植物学报 ›› 2016, Vol. 51 ›› Issue (5): 667-678.doi: 10.11983/CBB15190

• 技术方法 • 上一篇    下一篇

叶绿素的快速提取与精密测定

邱念伟1,, 王修顺1,, 杨发斌1, 杨晓刚1, 杨文1, 刁润洁1, 王秀1, 崔静1, 周峰2,*()   

  1. 1曲阜师范大学生命科学学院, 曲阜 273165
    2南京晓庄学院生物化工与环境工程学院, 南京 211171
  • 收稿日期:2015-10-23 接受日期:2016-02-27 出版日期:2016-09-01 发布日期:2016-09-27
  • 通讯作者: 邱念伟,王修顺,周峰 E-mail:zfibcas@163.com
  • 作者简介:

    # 共同第一作者

  • 基金资助:
    山东省自然科学基金(No.ZR2015DL009)、曲阜师范大学精品实验项目(No.JP2014006, No.SJ201409)和曲阜师范大学科技计划(No.XKJ201404)

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 E-mail:zfibcas@163.com
  • About author:

    # Co-first authors

摘要:

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: Ca (mg∙L-1)=12.27A663.6-2.52A646.6; Cb (mg∙L-1)=20.10A646.6-4.92A663.6; CT (mg∙L-1)=Ca+Cb=7.35A663.6+17.58A646.6

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.

图1

80%丙酮(A)和DMSO (B)在不同提取温度下的挥发性 不同小写字母表示差异显著(P<0.05)。"

表1

80%丙酮或DMSO浸提法在不同温度下的提取时间(单位: 小时)"

Plant material 80% acetone DMSO
4°C 20°C 30°C 40°C 50°C 65°C
Spinach leaf 79.3±6.7 a 71.2±5.4 b 47.6±3.6 c 38.7±4.2 d 25.6±3.7 e 1.08±0.04 f
Wheat leaf 28.7±3.1 a 23.1±2.3 b 17.9±1.8 c 14.8±1.2 d 8.3±0.7 e 0.82±0.07 f
Chinese pine leaf 66.7±4.8 a 42.4±3.4 b 27.3±2.5 c 23.6±2.1 d 17.5±1.6 e 2.08±0.14 f

图2

叶绿素在80%丙酮(A)和DMSO (B)中的吸收光谱"

图2

叶绿素在80%丙酮(A)和DMSO (B)中的吸收光谱"

图3

叶绿素在80%丙酮(A)和DMSO (B)中的稳定性 不同小写字母表示差异显著(P<0.05)。"

表2

叶绿素在80%丙酮与DMSO混合液中的吸收峰(平均值±标准差)"

DMSO:80% acetone (v/v)
0:5 1:4 1:1
Wavelength of Chla (nm) 663.7±0.03 (663.3-664.1) 663.2±0.03 (663.0-663.4) 663.9±0.05 (663.6-664.2)
Wavelength of Chlb (nm) 647.0±0.04 (646.6-647.4) 646.8±0.02 (646.4-647.2) 647.8±0.04 (647.5-651.1)

图4

色谱纯叶绿素在DMSO:80%丙酮=1:4 (v/v)混合溶剂中的吸收光谱"

图5

不同温度下DMSO浸提菠菜叶绿素所需时间 不同小写字母表示差异显著(P<0.05)。"

图6

不同温度下DMSO溶液中叶绿素的降解 不同小写字母表示差异显著(P<0.05)。"

表3

叶绿素在2种溶剂中吸收峰波长处吸光度的比较"

Chla solution Chlb solution
663.2 nm 663.6 nm 663.7 nm 646.6 nm 646.8 nm 647.0 nm
80% acetone 0.852 0.853 0.853 0.498 0.498 0.498
1:4 miscible solvent 0.853 0.852 0.852 0.501 0.501 0.501

