Selection of protocols for phytoplankton pigment analysis: a comparative study

Jinxiu WANG; Fanzhou KONG; Zhuang NIU; Rencheng YU

doi:10.1007/s00343-024-4025-9

Your Location：

Home >

Browse articles >

Selection of protocols for phytoplankton pigment analysis: a comparative study

Ecology | Updated：2025-05-27

- Selection of protocols for phytoplankton pigment analysis: a comparative study
- Journal of Oceanology and Limnology Vol. 43, Issue 3, Pages: 817-830(2025)
- Affiliations：
  
  1.CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
  2.Laboratory for Marine Ecology and Environmental Science, Qingdao Marine Science and Technology Center, Qingdao 266214, China
  3.University of Chinese Academy of Sciences, Beijing 100049, China
  4.Marine Monitoring and Forecasting Center of Zhejiang Province, Hangzhou 310007, China
- Author bio：
  
  fzkong@qdio.ac.cn
- Funds：
- DOI：10.1007/s00343-024-4025-9
  CLC：
- Received：22 January 2024，
  
  Online First：17 June 2024，
  
  Published：01 May 2025
- Accepted：
Scan QR Code
WANG Jinxiu,KONG Fanzhou,NIU Zhuang,et al.Selection of protocols for phytoplankton pigment analysis: a comparative study[J].Journal of Oceanology and Limnology,2025,43(03):817-830.
DOI：

WANG Jinxiu,KONG Fanzhou,NIU Zhuang,et al.Selection of protocols for phytoplankton pigment analysis: a comparative study[J].Journal of Oceanology and Limnology,2025,43(03):817-830. DOI： 10.1007/s00343-024-4025-9.

摘要

Abstract

Pigments are widely used as indices for estimation of phytoplankton biomass and composition

and many protocols have been developed to analyze pigments in phytoplankton. Different protocols were compared using four solvents (methanol

95% methanol

dimethylformamide

and 90% acetone) and two instruments (fluorometer and high-performance liquid chromatography (HPLC) coupled with diode array detector). Analysis of chlorophyll

(Chl

) with fluorometer could lead to over- or underestimation due to the interference from its derivatives in all probability. Among the four extractants

90% acetone had a high recovery for chlorophylls. In contrast

95% methanol was a poor extractant for chlorophylls due to the degradation of Chl

especially in diatoms. The 95% methanol

however

had high extraction efficiencies for most diagnostic xanthophylls. Therefore

the selection of pigment analytical protocols should follow the specific purpose of phytoplankton study. In addition to fluorometry

an HPLC method with 90% acetone as extractant shall be a good choice for the analysis of Chl

to estimate phytoplankton biomass

especially for diatom-dominated samples

while an HPLC method with 95% methanol as extractant be more suitable to characterize different taxa in phytoplankton communities.

关键词

Keywords

references

Airs R L , Atkinson J E , Keely B J . 2001 . Development and application of a high resolution liquid chromatographic method for the analysis of complex pigment distributions . Journal of Chromatography A , 917 ( 1-2 ): 167 - 177 , https://doi.org/10.1016/S0021-9673(01)00663-X https://doi.org/10.1016/S0021-9673(01)00663-X .

Arar E J , Collins G B . 1997 . Method 445.0 in Vitro Determination of Chlorophyll a and Pheophytin a in Marine and Freshwater Algae By Fluorescence . U.S. Environmental Protection Agency, Washington . 22 p.

Bianchi T S , Lambert C , Biggs D C . 1995 . Di stribution of chlorophyll a and phaeopigments in the northwestern Gulf of Mexico: a comparison between fluorometric and high-performance liquid chromatography measurements . Bulletin of Marine Science , 56 ( 1 ): 25 - 32 .

Bidigare R R , Van Heukelem L , Trees C C . 2002 . HPLC phytoplankton pigments: sampling, laboratory methods, and quality assurance procedures . Ocean Optics Protocols for Satellite Ocean Color Sensor Validation, Revision , 3 ( 2 ): 258 - 268 .

