N,N-Dimethylformamide와 Dimethylsulfoxide를 이용한 감귤 잎의 엽록소 측정방법 확립

Establishment of Analytical Method for Chlorophyll Using the N,N-Dimethylformamide and Dimethylsulfoxide in Citrus Leaves

  • ABSTRACT

    BACKGROUND:

    Purpose of the study was to establish the extinction coefficients of chlorophyll a and b in N,N-dimethylformamide(DMF) and Dimethylsulfoxide(DMSO) solvents and to find out the conditions of optimal extraction temperature and time in citrus leaves.

    METHODS AND RESULTS:

    Chlorophyll a and b standards were dissolved in DMF and DMSO. Extinction coefficients of chlorophyll pigments were determined and their contents were quantified using spectrophotometer. Chlorophyllous pigments of citrus(Citrus unshiu Marc. cv. Okitsu wase) leaves were extracted at 25, 40, 60 and 80℃ for 4, 6, 8, 24 and 48 hours to determine the optimal extraction condition.

    CONCLUSION:

    The extinction coefficients of chlorophyll a(Chl a) and chlorophyll b(Chl b) of DMF extracts for high extinction wavelength were 663.8 and 647.2 nm. Similarly, the high extinction wavelength of DMSO extracts were 665.8 and 649.0 nm for chl a and b respectively. Chl a, Chl b and total chlorophyll content of DMF extracts were Chl a = 12.10A663.8 - 2.74A647.2, Chl b = 21.94A647.2 - 5.06A663.8 and total Chl = 19.193A647.2 + 7.04A663.8. Similarly, Chl a, Chl b and total Chl of DMSO extracts were Chl a = 14.53A665.8 - 5.40A649.0, Chl b = 26.98A649.0 - 7.11A665.8 and total Chl = 21.58A649.0 + 7.43A665.8. The chlorophyll extracts of DMF and DMSO were very stable in dark. High chlorophyll contents of citrus leaves were found at 80℃ extraction for 6 hours in DMF and at 80℃ extraction for 24 hours in DMSO. However, the chlorophyll content was decreased significantly after 8 hours in DMF extraction while it was remained up to 30 hours in DMSO extraction.


  • KEYWORD

    Analysis , Nutrition , Pigment , Solvent

  • 1. Arnon D.I. 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxides in Beta vulgaris [Plant Physiol.] Vol.24 P.1-15 google doi
  • 2. Barnes J.D., Balaguer L., Manrique E., Elivira S., Davision A.W. 1992 A reappraisal of the use of DMSO for the extraction and determination of chlorophyll a and b in lichens and higher plants [Environ. Exp. Bot.] Vol.32 P.85-100 google doi
  • 3. Hiscox J.D., Israelstam G.F. 1979 A method for the extraction of chlorophyll from leaf tissue without maceration [Can. J. Bot.] Vol.57 P.1332-1334 google doi
  • 4. Inskeep W.P., Bloom P.R. 1985 Extinction coefficients of chlorophyll a and b in N,N -dimethylformamide and 80% acetone [Plant physiol.] Vol.77 P.483-485 google doi
  • 5. Knudson L.L., Tibbitts T.W., Edwards G.E. 1977 Measurement of ozone injury by determination of leaf chlorophyll concentration [Plant Physiol.] Vol.60 P.606-608 google doi
  • 6. Mackinney G. 1941 Absorption of light by chlorophyll solutions [J. Biol. Chem.] Vol.140 P.315-322 google
  • 7. Moran R., Porath D. 1980 Chlorophyll determination in intact tissues using N, N -dimethylformamide [Plant Physiol.] Vol.65 P.478-479 google doi
  • 8. Park J.M., Ro H.M., Kim Y.M., Seong K.C. 1996 Chlorophyll determination in horticultural crops using dimethyl sulphoxide [RDA. J. Agri. Sci.] Vol.38 P.552-557 google
  • 9. Seo K.Y., Yi M.J., Son Y.H., Koo J.W., Noh N.J., Yi M.J. 2005 Extraction and Determination of Chlorophyll Contents of Korean Pine Needles Using Acetone and DMSO(Dimethylsulfoxide) [J. Korean For. Soc.] Vol.94 P.264-268 google
  • 10. Shoaf T.W.T., Lium B.W. 1976 Improved extraction of chlorophyll a and b from algae using demethylsulfoxide [Limnol. Oceanogr.] Vol.21 P.926-928 google
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  • [Fig. 1.] Absorbance spectra of chlorophyll a and b in a solvents, in a N,N-dimethylformamide, chlorophyll a has approximate absorbance maxima of 663.8 nm(A), while chlorophyll b has approximate of 647.2 nm(B). In a Dimethylsulfoxide, chlorophyll a has approximate absorbance maxima 665.2 nm(C), while chlorophyll b has approximate of maxima of 649.0 nm(D).
    Absorbance spectra of chlorophyll a and b in a solvents, in a N,N-dimethylformamide, chlorophyll a has approximate absorbance maxima of 663.8 nm(A), while chlorophyll b has approximate of 647.2 nm(B). In a Dimethylsulfoxide, chlorophyll a has approximate absorbance maxima 665.2 nm(C), while chlorophyll b has approximate of maxima of 649.0 nm(D).
  • [Table 1.] Calculated extinction coefficients for chlorophyll a and b dissolved in N,N-dimethylformamide and Dimethylsulfoxide
    Calculated extinction coefficients for chlorophyll a and b dissolved in N,N-dimethylformamide and Dimethylsulfoxide
  • [Table 2.] Simultaneous equations for quantifying chlorophyll from measured absorbance dissolved in N,N-dimethylformamide and Dimethylsulfoxide
    Simultaneous equations for quantifying chlorophyll from measured absorbance dissolved in N,N-dimethylformamide and Dimethylsulfoxide
  • [Table 3.] Time-lapsed changes in the concentration of chlorophyll extracted by N,N-dimethylformamide and Dimethylsulfoxide in the light condition at 25℃
    Time-lapsed changes in the concentration of chlorophyll extracted by N,N-dimethylformamide and Dimethylsulfoxide in the light condition at 25℃
  • [Table 4.] Time-lapsed changes in the concentration of chlorophyll extracted by N,N-dimethylformamide and Dimethylsulfoxide in the dark condition
    Time-lapsed changes in the concentration of chlorophyll extracted by N,N-dimethylformamide and Dimethylsulfoxide in the dark condition
  • [Fig. 2.] Effects of heat treatments and solvents on the contents of chlorophyll in citrus leaves. A; 663.8 nm readings of chlorophyll a extracted by N,N-dimethylformamide, B; 649.0 nm readings of chlorophyll a extracted by Dimethylsulfoxide, C; 647.2 nm readings chlorophyll b extracted by N,N-dimethylformamide, D; 665.8 nm readings of chlorophyll b extracted by Dimethylsulfoxide.
    Effects of heat treatments and solvents on the contents of chlorophyll in citrus leaves. A; 663.8 nm readings of chlorophyll a extracted by N,N-dimethylformamide, B; 649.0 nm readings of chlorophyll a extracted by Dimethylsulfoxide, C; 647.2 nm readings chlorophyll b extracted by N,N-dimethylformamide, D; 665.8 nm readings of chlorophyll b extracted by Dimethylsulfoxide.