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Volume 62, Issue 3


Chlorophyll a extraction from freshwater algae — a reevaluation

Michael Schagerl / Gabriela Künzl
Published Online: 2007-06-01 | DOI: https://doi.org/10.2478/s11756-007-0048-x


In this study, two extracting methods (sonication and dispersing) and three solvents (90% acetone, N,N′-dimethylformamide and methanol) were compared for their ability to extract chlorophyll a of freshwater phytoplankton. Measurements were performed with both spectrophotometry and high-performance liquid chromatography. Results showed that (i) cell disruption is essential and that (ii) the method of cell disruption and solvent applied differed significantly. Dispersing in acetone surpassed all other combinations. Sonication in N,N′-dimethylformamide was found less effective. N,N′-dimethylformamide and methanol seem to promote the formation of degradation products (chlorophyllide a, allomer, epimer and phaeophytin a) which lead to overestimates of chlorophyll a of about 10% by means of spectrophotometry.

Keywords: chlorophyll a; extraction; phytoplankton; HPLC; spectrophotometry; sonication

  • [1] Abaychi J.K. & Riley J.P. 1979. The determination of phytoplankton pigments by high-performance liquid chromatography. Anal. Chim. Acta 107: 1–11. http://dx.doi.org/10.1016/S0003-2670(01)93190-3CrossrefGoogle Scholar

  • [2] Bidigare R.R., Kennicutt II M.C. & Brooks J.M. 1985. Rapid determination of chlorophylls and their degradation products by high-performance liquid chromatography. Limnology and Oceanogr. 30: 432–435. Google Scholar

  • [3] Braumann T. & Grimme L.H. 1979. Single-step separation and identification of photosynthetic pigments by high-performance liquid chromatography. J. Chromatogr. 170: 264–268. http://dx.doi.org/10.1016/S0021-9673(00)84264-8CrossrefGoogle Scholar

  • [4] Brotas V. & Plante-Cuny M.R. 2003. The use of HPLC pigment analysis to study microphytobenthos communities. Acta Oecolog. — Int. J. Ecol. 24: 109–115. CrossrefGoogle Scholar

  • [5] Buchaca T., Felip M. & Catalan J. 2005. A comparison of HPLC pigment analyses and biovolume estimates of phytoplankton groups in an oligotrophic lake. J. Plankton Res. 27: 91–101. http://dx.doi.org/10.1093/plankt/fbh154CrossrefGoogle Scholar

  • [6] Burnison B.K. 1980. Modified dimethyl sulfoxide (DMSO) extraction for chlorophyll analysis of phytoplankton. Can. J. Fish. Aquat. Sci. 37: 729–733. CrossrefGoogle Scholar

  • [7] Cartaxana P. & BROTAS V. 2003. Effects of extraction on HPLC quantification of major pigments from benthic microalgae. Arch. Hydrobiol. 157: 339–349. http://dx.doi.org/10.1127/0003-9136/2003/0157-0339CrossrefGoogle Scholar

  • [8] Fietz S., Bleiss W., Hepperle D., Koppitz H., Krienitz L. & Nicklisch A. 2005. First record of Nannochloropsis limnetica (Eustigmatophyceae) in the autotrophic picoplankton from Lake Baikal. J. Phycol. 41: 780–790. http://dx.doi.org/10.1111/j.0022-3646.2005.04198.xCrossrefGoogle Scholar

  • [9] Frigaard N.U., Larsen K.L. & Cox R.P. 1996. Spectrochromatography of photosynthetic pigments as a fingerprint technique for microbial phototrophys. FEMS Microbiol. Ecol. 20: 69–77. http://dx.doi.org/10.1111/j.1574-6941.1996.tb00306.xCrossrefGoogle Scholar

  • [10] Gerloff-Elias A., Spijkerman E. & Schubert H. 2005. Light acclimation of Chlamydomonas acidophila accumulating in the hypolimnion of an acidic lake (pH 2.6). Freshwater Biol. 50: 1301–1314. http://dx.doi.org/10.1111/j.1365-2427.2005.01400.xCrossrefGoogle Scholar

