however,is to separatethepigmentsby retentionsized forHA Milliporefilters,GF/C glass fi-
chromatography. Jeffrey(1976) has also discussedthe ber filters,and 984H glass fiberfiltersare close to 0.5,
advantagesof chromatographic separationsof pigments 0.7 and 0.5 [tmrespectively. The 984H glass fiberfilters
priorto theiranalysis. are very effectivein retainingmost bacterial cells we
have testedin thelaboratory,and hence it can safelybe
3.2.3. Influenceof chlorophylla on thedetermination assumed thattheywill also retainessentiallyall phyto-
of otherpigments plankton cells in natural populations. We have fre-
Chlorophyllc is foundin commongroupsof plankters quently filteredphytoplanktonsamples from diverse
like diatoms and dinoflagellates.Ratios of chlorophyll aquatic environments(eutrophicand oligotrophicwa-
c/a normally range between 0.5 in dinoflagellates ters,coral reefs,polar waters,etc.) throughmembrane
(Madgwick 1966) up to 1.0 in an Amphidinumsp. cul- filters(0.22 and 0.45 im pore size) and throughWhat-
ture (Jeffrey 1968). man GF/C glass fiberfilters;in all teststhe amountof
No attemptwas made in thisstudyto isolate chloro- chlorophyllrecovered fromthe glass fiberfilterswas
phyllc. Althoughthe developed zone of chlorophyllc equal to or greaterthan the membranefilters.These
can be seen, it is ratherclose to the centerof the chro- observationsare supportedby data of Long and Cooke
matogram,where otherpigmentslike chlorophyllidea (1971), who have shownthattheuse ofglassfiberfilters
and phaeophorbide a interfere.The same problem is resultsin slightlyhigherchlorophyllvalues as compared
found, when sucrose thin-layerchromatogramsare withmembranefilters.
made (Jeffrey1976), and additional problems arise There are some claims (Farooq Azam pers. comm.)
when elutionsfromthe chromatogramsare made, due that micro-fineglass fiberfilters(Reeve Angel 984H)
to the factthatchlorophyllc is reportedto-occurin two are more efficientfor chlorophyllretentionthan are
forms:chlorophyllc1 and c2 (Dougherty et al. 1966, GF/C filters.We tested these two filterswithtropical
Jeffrey 1968). lagoon waterswhichwererichin smallgreenflagellated
Loftus and Carpenter(1971) isolated chlorophyllc cells. Six replicate samples with GF/C filtersgave a
usingthe n-hexane-90%-acetonephase separationde- chlorophyllconcentrationof 4.38 tg I1, whilesix sam-
scribedby Parsons (1963) and theyreportedan over- ples with984H glass fiberfiltersgave 4.43 figchloro-
estimationofchlorophylla byas muchas 10% whenthe phylll1h.As therewas no significant loss of chlorophyll
ratio c/a was 1.0. throughthe GF/C glass fiberfilter,it apparentlyis not
Furtherstudieson the absorptionspectraand emis- necessary to use the micro-fineglass fiber filtersin
sion spectraof chlorophyllc and phaeophytinc in met- chlorophylldeterminations.
hanol are needed beforean exact evaluationof the in- The use of glass fiberfiltersoffersthe followingad-
fluenceof thesetwocomponentscan be done. Fromthe vantageswhen compared to the use of membranefil-
present knowledge the occurrence of chlorophyllc ters.(1) The glass fiberfiltersfiltermuchfasterand do
seems to have a minorinfluenceon both the fluorome- not clog as rapidly.(2) Aftercentrifugation thereis no
tric and spectrophotometric determinations of chloro- turbidityin theacetone or methanolextractswhenusing
phylla and phaeopigmenta. (Holm-Hansen et al. 1965, glass fiberfilters.(3) If one wants to rupturecells by
Loftus and Carpenter 1971). In regard to the fluoro- grinding, the glass fibersare excellentabrasivematerial
metricdeterminations, thisassumes thatthe instrument for aid in cell breakage. Even thoughthismay not be
is equipped withthe proper filtersas discussed in the essentialforpigmentwork,some investigatorsuse the
referencesabove. same procedureforrupturing cells forenzymaticstudies
(Packard et al. 1971). (4) In most field studies it is
desirable to have data on variouschemicalparameters
3.3. Methodologicalvariationsapplicable to both methanol
on the same water sample. For such comparisonsit is
and acetone
essentialthatall filtersused forthe variousdetermina-
3.3.1. Choice offilter tionsretainthe same size fractionof particulatemateri-
There has been some reluctanceby oceanographersand als. Althougholder methodsforparticulateorganicni-
limnologiststo use glass fiberfiltersinstead of mem- trogen and phosphorus utilized membrane filters
brane filtersforpigmentstudies.Part of thisreluctance (Stricklandand Parsons 1965), most investigators now
has undoubtlybeen due to thefactthatglassfiberfilters use glass fiberfiltersforthese determination as well as

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 forparticulateorganiccarbon. It is thereforeappropri- 3.5 -h- -2.5h- - 23 h - -7h - -280h -
ate thatpigmentdata also shouldbe obtainedbytheuse
of glass fiberfilters. 5.0 -

