The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY (한국해양학회지:바다)

The Korean Society of Oceanography (한국해양학회)
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Evaluation of Grinding Effects on the Extraction of Photosynthetic Pigments for HPLC Analysis

광합성 색소의 HPLC 분석을 위한 여과지 분쇄 효과 평가

  • Jang, Su Jin (Department of Oceanography, Pukyong National University) ;
  • Park, Mi Ok (Department of Oceanography, Pukyong National University)
  • Received : 2014.09.19
  • Accepted : 2015.05.11
  • Published : 2015.05.31
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Abstract

High-Performance Liquid Chromatography (HPLC) is a widely used method for measuring the concentration of chlorophyll a as an indicator for estimating phytoplankton biomass and primary production and also for identifying carotenoids to determine phytoplankton composition. However, tissue grinding procedure requires a lot of time and experience in the analysis of multiple sample. Accordingly, we measured the concentrations of photosynthetic pigments before and after the grinding, in order to understand the grinding effects on the quantitative analysis of chlorophylls and carotenoids using samples from southwestern East Sea. When tissue grinding procedure was omitted, we found that Chl a concentrations were underestimated up to 45% in average. Also, concentrations of Zeaxanthin, 19'-butanoyloxyfucoxanthin, 19'-hexanoyloxyfucoxanthin, biomarkers of pico and nano-size phytoplankton, were underestimated up to maximum 77~85% without grinding. We found that the smaller the phytoplankton, the bigger underestimation of their biomarker pigments concentration is likely to happen due to the incomplete extraction. Thus, tissue grinding procedure should be included for HPLC analysis in all cases, to prevent the underestimation of not only Chl a but also carotenoids pigments.

High-Performance Liquid Chromatography (HPLC) 분석방법은 식물플랑크톤의 생물량 및 일차생산력을 추정하기 위한 지시자로서 chlorophyll a 농도를 측정하고 carotenoids의 종류를 파악해 종조성을 확인하기 위해 널리 이용되고 있다. 그러나 대량시료의 분석에 요구되는 시료 전처리 과정 중 여과지 분쇄는 상당한 시간과 숙련이 요구된다. 본 연구에서는 엽록소 및 carotenoids의 정량분석에 대한 여과지 분쇄의 영향을 파악하고자 동해 남서부 해역의 시료를 이용해 여과지 분쇄 전후의 광합성 색소 농도를 비교 평가했다. HPLC 분석에서 여과지 분쇄 생략 시 Chl a의 경우 평균 45% 과소평가되었다. 또한 pico, nano 크기 식물플랑크톤의 지표색소인 Zeaxanthin, 19'-butanoyloxyfucoxanthin, 19'-hexanoyloxyfucoxanthin는 최대 77~85% 과소평가되었다. 크기가 작은 식물플랑크톤의 경우 여과지 분쇄가 생략될 경우 불완전한 추출로 지표색소의 농도가 실제보다 저평가될 가능성이 크다는 것을 확인하였다. 따라서 HPLC 분석에서 여과지 분쇄 생략 시 Chl a 뿐 아니라 carotenoids 또한 과소평가 되므로 모든 경우에서 여과지 분쇄 과정이 반드시 필요하다고 판단된다.