  • [11] Gieskes W.W.C. & Kraay G.W. 1982. Comparison of chromatographic (HPLC and TLC) conventional methods for the measurement of chlorophylls in oceanic waters. Arch. für Hydrobiol. Beihefte Ergebnisse der Limnol. 16: 123. Google Scholar

  • [12] Gieskes W.W.C. & Kraay G.W. 1983. Dominance of Cryptophyceae during the phytoplankton spring bloom in the central North Sea detected by HPLC analysis of pigments. Mar. Biol. 75: 179–185. http://dx.doi.org/10.1007/BF00406000CrossrefGoogle Scholar

  • [13] Gieskes W.W.C. & Kraay G.W. 1986. Analysis of phytoplankton pigments by HPLC before, during and after mass occurrence of the microflagellate Corymbellus aureus during the spring bloom in the open northern North Sea in 1983. Mar. Biol. 92: 45–52. http://dx.doi.org/10.1007/BF00392744CrossrefGoogle Scholar

  • [14] Havskum H., Schlüter L., Scharek R., Berdalet E. & Jacquet S. 2004. Routine quantification of phytoplankton groups—microscopy or pigment analyses? Mar. Ecol. Prog. Ser. 273: 31–42. Google Scholar

  • [15] Jacobsen T.R. 1978. A quantitative method for the separation of chlorophyll a and b from phytoplankton pigments by high pressure liquid chromatography. Mar. Sci. Commun. 4: 33–47. Google Scholar

  • [16] Jacobsen T.R. & Rai H. 1990. Comparison of spectrophotometric, fluorometric and high-performance liquid chromatography methods for determination of chlorophyll a in aquatic samples: effects of solvent and extraction procedures. Int. Rev. Hydrobiol. Hydrograp. 75: 207–217. http://dx.doi.org/10.1002/iroh.19900750207CrossrefGoogle Scholar

  • [17] Jeffrey S.W. & Humphrey G.F. 1975. New spectrophotometric equations for determing chlorophylls a, b, c1, and c2 in higher plants, algae and natural phytoplankton. Bioch. Physiol. Pflanzen 167: 191–194. Google Scholar

  • [18] Jeffrey S.W. 1981. An improved thin-layer chromatographic technique for marine phytoplankton pigments. Limnol. Oceanogr. 26: 191–197. Google Scholar

  • [19] Kusel-Fetzmann E. & Schagerl M. 1993. Verzeichnis der Sammlung von Algenkulturen an der Abteilung für Hydrobotanik am Institut für Pflanzenphysiologie der Universität Wien. Phyton 33: 209–234. Google Scholar

  • [20] Lionard M., Azémar F., Boulętreau S., Muylaert K., Tackx M. & Vyverman W. 2005. Grazing by meso-and microzooplankton on phytoplankton in the upper reaches of the Schelde estuary (Belgium/The Netherlands). Estuarine Coast. Shelf Sci. 64: 764–774. http://dx.doi.org/10.1016/j.ecss.2005.04.011CrossrefGoogle Scholar

  • [21] Llewellyn C.A., Fishwick J.R. & Blackford J.C. 2005. Phytoplankton community assemblage in the English Channel: a comparison using chlorophyll a derived from HPLC-CHEMTAX and carbon derived from microscopy cell counts. J. Plankt. Res. 27: 103–119. http://dx.doi.org/10.1093/plankt/fbh158CrossrefGoogle Scholar

  • [22] Lorenzen C.J. 1967. Determination of chlorophyll and pheopigments: spectrophotometric equations. Limnol. Oceanogr. 12: 343–346. Google Scholar

  • [23] Mackey M., Mackey D., Higgins H. & Wright S. 1996. CHEMTAX — a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton. Mar. Ecol. Prog. Ser. 144: 265–283. CrossrefGoogle Scholar

  • [24] Mantoura R.F.C. & 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: 297–314. http://dx.doi.org/10.1016/S0003-2670(00)80092-6CrossrefGoogle Scholar

  • [25] Marker A.F.H. 1972. The use of acetone and methanol in the estimation of chlorophyll in the presence of phaeophytin. Freshwater Biol. 2: 361–385. http://dx.doi.org/10.1111/j.1365-2427.1972.tb00377.xCrossrefGoogle Scholar

