PII: S0043-1354(97)00452-1

Wat. Res. Vol. 32, No. 7, pp. 22202223, 1998

# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0043-1354/98 $19.00 + 0.00

TECHNICAL NOTE
EXTRACTION AND QUANTIFICATION OF
CHLOROPHYLL A FROM FRESHWATER GREEN ALGAE
DAVID SIMON* and STUART HELLIWELL
School of Science and Technology, Charles Sturt University, P.O. Box 588, Wagga Wagga, NSW 2678,
Australia
(First received February 1997; accepted in revised form November 1997)
AbstractWith the renement of microanalytical methods useful for quantifying plant pigments, the
extraction procedure appears to be one of the key steps in chlorophyll analysis. Comparisons of two
solvents; methanol and acetone and four methods of extraction; probe sonication, bath sonication, tissue grinding and maceration by mortar and pestle were performed. Using methanol, a probe sonicator
was more ecient in chlorophyll a extraction than the other extraction methods ( p < 0.01). Methanol
was the better solvent in all but the maceration method. The maceration method was equally eective
for both solvents but was statistically lower ( p < 0.01) than achieved by methanol and probe sonicator.
# 1998 Elsevier Science Ltd. All rights reserved
Key wordschlorophyll a, HPLC, sample preparation, solvents, extraction method

INTRODUCTION

Accurate quantication of chlorophyll a is an important step in estimating phytoplankton biomass

in both marine and freshwater environments.
Determination of chlorophyll has been traditionally
based on extraction into a solvent followed by
quantication by either spectrophotometry or
uorometry or, more recently, by high-performance
liquid chromatography (HPLC) (Schwartz and von
Elbe, 1982) with some adding a clean-up step (e.g.
Sartory, 1985). HPLC allows separation of the pigments that may otherwise interfere with the spectral
determination of the pigment of interest, often
chlorophyll a. Should this occur, overestimation of
chlorophyll a will result (Sartory, 1985; Meyns et
al., 1994). Further, the determination of multiple
pigments can be used for a number of purposes
such as investigating algae population dynamics
(Meyns et al., 1994; Le Rouzic et al., 1995).
Although the method of detection and quantication of chlorophyll is well established there
appears to be conicting evidence as to the best
method of extracting the pigments from algae.
Removal of the pigment from the cells has been
done without disruption of the cell wall (Golterman
et al., 1978); or after disruption by sonication
*Author to whom all correspondence should be addressed.
Current address: Environmental Health Branch, South
Australian Health Commission, P.O. Box 6, Rundle
Mall, SA 5000, Australia.

(Nelson, 1960); or tissue grinding (APHA, 1992); or

both tissue grinding and sonication (Downes et al.,
1993). Holden (1976) in her review, reports other
methods including standing in water to allow swelling (e.g. marine phytoplankton) and freezing followed by thawing.
Most commonly acetone, methanol or ethanol
have been used as the solvent (Mantoura and
Llewellyn, 1983). However, other solvents such as
chloroform (Palmisano et al., 1988), dimethyl sulfoxide (Sartory and Grobbelaar, 1984) or petroleum
ether (Strain and Svec, 1966) have been used.
In our laboratory there appeared to be a signicant dierence in the amount of chlorophyll a
extracted depending upon the method of cell disruption and solvent used. This paper examines the
ability of a number of methods (probe sonication,
bath sonication, tissue grinding and maceration by
mortar and pestle) and two solvents (acetone and
methanol) to extract chlorophyll a from a freshwater green alga.
MATERIALS AND METHODS

Reagents
The solvents, acetone (Nanograde, Mallinckrodt) and
methanol (HiPerSov, BDH) were of highest purity, suitable for high performance liquid chromatography.
Chlorophyll a (Sigma, extracted from Anacystis nidular)
was reconstituted according the manufacturers instruction
by resuspending the chlorophyll a in acetone and diluting
to 50 mg/l. The nal solution was stored at 208C in an
air tight glass vial. Serial dilutions (usually ve) were