Chen J X , Ye X , Chen X P et al . 2005 . Study on extraction of photosynthetic pigments from phytoplankton by organic solvents . Journal of Xiamen University (Natural Science) , 44 ( 1 ): 102 - 106 , https://doi.‍‍org/10.3321/j.issn:0438-0479.2005.01.026. https://doi.‍‍org/10.3321/j.issn:0438-0479.2005.01.026. (in Chinese with English abstract)

Cheng J Y , Don-Paul M , Antia N J . 1974 . Isolation of an unusually stable Cis -isomer of alloxanthin from a bleached autolysed culture of Chroomonas salina grown photoheterotrophically on glycerol. observations on Cis - trans isomerization of alloxanthin . The Journal of Protozoology , 21 ( 5 ): 761 - 767 , https://doi.org/10.1111/j.1550-7408.1974.tb03748.x https://doi.org/10.1111/j.1550-7408.1974.tb03748.x .

Falkowski P , Kiefer D A . 1985 . Chlorophyll a fluorescence in phytoplankton: relationship to photosynthesis and biomass . Journal of Plankton Research , 7 ( 5 ): 715 - 731 , https://doi.‍org/10.‍1093/plankt/7.5.715 https://doi.‍org/10.‍1093/plankt/7.5.715 . https://do 10.1093/plankt/7.5.715 http://dx.doi.org/10.1093/plankt/7.5.715

Gibb S W , Cummings D G , Irigoien X et al . 2001 . Phytoplankton pigment chemotaxonomy of the northeastern Atlantic . Deep Sea Research Part II: Topical Studies in Oceanography , 48 ( 4-5 ): 795 - 823 , https://doi.org/10.1016/S0967-0645(00)00098-9 https://doi.org/10.1016/S0967-0645(00)00098-9 .

Gregg W W , Conkright M E , Ginoux P et al . 2003 . Ocean primary production and climate: global decadal changes . Geophysical Research Letters , 30 ( 15 ): 1809 , https://doi.org/10.1029/2003GL016889 https://doi.org/10.1029/2003GL016889 .

Hagerthey S E , William Louda J , Mongkronsri P . 2006 . Evaluation of pigment extraction methods and a recommended protocol for periphyton chlorophyll ‍ a determination and chemotaxonomic assessment . Journal of Phycology , 42 ( 5 ): 1125 - 1136 , https://doi.org/10.1111/j.1529-8817.2006.00257.x https://doi.org/10.1111/j.1529-8817.2006.00257.x .

Holm-Hansen O , Lorenzen C J , Holmes R W et al . 1965 . Fluorometric determination of chlorophyll . ICES Journal of Marine Science , 30 ( 1 ): 3 - 15 , https://doi.org/10.1093/icesjms/30.1.3 https://doi.org/10.1093/icesjms/30.1.3 .

Holm-Hansen O , Riemann B . 1978 . Chlorophyll a determination: improvements in methodology . Oikos , 30 ( 3 ): 438 - 447 , https://doi.org/10.2307/3543338 https://doi.org/10.2307/3543338 .

Hörtensteiner S . 2009 . Stay-green regulates chlorophyll and chlorophyll-binding protein degradation during senescence . Trends in Plant Science , 14 ( 3 ): 155 - 162 , https://doi.org/10.1016/j.tplants.2009.01.002 https://doi.org/10.1016/j.tplants.2009.01.002 .

Jeffrey S W . 1974 . Profiles of photosynthetic pigments in the ocean using thin-layer chromatography . Marine Biology , 26 ( 2 ): 101 - 110 , https://doi.org/10.1007/BF00388879 https://doi.org/10.1007/BF00388879 .

Jeffrey S W , Hallegraeff G M . 1987 . Chlorophyllase distribution in ten classes of phytoplankton: a problem for chlorophyll analysis . Marine Ecology Progress Series , 35 : 293 - 304 , https://doi.org/10.3354/meps035293 https://doi.org/10.3354/meps035293 .