  • [26] Marker A.F.H., Crowther C.A. & Gunn R.J.M. 1980. Methanol and acetone as solvents for estimating chlorophyll a and phaeopigments by spectrophotometry. Arch. Hydrobiol. Beih. 14: 52–69. Google Scholar

  • [27] Millie D.F., Paerl H.W. & Hurley J.P. 1993. Microalgal pigment assessments using high-performance liquid chromatography: a synopsis of organismal and ecological applications. Can. J. Fish. Aquat. Sci. 50: 2513–2527. http://dx.doi.org/10.1139/f93-275CrossrefGoogle Scholar

  • [28] Neveux J. 1988. Extraction of chlorophylls from marine phytoplankton. Verhandlungen Internationale Vereinigung für Theoretische und angewandte Limnologie 23: 928–932. Google Scholar

  • [29] Nijs, M., L. Nabben & K. Wernars, 1996. Isolation of Fusarium DNA for molecular analysis with and without mechanical cell disruption. J. Microbiol. Methods 27: 13–17. http://dx.doi.org/10.1016/0167-7012(96)00920-7CrossrefGoogle Scholar

  • [30] Norman A.B., Borchers M.T., Wachendorf T.J., Price A.L. & Sanberg P.R. 1989. Loss of D1 and D2 dopamine receptors and muscarinic cholinergic receptors in rat brain following in vitro polytron homogenisation. Brain Res. Bull. 22: 633–636. http://dx.doi.org/10.1016/0361-9230(89)90082-8CrossrefGoogle Scholar

  • [31] Nusch E.A. 1980. Comparison of different methods for chlorophyll and phaeopigment determination. Arch. Hydrobiol. Beih. Ergeb. Limnol. 17: 14–36. Google Scholar

  • [32] Otsuki A. & Takamura N. 1988. Comparison of chlorophyll a concentrations measured by fluorometric HPLC and spectrophotometric methods in highly eutrophic small Lake Kasumigaura. Verhandlungen Internationale Vereinigung für Theoretische und angewandte Limnologie 23: 944–951. Google Scholar

  • [33] Owens T.G. & Falkowski P.G. 1982. Enzymatic degradation of chlorophyll a by marine phytoplankton in vitro. Phytochem. 21: 979–984. http://dx.doi.org/10.1016/S0031-9422(00)82401-2CrossrefGoogle Scholar

  • [34] Parsons T.R., Maika Y. & Lalli C.M. 1984. A manual of chemical and biological methods for seawater analysis. Pergamon Press, 173pp. Google Scholar

  • [35] Pepe M., Giardino C., Borsani G., Cardoso A.C., Chiaudani G., Premazzi G., Rodari E. & Zilioli E. 2001. Relationship between apparent optical properties and photosynthetic pigments in the sub-alpine Lake Iseo. Sci. Total Environ. 268: 31–45. http://dx.doi.org/10.1016/S0048-9697(00)00691-4CrossrefGoogle Scholar

  • [36] Porra R.J., Thompson W.A. & Kriedemann P.E. 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. BBA 975: 384–394. Google Scholar

  • [37] Rai H. 1973. Methods involving the determination of photosynthetic pigments using spectrophotometry. Verhandlungen Internationale Vereinigung für Theoretische und angewandte Limnologie 18: 1864–1875. Google Scholar

  • [38] Rai H. 1980. Some problems in determination of photosynthetic planctonic pigments and their decomposition products. Arch. Hydrobiol. Beih. Ergeb. Limnol. 14: 3–13. Google Scholar

  • [39] Riemann B. 1980. A note on the use of methanol as an extraction solvent for chlorophyll a determination. Arch. Hydrobiol. Beih. Ergeb. Limnol. 14: 70–78. Google Scholar

  • [40] Richards F.A. & Thompson T.G. 1952. The estimation and characterisation of plankton populations by pigment analysis. II. A spectrophotometric method for the estimation of plankton pigments. J. Mar. Res. 11: 156–172. Google Scholar

  • [41] Sartory D.P. 1985. The determination of algal chlorophyllous pigments by high performance liquid chromatography and spectrophotometry. Water Res. 19: 605–610. http://dx.doi.org/10.1016/0043-1354(85)90066-1CrossrefGoogle Scholar

