Skip to main content
SpringerLink
Log in

HPLC separation of cyanobacterial and algal photosynthetic pigments

  • Original Article
  • Published:
Biologia Aims and scope Submit manuscript

Abstract

Photosynthetic pigments analysis has become a standard tool in ecological and physiological studies of photosynthetic organisms. With numerous methods previously published, there is no one ideal protocol that could satisfy all the research needs. Therefore, regarding the purpose of HPLC pigment analyses, a suitable method should be chosen. In this study, two C18 columns (i.e., LiChrospher 100 RP18e and Spherisorb ODS2) and three sets of eluents (based on acetone, acetonitrile, methanol, and water) were used to develop three separation protocols. They were then examined with respect to their resolution and sensitivity, by analyzing pigment extracts obtained from 10 cyanobacterial and algal cultures and two types of environmental samples, i.e. phytoplankton and microphytobenthos. All the protocols provided highly repeatable results and allowed for the separation of all taxonomically most relevant chlorophylls and carotenoids. They had similar resolution and sensitivity. The Syst1 method was the shortest, while Syst2 had better resolution of pigments in the middle part of the protocol when most of the diagnostic pigments are separated (i.e., alloxanthin, diatoxanthin, lutein, chlorophyll b). Syst3, on the other hand, enabled distinguishing the highest number of pigments, including their derivatives and degradation products. The results showed that all protocols may be used for routine analysis of cyanobacterial and algal pigments in various sample types.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Asencio AD, Hoffmann L (2013) Chemosystematic evaluation of the genus Scytonema (cyanobacteria) based on occurrence of phycobiliproteins, scytonemin, carotenoids and mycosporine-like amino acid compounds. Eur J Phycol 48:331–344

    Article  CAS  Google Scholar 

  • Brotas V, Plante Cuny MR (1998) Spatial and temporal patterns of microphytobenthic taxa of estuarine tidal flats in the Tagus estuary (Portugal) using pigment analysis by HPLC. Mar Ecol Prog Ser 171:43–57

    Article  Google Scholar 

  • Brotas V, Plante Cuny MR (2003) The use of HPLC pigment analysis to study microphytobenthos communities. Acta Oecolog. https://doi.org/10.1016/S1146-609X(03)00013-4

  • Cartaxana P, Jesus B, Brotas V (2003) Pheophorbide and pheophytin a-like pigments as useful markers for intertidal microphytobenthos grazing by Hydrobia ulvae. Estuar Coast Shelf Sci 58:293–297

    Article  CAS  Google Scholar 

  • Chai C, Jiang T, Cen J, Ge W, Lu S (2016) Phytoplankton pigments and functional community structure in relation to environmental factors in the Pearl River estuary. Oceanologia 58:201–211

  • Eisner LB, Twardowski MS, Cowles TJ, Perry MJ (2003) Resolving phytoplankton photoprotective: photosynthetic carotenoid ratios on fine scales using in situ spectral absorption measurements. Limnol Oceanogr 48:632–646

    Article  CAS  Google Scholar 

  • Goela PC, Danchenko S, Icely JD, Lubian LM, Cristina S, Newton A (2014) Using CHEMTAX to evaluate seasonal and interannual dynamics of the phytoplankton community off the south-west coast of Portugal. Estuar Coast Shelf Sci 151:112–123

    Article  CAS  Google Scholar 

  • Goss R, Jakob T (2010) Regulation and function of xanthophyll cycle-dependent photoprotection in algae. Photosynth Res 106:103–122

    Article  CAS  Google Scholar 

  • Hooker SB, Van Heukelem L, Thomas CS, Claustre H, Ras J, Barlow R, Sessions H, Schlüter L, Perl J, Trees C, Stuart V, Head E, Clementson L, Fishwick J, Llewellyn C, Aiken J (2005) The second SeaWiFS HPLC analysis round-Robin experiment (SeaHARRE-2). NASA TM-2005-212785, Washington

  • Jahns P, Holzwarth AR (2012) The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochim Biophys Acta 1817:182–193

    Article  CAS  Google Scholar 

  • Jayaraman S, Knuth ML, Cantwell M, Santos A (2011) High performance liquid chromatography analysis of phytoplankton pigments using a C16-amide column. J Chromatogr A 1218:3432–3438

    Article  CAS  Google Scholar 

  • Jeffrey SW, Vesk M (1997) Introduction to marine phytoplankton and their pigment signatures. In: Jeffrey SW, Mantoura RFC, Wright SW (eds) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO Publishing, Paris, pp 37–84

    Google Scholar 

  • Jodłowska S, Latała A (2003) Simultaneous separation of chlorophylls and carotenoids by RP-HPLC in some algae and cyanobacteria from the southern Baltic. Oceanol Hydrobiol Stud 2:81–89

    Google Scholar 

  • Kraay GW, Zapata M, Veldhuis MJW (1992) Separation of chlorophylls c1, c2, c3 of marine phytoplankton by reversed-phase-C18-high-performance liquid chromatography. J Phycol 28:708–712

    Article  CAS  Google Scholar 

  • Lawrenz E, Fedewa EJ, Richardson TL (2011) Extraction protocols for the quantification of phycobilins in aqueous phytoplankton extracts. J Appl Phycol 23:865–871

    Article  Google Scholar 

  • MacIntyre HL, Kana TM, Anning T, Geider RJ (2002) Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria. J Phycol 38:17–38

    Article  Google Scholar 

  • Mantoura RFC, Repeta DJ (1997) Calibration methods for HPLC. In: Jeffrey SW, Mantoura RFC, Wright SW (eds) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO Publishing, Paris, pp 407–428