Jeffrey S W , Mantoura R F C , Bjornland T . 1997 . Data for the identification of 47 key phytoplankton pigments . In: Jeffrey S W, Mantoura R F C, Wright S W eds. Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods . UNESCO, Paris. p. 449 - 559 , https://doi.org/10.1023/A:1007168802525 https://doi.org/10.1023/A:1007168802525 .

Jeffrey S W , Vesk M . 1997 . Introduction to marine phytoplankton and their pigment signatures . In: Jeffrey S W, Mantoura R F C, Wright S W eds. Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods . UNESCO, Paris. p. 37 - 84 , https://doi.org/10.1023/A:1007168802525 https://doi.org/10.1023/A:1007168802525 .

Karboune S , Neufeld R , Kermasha S . 2005 . Immobilization and biocatalysis of chlorophyllase in selected organic solvent systems . Journal of Biotechnology , 120 ( 3 ): 273 - 283 , https://doi.org/10.1016/j.jbiotec.2005.06.025 https://doi.org/10.1016/j.jbiotec.2005.06.025 .

Kong F Z , Yu R C , Zhang Q C et al . 2012 . Pigment characterization for the 2011 bloom in Qinhuangdao implicated "brown tide" events in China . Chinese Journal of Oceanology and Limnology , 30 ( 3 ): 361 - 370 , https://doi.org/10.1007/s00343-012-1239-z https://doi.org/10.1007/s00343-012-1239-z .

Kozlowski W A , Deutschman D , Garibotti I et al . 2011 . An evaluation of the application of CHEMTAX to Antarctic coastal pigment data . Deep Sea Research Part I: Oceanographic Research Papers , 58 ( 4 ): 350 - 364 , https://doi.org/10.1016/j.dsr.2011.01.008 https://doi.org/10.1016/j.dsr.2011.01.008 .

Kramer S J , Siegel D A , Maritorena S et al . 2022 . Modeling surface ocean phytoplankton pigments from hyperspectral remote sensing reflectance on global scales . Remote Sensing of Environment , 270 : 112879 , https://doi.org/10.1016/j.rse.2021.112879 https://doi.org/10.1016/j.rse.2021.112879 .

Kuczynska P , Jemiola-Rzeminska M , Strzalka K . 2015 . Photosynthetic pigments in diatoms . Marine Drugs , 13 ( 9 ): 5847 - 5881 , https://doi.org/10.3390/md13095847 https://doi.org/10.3390/md13095847 .

Kumari B . 2005 . Comparison of high performance liquid chromatography and fluorometric ocean colour pigments . Journal of the Indian Society of Remote Sensing , 33 ( 4 ): 541 - 546 , https://doi.org/10.1007/BF02990739 https://doi.org/10.1007/BF02990739 .

Latasa M , Bidigare R R , Ondrusek M E et al . 1996 . HPLC analysis of algal pigments: a comparison exercise among laboratories and recommendations for improved analytical performance . Marine Chemistry , 51 ( 4 ): 315 - 324 , https://doi.org/10.1016/0304-4203(95)00056-9 https://doi.org/10.1016/0304-4203(95)00056-9 .

Latasa M , Van Lenning K , Garrido J L et al . 2001 . Losses of chlorophylls and carotenoids in aqueous acetone and methanol extracts prepared for RPHPLC analysis of pigments . Chromatographia , 53 ( 7-8 ): 385 - 391 , https://doi.org/10.1007/BF02491072 https://doi.org/10.1007/BF02491072 .

Louda J W , Li J , Liu L et al . 1998 . Chlorophyll- a degradation during cellular senescence and death . Organic Geochemistry , 29 ( 5-7 ): 1233 - 1251 , https://doi.org/10.1016/S0146-6380(98)00186-7 https://doi.org/10.1016/S0146-6380(98)00186-7 .

Mackey M D , Mackey D J , Higgins H W et al . 1996 . CHEMTAX-a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton . Marine Ecology Progress Series , 144 : 265 - 283 , https://doi.org/10.3354/meps144265 https://doi.org/10.3354/meps144265 .

