Chinese Bulletin of Botany ›› 2016, Vol. 51 ›› Issue (5): 667-678.DOI: 10.11983/CBB15190

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Fast Extraction and Precise Determination of Chlorophyll

Nianwei Qiu1†, Xiushun Wang1†, Fabin Yang1, Xiaogang Yang1, Wen Yang1, Runjie Diao1, Xiu Wang1, Jing Cui1, Feng Zhou2*   

  1. 1College of Life Sciences, Qufu Normal University, Qufu 273165, China
    2School of Biochemical and Environmental Engineering, Nanjing Xiaozhuang University, Nanjing 211171, China
  • Received:2015-10-23 Accepted:2016-02-27 Online:2016-09-01 Published:2016-09-27
  • Contact: Qiu Nianwei,Wang Xiushun,Zhou Feng
  • About author:

    # Co-first authors

Abstract:

The Arnon method is the most classical and common method for extracting and determining chlorophyll. De- spite many improvements to this method, severe problems remain, such as inaccurate test wavelength, wrong content formula, low extraction speed, large errors in results, and tedious operation process. We present a fast two-step extraction and determination method for chlorophyll. The first step is extracting chlorophyll with dimethyl sulfoxide (DMSO) at high temperature, then diluting the chlorophyll solution with 80% acetone. Chlorophyll content determined by this method can be completed within 3 h. The optimal experimental conditions for extraction and the accurate formula for chlorophyll content were obtained by analyzing extraction temperature, extraction time, dilution ratio and absorption spectroscopy. The merits and reliability of this method were tested with some typical plant materials. The method is described as follows: Cut the plant material into a 1 mm wide filament or small pieces and place 50-100 mg plant material into a 10 mL gradu- ated test tube with a stopper. Then add 2 mL DMSO into the test tube and dip the plant material into DMSO. Place the tubes into a 65°C incubator away from the light until all plant material turns white or transparent. As the liquid cools, add 8 mL 80% (v/v) acetone to dilute DMSO, mix well, then determine absorbance at 663.6 and 646.6 nm by spectrophoto- metry. Chlorophyll concentration can be calculated with the following formulas: Ca (mg∙L-1)=12.27A663.6-2.52A646.6; Cb (mg∙L-1)=20.10A646.6-4.92A663.6; CT=Ca+Cb=7.35A663.6+17.58A646.6.