2220

Extraction and quantication of chlorophyll a

2221

made (10100 mg/l) using de-acidied methanol immediately prior to calibration of the HPLC. The methanol was
de-acidied by adding one gram of magnesium carbonate
(Analytical grade, Ajax) to each litre. A 1% magnesium
carbonate suspension was prepared and used to make a
90% acetone solution. Water used in all experiments was
``nano-pure'' produced by a Millipore Nano-pure water
system.
Sample preparation
The alga Selenastrum obliquus, cultured using the algal
assay bottle test method of Miller et al. (1978), was used
in all experiments. The phytoplankton were harvested by
ltering 25 ml of the culture through a 45 mm Whatman
GF/C glass bre lter under vacuum. Cell numbers were
determined in the usual way using a haemocytometer
(Improved Neubauer, Weber, U.K.). Following disruption
of the cells, the extract was adjusted to 25 ml with the
appropriate solvent and ltered through a 25 mm
Whatman GF/F glass bre lter. Samples were stored at
208C until quantied by HPLC, usually within 30 min or
if using the American Public Health Association (APHA)
Standard Method for the Examination of Water and
Waste Water, procedure 10200 (APHA, 1992), the sample
was stood for 2 h at 48C.
Cell disruption
Probe or bath sonication. Filters bearing phytoplankton
were cut into 0.5 cm squares, which aided sonication, and
placed into a 70 ml aluminium foil wrapped sonication
vessel with 25 ml of either 90% acetone or methanol. The
sonication vessel was kept cool by placing it in a beaker
containing ice. The sample was either sonicated for 2 min
with a 25 mm tipped probe (Sonier 450, Branson Ultrasonics, Danbury, CT, U.S.A.) with the output controller
set at number seven for continuous output (approximately
19 W/cm2) or by placing the vessel for 5 min in an ultrasonic bath (Ultrasonic Industries Pty. Ltd., Sydney, NSW,
Australia) with an average output of 60 W (approximately
0.3 W/cm2).
Tissue grinding. The APHA Standard Method for the
Examination of Water and Waste Water, procedure 10200
(APHA, 1992), was followed except when methanol was
used rather than 90% acetone. Filters were cut into 5
lengths and placed into a glass round bottom tissue grinder (Wheaton Scientic, Millville, NJ, U.S.A.) containing
3 ml of solvent. The cells were disrupted with a teon
grinding head rotating at 500 rpm for 2 min. The slurry
generated was placed into a glass container along with
two 3 ml washings of the grinder. After adjusting the
volume the samples were allowed to stand for two hours
at 48C in the dark prior to HPLC analysis.
Mortar and pestle. The lters were placed into a glass
mortar and macerated with a glass pestle in either 20 ml
of acetone or methanol. The process usually took 5 min.
The slurry generated was placed into a glass container
along with two 2 ml washings of the mortar. After adjustTable 1. Comparison of the quantity of chlorophyll a (mg)
extracted by various methods from a culture of Selenastrum obliquus
Method

Methanol
Mean

Probe sonication
Bath sonication
Tissue grinding
Mortar and pestle
Standing cells in
solvent for 24 h at 48C
a

Standard deviation, n = 6.

1.70
1.50
1.41
1.44
0.44

Acetone
a

SD

0.06
0.08
0.08
0.15
0.13

Mean
0.57
0.76
1.10
1.44

SDa
0.03
0.08
0.01
0.10

Fig. 1. Fluorescence chromatogram of an extract of pigment from Selenastrum obliquus, performed immediately
after extraction (A) or 12 d later after being stored at
room temperature in the dark (B).
ing the volume the samples were allowed to stand for two
hours at 48C in the dark prior to HPLC analysis.
Quantication
The rapid HPLC method of Murray et al. (1986) was
adapted to quantify the chlorophyll. Briey, a Perkin
Elmer LC 240 HPLC pump (Perkin Elmer, Norwalk, CN,
U.S.A.) attached to an ODS Sperisorb (2.5 mm)
250 mm  4.6 mm column (Alltech, Sydney, Australia) was
used with a Perkin Elmer LC 240 uorescence detector
(lex=431 nm; lem=667 nm). The detector was set to give
a one volt output range. Samples were injected on-column
by a Rheodyne loop injector (200 ml loop) and carried
with a mobile phase of ethylacetate, methanol and water
(44:49.5:6.5) owing at 2 ml/min. The typical time for
elution of chlorophyll a was about 4 min.
Peak area integration was performed by a Perkin
Nelson 1020 computer/software package (Perkin Elmer,
Norwalk, CN, U.S.A.). Peaks were integrated valley-tovalley with data collection rate automatically set to integrate peaks of greater than 1.4 to 3.2 s base width.
All samples were taken from the same batch of culture,
and all experiments including HPLC analysis were performed on the same day. Experiments were done in subdued light and all containers were wrapped in aluminium
foil. Each method of extraction was repeated six times.
RESULTS AND DISCUSSION