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 . Analytica Chimica Acta , 151 : 297 - 314 , https://doi.org/10.1016/S0003-2670(00)80092-6 https://doi.org/10.1016/S0003-2670(00)80092-6 .

Marrari M , Hu C M , Daly K . 2006 . Validation of SeaWiFS chlorophyll a concentrations in the Southern Ocean: a revisit . Remote Sensing of Environment , 105 ( 4 ): 367 - 375 , https://doi.org/10.1016/j.rse.2006.07.008 https://doi.org/10.1016/j.rse.2006.07.008 .

Mendes C R , Sá C , Vitorino J et al . 2011 . Spatial distribution of phytoplankton assemblages in the Nazaré submarine canyon region (Portugal): HPLC-CHEMTAX approach . Journal of Marine Systems , 87 ( 1 ): 90 - 101 , https://doi.org/10.1016/j.jmarsys.2011.03.005 https://doi.org/10.1016/j.jmarsys.2011.03.005 .

Mfnguez-Mosquera M I , Gandul-Rojas B , Gallardo-Guerrero L . 1994 . Measurement of chlorophyllase activity in olive fruit ( Olea europaea ) . The Journal of Biochemistry , 116 ( 2 ): 263 - 268 , https://doi.org/10.1093/oxfordjournals.jbchem.a124517 https://doi.org/10.1093/oxfordjournals.jbchem.a124517 .

Moisan T A H , Sathyendranath S , Bouman H A . 2012 . Ocean color remote sensing of phytoplankton functional types . In: Fatoyinbo L ed. Remote Sensing of Biomass—Principles and Applications . IntechOpen, Rijeka, Croatia. p. 101 - 122 . https://do 10.5772/17174 http://dx.doi.org/10.5772/17174

Neveux J . 1988 . Extraction of chlorophylls from marine phytoplankton: with 4 tables in the text . SIL Proceedings, 1922-2010, Internationale Vereinigung für theoretische und angewandte Limnologie : Verhandlungen , 23 ( 2 ): 928 - 932 , https://doi.org/10.1080/03680770.1987.11899744 https://doi.org/10.1080/03680770.1987.11899744 .

Neveux J , Seppälä J , Dandonneau Y . 2012 . Multivariate analysis of extracted pigments using spectrophotometric and spectrofluorometric methods . In: Roy S, Llewellyn C A, Egeland E S et al Eds. Phytoplankton Pigments : Characterization, Chemotaxonomy and Applications in Oceanography. Cambridge University Press, New York. p . 343 - 372 , https://doi.org/10.1017/CBO9780511732263.012 https://doi.org/10.1017/CBO9780511732263.012 .

Ohde T , Siegel H , Gerth M . 2007 . Validation of MERIS Level‐2 products in the Baltic Sea, the Namibian coastal area and the Atlantic Ocean . International Journal of Remote Sensing , 28 ( 3-4 ): 609 - 624 . https://doi.org/10.1080/01431160600972961 https://doi.org/10.1080/01431160600972961 .

Osório C , Machado S , Peixoto J et al . 2020 . Pigments content (chlorophylls, fucoxanthin and phycobiliproteins) of different commercial dried algae . Separations , 7 ( 2 ): 33 , https://doi.org/10.3390/separations7020033 https://doi.org/10.3390/separations7020033 .

Pasquet V , Chérouvrier J R , Farhat F et al . 2011 . Study on the microalgal pigments extraction process: performance of microwave assisted extraction . Process Biochemistry , 46 ( 1 ): 59 - 67 , https://doi.org/10.1016/j.procbio.2010.07.009 https://doi.org/10.1016/j.procbio.2010.07.009 .

Pinckney J , Papa R , Zingmark R . 1994 . Comparison of high-performance liquid chromatographic, spectrophotometric, and fluorometric methods for determining chlorophyll ‍ a concentrations in estaurine sediments . Journal of Microbiological Methods , 19 ( 1 ): 59 - 66 , https://doi.org/10.1016/0167-7012(94)90026-4 https://doi.org/10.1016/0167-7012(94)90026-4 .