  • [42] Sartory D.P. & Grobbelaar J.E. 1984. Extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis. Hydrobiol. 114: 177–187. CrossrefGoogle Scholar

  • [43] Shoaf W.T. & Lium B.W. 1976. Improved extraction of chlorophyll a and b from algae using dimethylsulfoxide. Limnol. Oceanogr. 21: 926–928. http://dx.doi.org/10.4319/lo.1976.21.6.0926CrossrefGoogle Scholar

  • [44] Siman D. & Helliwell S. 1998. Extraction and quantification of chlorophyll a from freshwater green algae. Water Res. 32: 2220–2223. http://dx.doi.org/10.1016/S0043-1354(97)00452-1CrossrefGoogle Scholar

  • [45] Speziale B.J., Schreiner S.P., Giamatteo P.A. & Schindler J.E. 1984. Comparison of N,N′-dimethylformamide, dimethylsulfoxide, and acetone for extraction of phytoplankton chlorophyll. Can. J. Fish. Aquatic Sci. 41: 1519–1522. http://dx.doi.org/10.1139/f84-187CrossrefGoogle Scholar

  • [46] Strain H.H., Cope B.T. & Svec W.A. 1971. Analytical procedures for the isolation, identification, estimation and investigation of chlorophylls. Methods Enzymol. 23: 452– CrossrefGoogle Scholar

  • [47] Trees C.C., Kennicutt M.C. & Brooks J.M. 1985. Errors associated with the standard fluorometric determination of chlorophylls and pheopigments. Mar. Chem. 17: 1–12. http://dx.doi.org/10.1016/0304-4203(85)90032-5CrossrefGoogle Scholar

  • [48] Veldhuis M.J.W. & Kraay G.W. 1990. Vertical distribution and pigment composition of a picoplanctonic prochlorophyte in the subtropical North Atlantic: a combined study of HPLC-analysis of pigments and flow cytometry. Mar. Ecol. Prog. Ser. 68: 121–127. CrossrefGoogle Scholar

  • [49] Wright S.W., Jeffrey S.W. & Mantoura R.F.C. 1997. Evaluation of methods and solvents for pigment extraction, pp. 261–282. In: Jeffrey S.W., Mantoura R.F.C. & Wright S.W. (eds), Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO Publishing, Paris. Google Scholar

  • [50] Wright S.W., Jeffrey S.W., Mantoura R.F.C., Llewellyn C.A., Bjorland T., Repeta D. & Welschmeyer N. 1991. An improved HPLC method for the analysis of chlorophyll and carotenoids from marine phytoplankton. Mar. Ecol. Progr. Ser. 77: 183–196. CrossrefGoogle Scholar

  • [51] Wright S.W. & Shearer J.D. 1984. Rapid extraction and high-performance liquid chromatography of chlorophylls and carotenoids from marine phytoplankton. J. Chromatogr. 294: 281–295. http://dx.doi.org/10.1016/S0021-9673(01)96134-5CrossrefGoogle Scholar

  • [52] Wulff A., Vilbaste S. & Truu J. 2005. Depth distribution of photosynthetic pigments and diatoms in the sediments of a microtidal fjord. Hydrobiol. 534: 117–130. http://dx.doi.org/10.1007/s10750-004-1417-xCrossrefGoogle Scholar

  • [53] Zapata M., Ayala A.M. & Garrido J.L. 1987. Separation of chlorophylls and their degradation products in marine phytoplankton by reversed-phase high-performance liquid chromatography. Chromatographia 23: 26–30. http://dx.doi.org/10.1007/BF02310413CrossrefGoogle Scholar

  • [54] Zapata M. & Garrido J.L. 1991. Influence of injection conditions in reversed-phase high performance liquid chromatography of chlorophylls and carotenoids. Chromatographia 31: 589–594. http://dx.doi.org/10.1007/BF02279480CrossrefGoogle Scholar

About the article

Published Online: 2007-06-01

Published in Print: 2007-06-01

Citation Information: Biologia, Volume 62, Issue 3, Pages 270–275, ISSN (Online) 1336-9563, ISSN (Print) 0006-3088, DOI: https://doi.org/10.2478/s11756-007-0048-x.

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© 2007 Institute of Botany, Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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