Journal of Applied Phycology

, Volume 23, Issue 1, pp 1–6 | Cite as

Single-step chromatography for simultaneous purification of C-phycocyanin and allophycocyanin with high purity and recovery from Spirulina (Arthrospira) platensis

  • Shi-Gan Yan
  • Li-Ping Zhu
  • Hai-Nan Su
  • Xi-Ying Zhang
  • Xiu-Lan Chen
  • Bai-Cheng Zhou
  • Yu-Zhong ZhangEmail author


The cyanobacterium Spirulina (Arthrospira) platensis is a good source of phycobiliprotein purification. C-phycocyanin (C-PC) is the major phycobiliprotein, while allophycocyanin (APC) is less abundant in S. platensis. Previously reported methods for C-PC purification are only able to offer either high purity or high efficiency. This paper describes one-step anion exchange chromatography method with continuous pH gradient elution for simultaneous purification of C-PC and APC with high purity and high recovery. Crude C-PC and APC were extracted and concentrated by ammonium sulfate fractionation at saturation of 25% and 60%, then purified on a DEAE-Sepharose Fast Flow chromatography column with continuous pH gradient elution from pH 5.0 to 3.6. After this single-step chromatography, C-PC and APC with high purity and recovery were simultaneously obtained. The purity ratios of C-PC and APC reached 5.59 (A620/A280) and 5.19 (A650/A280), respectively. Their purity was further demonstrated by electrophoresis and fluorescence emission spectroscopy. Moreover, the total recovery yield of pure C-PC and APC were 67.04% and 80.0%, representing 111.83 and 29.28 mg·g−1 lyophilized weight, respectively. The obtained C-PC and APC remained stable over a pH range of 4–9. This purification method for high purity and recovery of C-PC and APC proved to be fairly efficient compared with previously reported methods.


C-phycocyanin Allophycocyanin Anion exchange chromatography pH gradient Purification 



The work was supported by the Hi-Tech Research and Development program of China (2008AA09Z404), the National Natural Science Foundation of China (40676078), and the Scientific and Technological Development Program of Shandong Province (2006GG2205009).