Porra R J . 1991 . Recent advances and re-assessments in chlorophyll extraction and assay procedures for terrestrial, aquatic and marine organisms, including recalcitrant algae . In: Scheer H ed. Chlorophylls . CRC Press , Boca Raton, Ann Arbor, Boston, London . p. 32 - 57 .

Richards F A , Thompson T G . 1952 . The estimation and characterization of plankton populations by pigment analyses II. A spectrophotometric method for the estimation of plankton pigments . Journal of Marine Research , 11 ( 2 ): 156 - 172 .

Ritchie R J . 2008 . Universal chlorophyll equations for estimating chlorophylls a , b , c , and d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone, methanol, or ethanol solvents . Photosynthetica , 46 ( 1 ): 115 - 126 , https://doi.‍org/10.1007/s11099-008-0019-7 https://doi.‍org/10.1007/s11099-008-0019-7 . https://do 10.1007/s11099-008-0019-7 http://dx.doi.org/10.1007/s11099-008-0019-7

Saini R K , Keum Y S . 2018 . Carotenoid extraction methods: a review of recent developments . Food Chemistry , 240 : 90 - 103 , https://doi.org/10.1016/j.foodchem.2017.07.099 https://doi.org/10.1016/j.foodchem.2017.07.099 .

Schagerl M , Künzl G . 2007 . Chlorophyll a extraction from freshwater algae—a reevaluation . Biologia , 62 ( 3 ): 270 - 275 , https://doi.org/10.2478/s11756-007-0048-x https://doi.org/10.2478/s11756-007-0048-x .

Soares A T , Júnior J G M , Lopes R G et al . 2016 . Improvement of the extraction process for high commercial value pigments from Desmodesmus sp. microalgae . Journal of the Brazilian Chemical Society , 27 ( 6 ): 1083 - 1093 , https://doi.org/10.5935/0103-5053.20160004 https://doi.org/10.5935/0103-5053.20160004 .

Suzuki R , Takahashi M , Furuya K et al . 1993 . Simplified technique for the rapid determination of phytoplankton pigments by reverse-phase high-performance liquid chromatography . Journal of Oceanography , 49 ( 5 ): 571 - 580 , https://doi.org/10.1007/BF02237463 https://doi.org/10.1007/BF02237463 .

Suzuki T , Kunieda T , Murai F et al . 2005 . Mg-dechelation activity in radish cotyledons with artificial and native substrates, Mg-chlorophyllin a and chlorophyllide a . Plant Physiology and Biochemistry , 43 ( 5 ): 459 - 464 , https://doi.org/10.1016/j.plaphy.2005.03.009 https://doi.org/10.1016/j.plaphy.2005.03.009 .

Terpstra W . 1981 . Identification of chlorophyllase as a glycoprotein . FEBS Letters , 126 ( 2 ): 231 - 235 , https://doi.org/10.1016/0014-5793(81)80249-9 https://doi.org/10.1016/0014-5793(81)80249-9 .

Tett P , Kelly M G , Hornberger G M . 1975 . A method for the spectrophotometric measurement of chlorophyll a and pheophytin a in benthic microalgae . Limnology and Oceanography , 20 ( 5 ): 887 - 896 , https://doi‍.org/10.4319/lo.1975.20.5.0887 https://doi‍.org/10.4319/lo.1975.20.5.0887 . https://do 10.4319/lo.1975.20.5.0887 http://dx.doi.org/10.4319/lo.1975.20.5.0887

Torres P B , Chow F , Furlan C M et al . 2014 . Standardization of a protocol to extract and analyze chlorophyll a and carotenoids in Gracilaria tenuistipitata Var. Liui. Zhang and Xia (Rhodophyta) . Brazilian Journal of Oceanography , 62 ( 1 ): 57 - 63 , https://doi.org/10.1590/s1679-87592014068106201 https://doi.org/10.1590/s1679-87592014068106201 .