3.3.2. Use of MgCO3 duringfiltration 2.5

Fig. 5 and Tab. 2 show the effectof additionof MgCO3
to the filteror to the sample before filtrationin test 2.0

samples containingvaryingamountsof chlorophylland

phaeopigments.In the sea water samples fromScripps 1.5- SEA WATER
pier (Fig. 5 upper figure)no significant effecton con-
centrationsof chlorophylla or phaeopigmenta was
found.In thefreshwatersamplesshownin Fig. 5 (lower
figure),althoughnot significantthe concentrationsof [0

phaeopigmenta are slightlyhigherin 3 of the 5 sam-

ples withoutadditionof MgCO3. This tendencyis signi- C 0 ABC ABC DEFGHI DEFGHI DEFGHI
0)
ficantin Tab. 2 in the testsamplesfromJuly.Here 8% E
CJ)
more phaeopigmenta is found in the samples without - i.O h - -2.0 h - - 25 h - -170 h - -560h -
MgCO3 than withit. 14 -

Both Pattersonand Parsons (1963) and Daley et al. 1I2

(1973) reportedreductionof bothphaeophytina (up to
100%) and phaeophorbidea (up to 70%) when either I0

MgCO3 or dimethylaniline was added to the filter.This

8-
phenomenoncould explain the increasein the amount
of phaeopigments found in our samples without 6- FRESH WATER
MgCO3. In the samples with MgCO3 some of the
4-
phaeopigmentswere probablyadsorbed by the MgCO3
and thus not detected by subsequent analyses. In the 2-
marine samples fromScripps pier and in the samples
fromMarch (Tab. 2), whereno differences were found, 0
ABC ABC DEFGHI DEFGHI DEFGH1
the actual amountof phaeopigmentswere so small that
adsorptionof some of the phaeopigmentsby MgCO3 Fig. 5. Recoveryof chlorophyll a (clear verticalbar) and
phaeopigment a (solidbar) fromphytoplankton as a function
would not alter the results. oflengthofextraction period,storageconditions, presenceor
As no significant loss of chlorophylla was foundin absenceOfMgCO3,and whether or notsamplewas ground.
any of the sampleswithoutMgCO3, and as the addition Whatman GF/CGlassfiberfilters wereusedforall samples.
of MgCO3 slows down the filtrationrate appreciably, Thetimesshowninthefigures refertothelength oftheperiod
betweenfiltration ofthesampleand measurement ofpigment
and reduces the amountof phaeopigmentsin the sam- fluorescence.
All measurements weredonebyfluorometry as
ple, it is suggestedthatthe use of MgCO3 be discontin- describedbyHolm-Hansen etal. (1965). Upperfigure: coastal
ued. Difficultiesassociatedwiththe use of MgCO3 have watera shortdistancefromScrippsInstitution of Oceano-
also beeen describedbySand-Jensen(1976), who attri- graphy. Lowerfigure: freshwatersamplefroma pondin the
coastalmountains eastofSan Diego.
butes the loss of chlorophylla duringstorageto forma- A - Without MgCO3,groundinacetone,roomtemperature.
B - WithMgCO3,groundinacetone,roomtemperature.
C - WithMgCO3,acetoneaddedbutfilter notgrund, room
temperature.
Tab. 2. Theeffect ofadditionof 1 ml1% MgCO3tothefilter, D - WithoutMgCO3,filterstoredfrozen,thengroundin
beforefiltration,on theamountsof chlorophylla and phae- acetone.
- With MgCO3, filterstored frozen,then groundin ace-
opigment a extractedfromdifferent popula- E
phytoplankton
tions in Lake Moss0. Diatoms were dominantin March and tone.
bluegreenalgae were mostabundantin July.Standarderroris F - Without MgCO3,groundin acetone,storedfrozen.
given for n = 10. Chlorophylla and phaeopigmenta in gg. G - WithMgCO3,groundin acetone,storedfrozen.
Pigmentlevelsweredeterminedspectrophotometrically H - Without MgCO3,acetoneaddedbutfilternotground,
accord-
ing to Moss (1967a, b) and Riemann (1976). storedfrozen.
I - WithMgCO3, acetoneadded but filternot ground,
Samples fromMarch storedfrozen.
+MgCO3 - MgCO3
Chl. a Phaeo. a Chl. a Phaeo. a
70.7 ? 1.1 10.5 ? 0.3 71.3 ? 0.8 10.0 ? 0.2
tionof aggregatesof algae and MgCO3; theseaggrega-
Samples fromJuly tes supposedly retain chlorophyllduring subsequent
+ MgCO3 - MgCO3 extractionattempts,causing a significant
Chl. a
loss of chlo-
Phaeo. a Chl. a Phaeo. a
72.5 ? 0.9 40.8 ? 0.7 69.3 ? 1.1 44.1 ? 0.5 rophyll.Such algae-MgCO3 aggregatesmay be spe-
cies-specific,as our studieswithmarineand freshwater

OIKOS 30:3 (1978) 445

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plankton(Fig. 5) didnotshowanylossduringstorage 100 -
associatedwithadditionofMgCO3to thesamples.
-HOCHOROPHYLL