    Google Scholar 

  • Masjídek J, Kopecký J, Koblížek M, Torzillo G (2004) The xanthophyll cycle in green algae (Chlorophyta): its role in the photosynthetic apparatus. Plant Biol 6:342–349

    Article  Google Scholar 

  • Mateos R, García-Mesa JA (2006) Rapid and quantitative extraction method for the determination of chlorophylls and carotenoids in olive oil by high-performance liquid chromatography. Anal Bioanal Chem 385:1247–1254

    Article  CAS  Google Scholar 

  • Mendes CR, Cartaxana P, Brotas V (2007) HPLC determination of phytoplankton and microphytobenthos pigments: comparing resolution and sensitivity of a C18 and C8 method. Limnol Oceanogr Methods 5:363–370

    Article  CAS  Google Scholar 

  • Pinckney JL, Richardson TL, Millie DF, Paerl HW (2001) Application of photopigment biomarkers for quantifying microalgal community composition and in situ growth rates. Org Geochem 32:585–595

    Article  CAS  Google Scholar 

  • Pniewski F, Sylwestrzak Z (2018) Influence of short periods of increased water temperature on species composition and photosynthetic activity in the Baltic periphyton communities. Biologia. https://doi.org/10.2478/s11756-018-0122-6

  • Pniewski FF, Biskup P, Bubak I, Richard P, Latała A, Blanchard G (2015) Photo-regulation in microphytobenthos from intertidal mudflats and non-tidal coastal shallows. Estuar Coast Shelf Sci 152:152–161

    Article  Google Scholar 

  • Rodríguez F, Zapata M, Garrido JL (1998) High performance liquid chromatographic separation of chlorophyll c forms from marine phytoplankton on octylsilica bonded phases. Chromatographia 48:677–680

    Article  Google Scholar 

  • Schlüter L, Garde K, Kaas H (2004) Detection of the toxic cyanobacteria Nodularia spumigena by means of a 4-keto-myxoxanthophyll-like pigment in the Baltic Sea. Mar Ecol Prog Ser 275:69–78

    Article  Google Scholar 

  • Schlüter L, Mohlenberg F, Havskum H, Larsen S (2000) The use of phytoplankton pigments for identifying and quantifying phytoplankton groups in coastal areas: testing the influence of light and nutrients on pigment/chlorophyll a ratios. Mar Ecol Prog Ser 192:49–63

    Article  Google Scholar 

  • Schlüter L, Mohlenberg F, Kaas H (2014) Temporal and spatial variability of phytoplankton monitored by a combination of monitoring buoys, pigment analysis and fast screening microscopy in the Fehmarn Belt estuary. Environ Monit Assess 186:5167–5184

    Article  Google Scholar 

  • Schmid H, Stich HB (1995) HPLC-analysis of algal pigments: comparison of columns, column properties and eluents. J Appl Phycol 7:487–494

    Article  CAS  Google Scholar 

  • Stoń J, Kosakowska A (2000) Qualitative and quantitative analysis of Baltic phytoplankton pigments. Oceanologia 42:449–471

    Google Scholar 

  • Stoń J, Kosakowska A (2002) Phytoplankton pigments designation – an application of RP-HPLC in qualitative and quantitative analysis. J Appl Phycol 14:205–210

    Article  Google Scholar 

  • Stoń J, Kosakowska A, Łotocka M, Łysiak-Pastuszak E (2002) Pigment composition in relation to phytoplankton community structure and nutrient content in the Baltic Sea. Oceanologia 44:419–437

    Google Scholar 

  • Stoń-Egiert J, Kosakowska A (2005) RP-HPLC determination of phytoplankton pigments – comparison of calibration results for two columns. Mar Biol 147:251–260

    Article  Google Scholar 

  • Stoń-Egiert J, Łotocka M, Ostrowska M, Kosakowska A (2010) The influence of biotic factors on phytoplankton pigment composition and resources in Baltic ecosystems: new analytical results. Oceanologia 52:101–125

    Article  Google Scholar 

  • Tamm M, Freiberg R, Tõnno I, Nõges P, Nõges T (2015) Pigment-based chemotaxonomy – a quick alternative to determine algal assemblages in large shallow eutrophic lake. PLoS One. https://doi.org/10.1371/journal.pone.0122526

  • Van Heukelem L, Lewitus AJ, Kana TM, Craft NE (1994) Improved separations of phytoplankton pigments using temperature-controlled high performance liquid chromatography. Mar Ecol Prog Ser 114:303–313

    Article  Google Scholar 

  • Van Heukelem L, Thomas CS (2001) Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments. J Chromatogr A 910:31–49

    Article  Google Scholar 

  • Van Leeuwe MA, Villerius LA, Roggeveld J, Visser RJW, Stefels J (2006) An optimized method for automated analysis of algal pigments by HPLC. Mar Chem 102:267–275

    Article  Google Scholar 

  • Wright SW, Jeffrey SW, Mantoura RFC, Llewellyn BT, Repeta D, Welschmeyer N (1991) Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar Ecol Prog Ser 77:183–196

    Article  CAS  Google Scholar 

  • Zapata M, Rodríguez F, Garrido JL (2000) Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases. Mar Ecol Prog Ser 195:29–45

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by the statutory funds of the Department of Marine Ecosystems Functioning at the Institute of Oceanography UG.

Author information

Authors and Affiliations

  1. Institute of Oceanography, University of Gdańsk, Al. Piłsudskiego 46, 81-590, Gdynia, Poland

    Filip Pniewski

Authors
  1. Filip Pniewski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Filip Pniewski.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pniewski, F. HPLC separation of cyanobacterial and algal photosynthetic pigments. Biologia 75, 223–233 (2020). https://doi.org/10.2478/s11756-019-00407-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11756-019-00407-8

Keywords

Search

Navigation