Data from the recovery experiments are shown in

Table 1 with a typical chromatogram shown in
Fig. 1(A). These data conrm the ability of HPLC
to provide a rapid and sensitive method to quantify
chlorophyll a extracted from an alga species (e.g.
Mantoura and Llewellyn, 1983; Sartory, 1985;
Murray et al., 1986). Changing the ratios of the
mobile phase from those used by Murray et al.
(1986) allowed better separation of the peaks surrounding the chlorophyll a peak. This was done by
adding 1% more water. Detector linearity was good
(r2>0.99, n = 10) for two ranges of chlorophyll a

2222

David Simon and Stuart Helliwell

(010 and 10100 mg/l). There was also an excellent

correlation between cell number and chlorophyll a
(r2=0.99, n = 10). The detection limit, dened as
three times the standard deviation of the blank, was
60 pg (n = 30) for chlorophyll a.
Methanol proved to be signicantly better as the
extraction solvent in the sonication and tissue
grinding methods. The use of methanol in the
sonication method removed three times more pigment than acetone, which was highly signicant
( p < 0.005), but only removed 20% more pigment
in the tissue grinding method ( p < 0.005). This
result is consistent with some others who have also
found methanol to be better at extracting chlorophyll a from green algae (Sartory and Grobbelaar,
1984). In contrast, the choice of solvent did not inuence the outcome when the mortar and pestle
technique was used to disrupt the cells. Although
more time consuming than sonication, mortar and
pestle maceration produced similar results, for both
solvents, however, was more variable compared to
probe sonication. These results concur with Wright
et al. (1997) who recommend that sonication with
methanol be used for removal of pigments from
marine algae.
The reason why acetone was such a poor solvent
in all methods except extraction using a mortar and
pestle is dicult to explain. Literature reports
suggest the use of methanol as a solvent can cause
severe problems. For instance, using methanol
allows the activation of chlorophyllase causing loss
of chlorophyll a (Mantoura and Llewellyn, 1983).
Other degradation pathways can be expected if left
standing at room temperature (Strain and Svec,
1966; Otsuki et al., 1987). This was demonstrated
when samples of extracted pigments in both solvents were allowed to remain at room temperature
for 12 d in the dark. While acetone extractions
showed no reduction in concentration ( p < 0.005)
there was clearly degradation of chlorophyll a in
the methanol samples, with an extra peak appearing
in the chromatogram (Fig. 1(B)). This extra peak
was consistent with the reduction in the concentration of chlorophyll a by 32%. Although not
identied it is suggested that the degradation peak
is 10-hydroxychlorophyll a, which Sartory (1985)
prepared by standing chlorophyll a in methanol for
18 h.
In a separate experiment it was found that merely
standing the cells in solvent for 24 h removed only
one quarter of the chlorophyll a that could be
extracted by the best method (methanol with probe
sonication). This result highlights the need to physically disrupt the cells. The probe sonicator was
more eective than the bath in aiding chlorophyll a
extraction. This may be because the probe is more
ecient in transferring energy, even though the
power output of the probe is a quarter of the bath's
output. The probe dissipates the energy directly
from a small surface area at the centre of the

sample to the edges rather than from the bath walls

via uid to the sample. This is further emphasized
when comparing the power output per unit surface
area: probe, 19 W/cm2; bath, 0.3 W/cm2.
At least two processes are needed to extract the
pigments into the solvent. The cell needs to be disrupted followed by elution of the pigments from the
cellular components. One reason why acetone
proved to be a poor solvent is perhaps because it
did not aid, to the same extent, the disruption of
the cells. It could also be argued that methanol is a
better eluting agent for pigments then acetone.
Further work will be required to test these hypothesis.
CONCLUSIONS

To optimize pigment recovery from algae cells

mechanical disruption is needed. Probe sonication
allows maximal extraction of chlorophyll a from
green algae. Tissue grinding improves extraction
over simple diusion but is not as ecient as sonication. Methanol is a more ecient solvent than
acetone, provided care is exercised preventing the
sample heating during sonication and the analysis
of the pigment is done as soon as possible after
extraction. Care also is needed in handling methanol because it is more toxic to humans than acetone
(American Conference of Governmental Industrial
Hygienists, 1995). Methanol, has in its favour, however, a lower vapour pressure, at room temperature,
and a much higher ash point than acetone.
AcknowledgementsThis paper has been supported in
part with a grant from the Murray Darling Freshwater
Research Council and a postgraduate grant from Charles
Sturt University.