Keywords

References

  1. Moon C.R., D.J. Kang, M.O. Park, J.H. Noh, S.J. Yoo, J.E. Moon, K.H. Shin, Y.S. Kim, J.K. Choi, Y.S. Suh, 2014. An Inter-Laboratory Comparison Study on Chlorophyll a Determination in Seawater. Journal of the Korean Society of Oceanography, 19: 76-87.
  2. Downes, M.T., L. Hrstich and W.F. Vincent, 1993. Extraction of chlorophyll and carotenoid pigments from Antarctic benthic mats for analysis by HPLC. Journal of Applied Phycology, 5(6): 623-628. https://doi.org/10.1007/BF02184641
  3. Downes, M.T., J.A. Hall, 1998. A sensitive fluorometric technique for the measurement of phycobilin pigments and its application to the study of marine and freshwater picoplankton in oligotrophic environments. Journal of Applied Phycology, 10: 357-363. https://doi.org/10.1023/A:1008085719486
  4. Gibb, S.W., D.G. Cummings, X. Irigoien, R.G. Barlow, R. Fauzi and C. Mantoura, 2001. Phytoplankton pigment chemotaxonomy of northeastern Atlantic. Deep Sea Res. II, 48: 795-823. https://doi.org/10.1016/S0967-0645(00)00098-9
  5. Gieskes, W.W. and G.W. Kraay, 1983. Unknown chlorophyll a derivatives in the North Sea and the tropical Atlantic Ocean revealed by HPLC analysis. Limnol. Oceanogr., 28: 757-766. https://doi.org/10.4319/lo.1983.28.4.0757
  6. Holm-Hansen, O., C.J. Lorenzen, R.W. Holmes and J.D.H. Strickland, 1965. Fluorometric determination of chlorophyll. J. Cons. perm. int. Explor. Mer., 30: 3-15. https://doi.org/10.1093/icesjms/30.1.3
  7. Iriarte A, Purdie D.A., 1994. Size distribution of Chl a biomass and primary production in a temperate estuary (Southampton Water): The contribution of photosynthetic picoplankton. Marine Ecology Progress Series, 115: 283-297. https://doi.org/10.3354/meps115283
  8. Jeffrey, S.W. and M. Vesk, 1997. Introduction to marine phytoplankton and their pigment signatures. In: Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods. edited by Jeffrey, S.W., R.F.C. Mantoura and S.W. Wright, UNESCO, Paris, 37-84 pp.
  9. Jeffrey, S.W. and R.F.C. Mantoura, 1997. Development of pigment methods for oceanography: SCOR-supported working groups and objectives. In: Phytoplankton Pigments in Oceanography, edited by Jeffrey, S.W., R.F.C. Mantoura, S.W. Wright, UNESCO, Paris, 19-36 pp.
  10. Jeffrey, S.W., R.F.C. Mantoura and Wright, S.W. (eds.), 1997. Phytoplankton Pigments in Oceanography. UNESCO Publishing, Paris.
  11. Legendre, L. and J. Le Fevre, 1995. Microbial food webs and the export of biogenic carbon in oceans. Aquat. Microb. Ecol., 9: 69-77. https://doi.org/10.3354/ame009069
  12. Legendre, L. and J. Michaud, 1998. Flux of biogenic carbon in oceans: size dependant regulation by pelagic food webs. Mar. Ecol. Prog. Ser., 164: 1-11. https://doi.org/10.3354/meps164001
  13. Lorenzen, C.J. and S.W. Jeffrey, 1980. Determination of chlorophyll in seawater. UNESCO Tech. pp. Mar. Sci., 35: 1-20.
  14. Longhurst, A.R., 1991. Role of the marine biosphere in the global carbon cycle. Limnol. Oceanogr., 36: 1507-1526. https://doi.org/10.4319/lo.1991.36.8.1507
  15. Mackey, D.J., H.W. Higgins, M.D. Mackey and D. Holdsworth, 1998. Algal class abundances in the western equatorial Pacific: Estimation from HPLC measurements of Chloroplast pigments using CHEMTAX. Deep-sea Res. I, 45: 1441-1468. https://doi.org/10.1016/S0967-0637(98)00025-9
  16. Mantoura, R.F.C. and C.A. Llewellyn, 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. https://doi.org/10.1016/S0003-2670(00)80092-6
  17. Mantoura, R.F.C., S.W. Wright, S.W. Jeffrey, R.G. Barlow and D.E. Cummings, 1997. Filtration and storage of pigments from microalgae. In: Phytoplankton Pigments in Oceanography, edited by Jeffrey, S.W., R.F.C. Mantoura, S.W. Wright, UNESCO, Paris, 283-305 pp.