3.3.3. Grinding and storageoffilters CLROPHYL

In some of our fieldstudieswe have frozensamples
filtered on glassfiberfiltersfor2-3 wk(-20'C) before
extraction with acetone (Holm-Hansen 1969,
Holm-Hansenet al. 1970). Thereis a questionof the 50
reliabilityofpigment concentrationswhensampleshave < PHAEO
been storedin thismanner,as Strickland and Parsons
(1968) statethatfilters shouldbe extracted without de-
layifat all possibleas storagealwaysleadsto low re- - ~ . A ~ PHAEO
sults.
Data in Fig. 5 showour observations on measure- 10 _ A _ B
mentsof chlorophyll a and phaeopigment a in both 0 1 2 3 0 1 2 3
freshwaterand marinesamplesextracted in 90% ace-
tonewithrespectto theeffects ofgrinding andstorage MIN. HOMOGENIZATION
at-20'C. BothsetsofdatainFig.5 support thefollow- Fig.6. Effectof homogenization on concentration ofchloro-
ingconclusions. (a) Thereis neither lossofpigment nor phylla ( ) andphaeopigment a (-----) Watersamples
anysignificant changeinthechlorophyll/phaeopigment fromLake Moss0werefiltered and extracted in either90%
ratiowhenextracted samplesare storedthreeweeksat acetonefor24 h (A) or in absolutemethanol for1 hour(B),
-20'C. It thusseemssafe to storeextractsat -20'C both withandwithout
confidence limits.
homogenization. Barsindicate the95%
duringfieldstudiesand to analyzethemat one timein
thebase laboratory, evenifthesampling program ex-
tendsoverseveralweeks.(b) Thereis no lossofchlo-
rophyll in sampleswhichhave been frozenin thewet (Yentsch and Menzel 1963) have published data in-
state,andwhicharesubsequently extracted withaceto- dicatingthatgrindingof samplesresultsin significantly
nepriortodetermination ofpigment content. Thisis an higherchlorophyllconcentrations. These studiesutilized
advantage infieldwork,as one doesnothaveto havea acetone as the extractant,however,and hence merely
supplyofacetoneanditalsoeliminates thepossibilityof confirmour data (Fig. 5) thatforsome environmental
loss of extracted pigment duringtransport of samples. samples,grindingdoes enhance the rate of chlorophyll
(c) If samplesare to be analyzedwithina fewhours extractionwhenacetoneis used as theextractant.As far
afterfiltration, it appearsthatit is necessary to grind as we are aware,thereare no publishedreportsshowing
filters inthehomogenization tube.Whentheextraction thebeneficialeffectofgrindingwhenabsolutemethanol
timeexceedsa fewhours,thedatainFig.5 indicatethat is used as the extractant.The investigatormust be
thereis no difference betweensampleswhichhavebeen aware, however,that some samples of phytoplankton
extracted withor without grinding. maypossiblyshow slow extractioneven whenmethanol
Subsequentstudieshave shownthatno detectable is used. An example of thisis the reportby Jeffrey and
differences in the amountof chlorophyll werefound Haxo (1968) in whichtheyused an initialfreezingpro-
whenfilters wereextracted in absolutemethanolafter cedure to facilitatesubsequentextractionof pigments
threeweeksofstorageat -20'C. fromzooxanthellae into methanol.Althoughwe have
Fig. 6 showsdata on theeffect of grinding offilters never obtained any beneficialeffectof grindingwhen
whichhavebeenextracted 24 h in 90% acetone(A) or methanol is the extractant,it would behoove the in-
1 h in absolutemethanol(B). Sampleswerefromthe vestigatorto verifythisforwhateveralgal samples are
eutrophicLake Moss0,withthe phytoplankton con- being studied.Our conclusionsare thatgrindingsome-
sisting mostly ofdiatomsandsomeblue-green algae.In timesmay be importantwhen extractingwithacetone,
bothextraction solventsno significant effect of homo- but is superfluouswhen usingmethanol.
genization was foundwhengroundsampleswerecom-
paredto theunhomogenized ones.Whytheamountof
phaeopigments werehigherin theacetoneextract than Acknowledgements - This workwas supportedin partby con-
inthemethanol extract seemsobscure,becauseno cor- tractEY-76-C-03-0010P.A. 20 fromtheU.S. EnergyRese-
arch and DevelopmentAdministration and by NSF grants
responding differences were foundin the amountof GB36173,GA 34948andGD34463 totheScripps Institution
chlorophyll a in thetwosolvents.Further experimentsof Oceanography foroperationsoftheAlphaHelixResearch
in Lake Moss0withphytoplankton populations domi- Program.One of theauthors(BR) was supported fromThe
natedbyblue-green algaeshowedno decreaseinchlor- Danish
511-7084.
Natural Science Research Council, grant no.
ophyllor phaeopigment concentration in methanolas
comparedto acetone.
It shouldbe noted,however, thatsomeinvestigators

446 OIKOS 30:3 (1978)

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All use subject to JSTOR Terms and Conditions
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