Van Leeuwe M A , Villerius L A , Roggeveld J et al . 2006 . An optimized method for automated analysis of algal pigments by HPLC . Marine Chemistry , 102 ( 3-4 ): 267 - 275 , https://doi.org/10.1016/j.marchem.2006.05.003 https://doi.org/10.1016/j.marchem.2006.05.003 .

Wang G Q , Lee Z , Mouw C B . 2018 . Concentrations of multiple phytoplankton pigments in the global oceans obtained from satellite ocean color measurements with MERIS . Applied Sciences , 8 ( 12 ): 2678 , https://doi.org/10.3390/app8122678 https://doi.org/10.3390/app8122678 .

West R , Keşan G , Trsková E et al . 2016 . Spectroscopic properties of the triple bond carotenoid alloxanthin . Chemical Physics Letters , 653 : 167 - 172 , https://doi.org/10.1016/j.cplett.2016.04.085 https://doi.org/10.1016/j.cplett.2016.04.085 .

Wright S W . 1997 . Evaluation of methods and solvents for pigment extraction . In: Jeffrey S W, Mantoura R F C, Wright S W eds. Phytoplankton Pigments in Oceanography: guidelines to modern methods . UNESCO, Paris. p. 261 - 282 , https://doi.org/10.1023/A:1007168802525 https://doi.org/10.1023/A:1007168802525 .

Yoo S J , Kim H C , Lee J et al . 2002 . Validation of chlorophyll algorithm in Ulleung Basin, East/Japan Sea . Korean Journal of Remote Sensing , 18 ( 1 ): 35 - 42 , https://doi.‍org/10.7780/kjrs.2002.‍18.1.35 https://doi.‍org/10.7780/kjrs.2002.‍18.1.35 .

Zapata M , Ayala A M , Franco J M et al . 1987 . Separation of chlorophylls and their degradation products in marine phytoplankton by reversed-phase high-performance liquid chromatography . Chromatographia , 23 ( 1 ): 26 - 30 , https://doi.org/10.1007/BF02310413 https://doi.org/10.1007/BF02310413 .

Zapata M , Rodríguez F , Garrido J L . 2000 . Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C 8 column and pyridine-containing mobile phases . Marine Ecology Progress Series , 195 : 29 - 45 , https://doi.org/10.3354/meps195029 https://doi.org/10.3354/meps195029 .

Zhu Z Y . 2007 . Hypoxia in the Changjiang estuary and its adjacent area—started with phytoplankton pigments . East China Normal University , Shanghai , https://doi.org/10.7666/d.y1073832. https://doi.org/10.7666/d.y1073832. (in Chinese with English abstract)

Views

Downloads

CSCD

Alert me when the article has been cited

Submit

Tools

Publicity Resources

Phytoplankton pigment patterns and community composition in the northern South China Sea during winter

The summer pattern of phytoplankton pigment assemblages in response to water masses in the Yellow Sea

Long-term dynamics of net-phytoplankton community under environmental drivers in Jiaozhou Bay, China

Standing crop and size structure of phytoplankton are strongly regulated by the turbidity maximum in the macrotidal Qiantang River estuary-Hangzhou Bay

Wall Corrosion Morphology Prediction of Hall ThrustersUsing Semi-Empirical Method

Related Author

Fanzhou KONG

Rencheng YU

Hongchang Zhai

Xiuren Ning

Xuexi Tang

Qiang Hao

Fengfeng Le

Jing Qiao

Related Institution

University of Chinese Academy of Sciences

College of Marine Life Science Ocean University of China

Second Institute of Oceanography SOA

State Key Lab of Satellite Ocean Environment Dynamics

State Key Laboratory of Estuarine and Coastal Research, Institute of Eco-Chongming, East China Normal University

Address：Editorial Office of JOL, 88 Haijun Rd. Qingdao, 266000, China
Tel：+86-532-82898754 Email：jol@qdio.ac.cn
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备09064830号-19 京公网安备11010802024621
It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.

⁰