Photosynthesis Research

, Volume 62, Issue 1, pp 107–116 | Cite as

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

  • Roman Kouril
  • Petr Ilík
  • Jan Naus
  • Benoit Schoefs


The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the QY) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.

Chl a/b ratio determination chlorophyll concentration chlorophyll b fluorescence greening protochlorophyll(ide) spectrofluorimetry spectrophotometry 


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  1. Argyroudi-Akoyunoglou JH and Akoyunoglou G (1970) Photoinduced changes in the chlorophyll a to chlorophyll b ratio in young bean plants. Plant Physiol 46: 247–249Google Scholar
  2. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol 24: 1–15Google Scholar
  3. Bertrand M and Schoefs B (1997) Working with photosynthetic pigments: problems and precautions. In: Pessarakli M (ed) Handbook of Photosynthesis, pp 151–172. Marcel Dekker: New York/Basel/Hong KongGoogle Scholar
  4. Boardman NK and Thorne SW (1971) Sensitive fluorescence method for the determination of chlorophyll a / chlorophyll b ratios. Biochim Biophys Acta 253: 222–231Google Scholar
  5. Brouers M and Michel-Wolwertz MD (1983) Estimation of protochlorophyll (ide) contents in plant extracts: Re-evaluation of the molar absorption coefficient of protochlorophyll(ide). Photosynth Res 4: 54–75Google Scholar
  6. Burkey KO (1986) Chlorophyll–protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings. Photosynth Res 10: 37–49Google Scholar
  7. Grover A and Mohanty P (1993) Leaf senescence – induced alterations in structure and function of higher plant chloroplasts In: Abrol YP, Mohanty P and Govindjee (eds) Photosynthesis: Photoreactions to Plant Productivity, pp 225–256. Kluwer Academic Publishers, Dordecht/Boston/LondonGoogle Scholar
  8. Jäkel N (1981) Zur Fluorometrischen Bestimmung von Chlorophyll a und Chlorophyll b. Kulturpflanze 29: 417–423Google Scholar
  9. Knox RS (1973) IV. Transfer of electronic excitation energy in condensed systems. In: Checcucci A and Weate RA (eds) Primary Molecular Events in Photobiology, pp 45–77. Elsevier, AmsterdamGoogle Scholar
  10. Lichtenthaler HK (1987) Chlorophylls and carotenoids. Pigments of photosynthetic biomembranes. Meth Enzymol 148: 350–382Google Scholar
  11. Mader P. Chlad F, Chladová J, Novák V, Sofrová D, Nauš J, Kohlová Švábová M and Makovec P (1980) Development of photosynthetic apparatus of etiolated seedlings of spring barley grown under various temperature, nitrogen and NaCl concentration in the nutrient solution. Photosynthetica 14(2): 222–235Google Scholar
  12. Meister A (1992) New fluorometric method for determination of chlorophyll a/b ratio. Photosynthetica 26: 533–539Google Scholar
  13. Nauš J (1983) A simple model for evaluation of optical effects in the fluorescence spectrum of planparallel layer samples. I. Theory. II. Applications. Acta Univ Palacki Olom, Fac.rer.nat. 76, Physica XXII: 9–34Google Scholar
  14. Nauš J, Klinkovský T, Ilík P and Cikánek D (1994) Model studies of chlorophyll fluorescence reabsorption at chloroplast level under different exciting light conditions. Photosynth Res 40: 67–74Google Scholar
  15. Neveux J and Panouse M (1987) Spectrofluorometric determination of chlorophylls and pheophytins. Arch Hydrobiol 109(4): 567–581Google Scholar
  16. Ogawa T and Shibata K (1965) A sensitive method for determining chlorophyll b in plant extracts. Photochem Photobiol 4: 193–200Google Scholar
  17. Porra RJ (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, pp 31–57. CRC Press, Boca Raton, FLGoogle Scholar
  18. Porra RJ, Thompson WA and Kriedemann PE (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. Biochim Biophys Acta 975: 384–394Google Scholar
  19. Schoefs B and Bertrand M (1997) Chlorophyll Biosynthesis. In: Pessarakli M (ed) Handbook of Photosynthesis, pp 49–70. Marcel Dekker: New York/Basel/Hong KongGoogle Scholar
  20. Schoefs B, Bertrand M and Lemoine Y (1998) Changes in the photosynthetic pigments in bean leaves during the first photoperiod of greening and the subsequent dark-phase. Comparison between old (l0-d-old) leaves and young (2-d-old) leaves. Photosynth Res 57: 203–213Google Scholar
  21. Shubin VV and Sabat SC (1990) A simple room temperature spectrofluorimetric method for determination of chlorophyll a/b ratio. Indian J Exp Biol 28: 87–90Google Scholar
  22. Smith JHC and Benitez A (1955) Chlorophylls: Analysis in Plant Materials. In: Paech K and Tracey MV (eds) Modern Methods of Plant Analysis, Vol 4, pp 142–196. Springer-Verlag, BerlinGoogle Scholar
  23. Šesták Z (1971) Determination of Chlorophylls a and b In: Šesták Z, Čatský J and Jarvis PG (eds) Plant Photosynthetic Production. Manual of Methods, pp 672–702. DrWJunk NV Publishers, The HagueGoogle Scholar
  24. Talbot MFJ and Sauer K (1997) Spectrofluorimetric method for the determination of large chlorophyll a/b ratios. Photosynth Res 53: 73–79Google Scholar
  25. Weber G and Teale FWJ (1957) Determination of the absolute quantum yield of fluorescent solutions. Trans Faraday Soc 53: 646–655Google Scholar
  26. Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144: 307–313Google Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Roman Kouril
    • 1
  • Petr Ilík
    • 1
  • Jan Naus
    • 1
  • Benoit Schoefs
    • 2
  1. 1.Department of Experimental Physics, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  2. 2.Laboratory of BiomembranesUniversity of South BohemiaCeské BudejoviceCzech Republic

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