叶绿素的快速提取与精密测定

Arnon法是叶绿素提取和测定最经典、最常用的方法, 此法虽经多次改进, 但仍存在着检测波长误差大、计算公式有误、提取速度慢、测定结果不够准确以及操作步骤繁琐等缺陷。该文提出了二甲基亚砜(DMSO)高温提取、80%丙酮稀释的两步快速浸提法, 使叶绿素提取和测定过程缩短至3小时以内。通过对提取温度、提取时间、稀释比例及吸收光谱等进行系统分析, 筛选出了叶绿素的最佳提取条件和叶绿素含量的准确计算公式, 并用多种类型的植物材料验证了改进后的提取方法, 证明该方法具优越性和可靠性。具体测定方法是将植物材料切成1 mm宽的细丝或细段, 取50-100 mg材料于10 mL具塞试管中; 加入2 mL DMSO, 使植物材料浸没其中, 65°C高温避光提取至植物材料变白或透明; 冷却后加入8 mL 80%丙酮, 混匀, 测定663.6和646.6 nm处的吸光度。用公式计算叶绿素浓度: C_a (mg?L^-1)=12.27A_663.6-2.52A_646.6; C_b (mg?L^-1)=20.10A_646.6-4.92A_663.6; C_T (mg?L^-1)=C_a+C_b=7.35A_663.6+17.58A_646.6。

Fast Extraction and Precise Determination of Chlorophyll

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: C_a (mg?L^-1)=12.27A_663.6-2.52A_646.6; C_b (mg?L^-1)=20.10A_646.6-4.92A_663.6; C_T=C_a+C_b=7.35A_663.6+17.58A_646.6.