  18. Mueller, J.L., R.R. Bidigare, C. Trees, W.M. Balch, J. Dore, D.T. Drapeau, D. Karl, L. Van Heukelem, J. Perl, 2003. Ocean optics protocols for satellite ocean color sensor validation, revirsion 5, volume ?: HPLC Phytoplankton pigments: Sampling, Laboratory Methods, and Quality Assurance Procedures. edited by Mueller, J.L., Fargion, G.S., McClain, C.R., NASA Tech. Rev. 5, vol. V. NASA Goddard Space Flight Center, 5-14 pp.
  19. Park, M.O., 2006. Composition and distribution of phytoplankton with size fraction results at Southwestern East/Japan Sea. Ocean Science Journal, 41(4): 301-313. https://doi.org/10.1007/BF03020632
  20. Park, M.O. and J.S. Park, 1997. HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. J. Korean Soc. Oceanogr., 32: 46-55.
  21. Pinckney, J.R.P. and R. Zingmark, 1994. Comparison of high performance liquid chromatographic, spectrophotometric and fluorometric methods for determining chlorophyll a concentrations in estuarine sediments. J. Microbiol. Methods, 19: 59-66. https://doi.org/10.1016/0167-7012(94)90026-4
  22. Plante-Cunny, M.R., C. Barranguet, D. Bonin and C. Grenz, 1993. Does chlorophyllide a reduce reliability of chlorophyll a measurements in marine coastal sediments? Aquat. Sci., 55: 19-30. https://doi.org/10.1007/BF00877256
  23. Riaux-Gobin, C., C.A. Llewellyn and B. Klein, 1987. Microphytobenthos from two subtidal sediments from North Brittany. II. Variations of pigment compositions and concentrations determined by HPLC and conventional techniques. Mar. Ecol. Prog. Ser., 40: 275-283. https://doi.org/10.3354/meps040275
  24. Rowan, K.S., 1991. Photosynthetic Pigments of Algea. Campridge University Press, 95-110 pp.
  25. Schlutter, L., F. Mohlenberg, H. Havskum and S. Larsen, 2000. The use of phytoplankton pigments for identifying and quantifying phytoplankton groups in costal areas: testing the influence of light and nutrients on pigment/chlorophyll a ratio. Marine Ecology Progress Series, 192: 49-63. https://doi.org/10.3354/meps192049
  26. SCOR-UNESCO Working Group 17, 1966. Determination of Photosynthetic Pigments in Seawater. UNESCO Monographs on Oceanographic Methodology, I, Paris, 69pp.
  27. Strickland, J. D. and T. R. Parsons, 1972. A practical handbook of seawater analysis. 2nd ed. Bull. Fish. Res. Bd. Can., pp. 167.
  28. Tester, P.A., M.E. Greesey, C. Guo, H.W. Paerl and D.F. Millie, 1995. Evaluating phytoplankton dynamics in the Newport River estuary (North Carolina, USA) by HPLC-derived pigment profiles. Mar. Ecol. Prog. Ser., 124: 237-245. https://doi.org/10.3354/meps124237
  29. Trees, C.C., R.R. Bidigare and J.M., Brooks, 1986. Distributions of chlorophylls and phaeopigments in the Northwestern Atlantic Ocean. J. Plan. Res., 8: 447-458. https://doi.org/10.1093/plankt/8.3.447
  30. Wright, S.W. and J.D. Shearer, 1984. Rapid extraction and high performance liquid chromatography of chlorophylls and carotenoids from marine phytoplankton. J. Chromatogr., 294: 281-295. https://doi.org/10.1016/S0021-9673(01)96134-5
  31. Wright, S.W., S.W. Jeffrey, R.F.C. Mantoura, 1997. Evaluation of methods and solvents for pigment extraction. In: Phytoplankton Pigments in Oceanography, edited by Jeffrey, S.W., R.F.C. Mantoura, S.W. Wright, UNESCO, Paris, 261-282 pp.
  32. Wright, S.W., S.W. Jeffrey, R.F.C. Mantoura, C.A. Llewellyn, T. Bjornland, D. Repeta, N. Welschmeyer, 1991. Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar. Ecol. Prog. Ser., 77: 183-196. https://doi.org/10.3354/meps077183

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