  1. Benedetti S, Rinalducci S, Benvenuti F, Francogli S, Pagliarani S, Giorgi L, Micheloni M, D’Amici GM, Zolla L, Canestrari F (2006) Purification and characterization of phycocyanin from the blue-green alga Aphanizomenon flos-aquae. J Chromatogr B 833:12–18CrossRefGoogle Scholar
  2. Bennett A, Bogorad L (1973) Complementary chromatic adaptation in a filamentous blue-green alga. J Cell Biol 58:419–435CrossRefPubMedGoogle Scholar
  3. Bermejo R, Felipe MA, Talavera EM, Alvarez-Pez JM (2006) Expanded bed absorption chromatography for recovery of phycocyanins from the microalga Spirulina platensis. Chromatographia 63:59–66CrossRefGoogle Scholar
  4. Boussiba S, Richmond AE (1979) Isolation and characterization of phycocyanins from the blue-green alga Spirulina platensis. Arch Microbial 120:155–159CrossRefGoogle Scholar
  5. Chen F, Zhang Y, Guo S (1996) Growth and phycocyanin formation of Spirulina platensis in photoheterotrophic culture. Biotechnol Lett 18:603–608CrossRefGoogle Scholar
  6. Chen T, Wong YS, Zheng W (2006) Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis. Phytochem 67:2424–2430CrossRefGoogle Scholar
  7. Glazer AN (1981) Photosynthetic accessory proteins with bilin prosthetic groups. In: Hatch MD, Boardman NK (eds) The biochemistry of plants, vol. 8, Photosynthesis. Academic, New York, pp 51–96Google Scholar
  8. Glazer AN, Stryer L (1984) Phycofluor probes. Trends Biochem Sci 9:423–427Google Scholar
  9. Haugland RP (1996) Handbook of fluorescent probes and research chemicals, 6th edn. Molecular Probes, EugeneGoogle Scholar
  10. Liu LN, Chen XL, Zhang XY, Zhang YZ, Zhou BC (2005) One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata. J Biotechnol 116:91–100CrossRefPubMedGoogle Scholar
  11. MacColl R, Csatorday K, Berns DS, Traeger E (1980) Chromophore interactions in allophycocyanin. Biochem 19:2817–2820CrossRefGoogle Scholar
  12. Market Corporation (2005). (consult date: October 2005)
  13. Minkova KM, Tchernov AA, Tchorbadjieva MI, Fournadjieva ST, Antova RE, Busheva MC (2003) Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis. J Biotechnol 102:55–59CrossRefPubMedGoogle Scholar
  14. Minkova K, Tchorbadjieva M, Tchernov A, Stojanova M, Gigova L, Busheva M (2007) Improved procedure for separation and purification of Arthronema africanum phycobiliproteins. Biotechnol Lett 29:647–651CrossRefPubMedGoogle Scholar
  15. Niu JF, Wang GC, Lin XZ, Zhou BC (2007) Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography. J Chromatogr B 850:267–276CrossRefGoogle Scholar
  16. Padgett MP, Krogmann DW (1987) Large scale preparation of pure phycobiliproteins. Photosynth Res 11:225–235CrossRefGoogle Scholar
  17. Patel A, Mishra S, Pawar R, Ghosh PK (2005) Purification and characterization of C-phycocyanin from cyanobacterial species of marine and freshwater habitat. Protein Expr Purif 40:248–255CrossRefPubMedGoogle Scholar
  18. Patil G, Raghavarao KSMS (2007) Aqueous two phase extraction for purification of C-phycocyanin. Biochem Eng J 34:156–164CrossRefGoogle Scholar
  19. Patil G, Chethana S, Sridevi AS, Raghavarao KS (2006) Method to obtain C-phycocyanin of high purity. J Chromatogr A 1127:76–81CrossRefPubMedGoogle Scholar
  20. Qureshi MA, Garlich JD, Kidd MT (1996) Dietary Spirulina platensis enhances humoral and cell-mediated immune functions in chickens. Immunopharmacol Immunotoxicol 18:465–476Google Scholar
  21. Romay C, Gonzalez R (2000) Phycocyanin is an antioxidant protector of human erythrocytes against lysis by peroxyl radicals. J Pharm Pharmacol 52:367–368Google Scholar
  22. Rito-Palomares M, Nunez L, Amador D (2001) Practical application of aqueous two-phase systems for the development of a prototype process for C-phycocyanin recovery from Spirulina maxima. J Chem Technol Biotechnol 76:1273–1280CrossRefGoogle Scholar
  23. Santiago-Santos MC, Ponce-Noyola T, Olvera-Ramirez R, Ortega-Lopez J, Canizares-Villanueva RO (2004) Extraction and purification of phycocyanin from Calothrix sp. Process Biochem 39:2047–2052CrossRefGoogle Scholar
  24. Siegelman HW, Kycia JH (1978) Algal biliproteins. In: Hellebust JA, Craigie JS (eds) Handbook of phycological methods. Physiological and biochemical methods. Cambridge University Press, Cambridge, pp 71–79Google Scholar
  25. Silveira ST, Quines LKM, Burkert CAV, Kalil SJ (2008) Separation of phycocyanin from Spirulina platensis using ion exchange chromatography. Bioprocess Biosyst Eng 31:477–482CrossRefPubMedGoogle Scholar
  26. Soni B, Trivedi U, Madamwar D (2008) A novel method of single step hydrophobic interaction chromatography for the purification of phycocyanin from Phormidium fragile and its characterization for antioxidant property. Bioresour Technol 99:188–194CrossRefPubMedGoogle Scholar
  27. Stewart DE, Farmer FH (1984) Extraction, identification, and quantitation of phycobiliprotein pigments from phototrophic plankton. Limnol Oceanogr 29:392–397Google Scholar
  28. Su HN, Xie BB, Chen XL, Wang JX, Zhang XY, Zhou BC, Zhang YZ (2010) Efficient separation and purification of allophycocyanin from Spirulina (Arthrospira) platensis. J Appl Phycol 22:65–70CrossRefGoogle Scholar
  29. Wyman M (1992) An in vivo method for the estimation of phycoerythrin concentration in marine cyanobacteria (Synechococcus spp.). Limnol Oceanogr 37:1300–1306Google Scholar
  30. Zhang YM, Chen F (1999) A simple method for efficient separation and purification of C-phycocyanin and allophycocyanin from Spirulina platensis. Biotechnol Tech 13:601–603CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Shi-Gan Yan
    • 1
    • 3
  • Li-Ping Zhu
    • 2
  • Hai-Nan Su
    • 1
  • Xi-Ying Zhang
    • 1
  • Xiu-Lan Chen
    • 1
  • Bai-Cheng Zhou
    • 1
  • Yu-Zhong Zhang
    • 1
    Email author
  1. 1.State Key Lab of Microbial TechnologyShandong UniversityJinanChina
  2. 2.School of Food and BioengineeringShandong Institute of Light IndustryJinanChina
  3. 3.Shandong Academy of Agricultural SciencesJinanChina

Personalised recommendations