图7

叶绿素(Ca/Cb=3:1)混合溶液在80%丙酮和DMSO:80%丙酮=1:4 (v/v)混合溶剂中的吸收光谱"

表4

叶绿素浓度计算公式(A)及计算结果的比较(B) (平均值±标准差)"

(A)
No. Formulas References
1 Ca (mg·L-1)=12.7A 663-2.69A645; Cb (mg·L-1)=22.9A645-4.68A663 Arnon, 1949
2 Ca (mg·L-1)=12.72A663-2.59A645; Cb (mg·L-1)=22.88A645-4.67A663 陈毓荃, 1986
3 Ca (mg·L-1)=12.63A664.5-2.52A647; Cb (mg·L-1)=20.47A647-4.73A664.5 Inskeep and Bloom, 1985
4 Ca (mg·L-1)=12.21A663-2.81A646; Cb (mg·L-1)=20.13A646-5.03A663 Wellburn and Lichtenthaler, 1984
5 Ca (mg·L-1)=12.25A663.2-2.79A646.8; Cb (mg·L-1)=21.50A646.8-5.10A663.2 Lichtenthaler, 1987
6 Ca (mg·L-1)=12.25A663.6-2.55A646.6; Cb (mg·L-1)=20.31A646.6-4.91A663.6 Porra et al., 1989
7 Ca (mg·L-1)=12.27A663.6-2.52A646.6; Cb (mg·L-1)=20.10A646.6-4.92A663.6
(correction formula of DMSO:80% acetone=1:4 (v/v) mixed solvent)
-
(B)
Wavelength (nm) Absorbance and concentration in 80% acetone Absorbance and concentration in DMSO:80%
acetone=1:4 (v/v) mixed solvent
663.0 0.6710±0.0017 0.6724±0.0028
663.2 0.6716±0.0013 0.6732±0.0028
663.6 0.6734±0.0017 0.6721±0.0024
664.5 0.6698±0.0026 0.6673±0.0035
645.0 0.2626±0.0019 0.2658±0.0016
646.0 0.2768±0.0013 0.2804±0.0019
646.6 0.2869±0.0014 0.2899±0.0015
646.8 0.2886±0.0017 0.2918±0.0016
647.0 0.2918±0.0014 0.2952±0.0018
No. Ca (mg·L-1) Cb (mg·L-1) CT (mg·L-1) Ca/Cb Ca (mg·L-1) Cb (mg·L-1) CT (mg·L-1) Ca/Cb
1 7.82 2.87 10.69 2.72 7.82 2.94 10.76 2.66
2 7.85 2.87 10.73 2.73 7.86 2.94 10.81 2.67
3 7.72 2.80 10.53 2.75 7.68 2.89 10.57 2.66
4 7.42 2.20 9.61 3.38 7.42 2.26 9.68 3.28
5 7.42 2.78 10.20 2.67 7.43 2.84 10.27 2.62
6 7.52 2.52 10.04 2.98 7.49 2.59 10.08 2.90
7 - - - - 7.52 2.50 10.02 3.00

表5

3种方法提取测定叶绿素浓度及Ca/Cb的比较(平均值±标准差)"

Methods Extracted by 80% acetone Fast two-step extraction Grinding with 80% acetone
CT Ca/Cb CT Ca/Cb CT Ca/Cb
Spinach leaf 8.60±0.22 a 3.19±0.07 a 8.50±0.24 a 3.28±0.10 a 8.48±0.41 a 3.12±0.11 a
Rape leaf 3.61±0.23 a 2.74±0.11 a 3.67±0.21 a 2.72±0.12 a 3.55±0.36 a 2.71±0.14 a
Poplar leaf 5.88±0.29 b 3.57±0.10 a 7.27±0.19 a 3.50±0.08 a 7.24±0.44 a 3.51±0.15 a
Chinese pine leaf 1.19±0.04 b 3.13±0.06 a 1.33±0.07 a 3.17±0.06 a 1.27±0.11 a 3.19±0.09 a
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