REFERENCES

American Conference of Governmental Industrial

Hygienists (1995) Threshold Limit Values (TLVs) for
Chemical Substances and Physical Agents and Biological
Exposure Indices (BEIs). ACGIH, Cincinnati.
APHA (1992) Standard Methods of the Examination of
Water And Wastewater, 18th edition. American Public
Health
Association,
American
Water
Works
Association, Washington, DC.
Downes M. T., Hrstich L. and Vincent W. F. (1993)
Extraction of chlorophyll and carotenoid pigments from
antarctic benthic mats for analysis by HPLC. J. Appl.
Phycol. 5, 623628.
Golterman, H. L., Clymo, R. S. and Ohnstad, M. A. M.
(1978) Methods for Physical and Chemical Analysis of
Freshwaters, 2nd edition, I.B.P. Handbook 8. Blackwell,
Oxford.
Holden, M. (1976) Chlorophylls. In Chemistry and
Biochemistry of Plant Pigments, ed. T. W. Goodwin,
Vol. 2, 2nd edition, pp. 237. Academic Press, New
York.
Le Rouzic B., Bertru G. and Brient L. (1995) HPLC
analysis of chlorophyll a breakdown products to interpret microalgae dynamics in a shallow bay.
Hydrobiologia 302, 7180.

Extraction and quantication of chlorophyll a

Mantoura R. F. C. and Llewellyn C. A. (1983) The rapid
determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters
by reverse phase high performance liquid chromatography. Anal. Chim. Acta 151, 297314.
Meyns S., Illi R. and Ribi B. (1994) Comparison of chlorophyll a analysis by HPLC and spectrophotometry:
where do the dierences come from? Arch. Hydrobiol.
132, 129139.
Miller, W. E., Greene, J. C. and Shiroyama, T. (1978) The
Selenastrum capricornutum Printz algal assay bottle test.
Corvallis Environmental Research Laboratory, U.S.
EPA, Corvallis, OR, EPA-600/9-78-018.
Murray A. P., Gibbs C. F., Longmore A. R. and Flett D.
J. (1986) Determination of chlorophyll in marine waters:
intercomparison of a rapid HPLC method with full
HPLC, spectrophotometric and uorometric methods.
Mar. Chem. 19, 211227.
Nelson D. L. (1960) Improved chlorophyll extraction
method. Science 132, 351.
Otsuki A., Watanabe M. M. and Sugahara K. (1987)
Chlorophyll pigments in methanol extracts from ten
axenic cultured diatoms and three green algae as determined by reverse phase HPLC with uorometric detection. J. Phycol. 23, 406414.

2223

Palmisano A. C., Cronin S. E. and Des Marais D.

J. (1988) Analysis of lipophilic pigments from a
phototrophic microbial mat community by high performance liquid chromatography. J. Microb. Methods 8,
209217.
Sartory D. P. (1985) The determination of algal chlorophyllous pigments by high performance liquid chromatography and spectrophotometry. Water. Res. 19, 605
610.
Sartory D. P. and Grobbelaar J. U. (1984) Extraction of
chlorophyll a from freshwater phytoplankton for spectrophotometric analysis. Hydrobiologia 114, 177187.
Schwartz S. J. and von Elbe J. H. (1982) High performance liquid chromatography of plant pigments. A
review. J. Liq. Chromatogr. 5(Suppl. 1), 4373.
Strain, H. H. and Svec, W. A. (1966) Extraction, separation, estimation, and isolation of the chlorophylls. In
The chlorophylls, ed. L. P. Veron and R. S. Gilbert, pp.
2261. Academic Press, New York.
Wright, S. W., Jerey, S. W. and Mantoura, R. F. C.
(1997) Evaluation of methods and solvents for pigment
extraction. In Phytoplankton pigments in oceanography:
guidelines to modern methods, ed. S. W. Jerey, R. F. C.
Mantoura and S. W. Wright, pp. 261282. UNESCO,
Paris.