Chin Bull Bot ›› 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:2016-09-27 Published:2016-09-01
  • Contact: Qiu Nianwei,Wang Xiushun,Zhou Feng E-mail:zfibcas@163.com
  • 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.

Figure 1

The volatility of 80% acetone (A) and DMSO (B) under different temperatures Different lowercase letters indicated significant difference at P<0.05."

Table 1

The extraction time of immersion extraction with DMSO and 80% acetone under different temperatures (unit: h)"

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

Figure 2

The absorption spectrum of chlorophyll a and chlorophyll b in 80% acetone (A) and DMSO (B)"

Figure 2

The absorption spectrum of chlorophyll a and chlorophyll b in 80% acetone (A) and DMSO (B)"

Figure 3

The stability of chlorophyll in 80% acetone (A) and DMSO (B) under different temperatures Different lowercase letters indicated significant difference at P<0.05."

Table 2

The absorption peak (nm) of chlorophyll in miscible liquids of DMSO and 80% acetone (means±SD)"

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)

Figure 4

The absorption spectrum of chlorophyll a and chlorophyll b in 1:4 (v/v) miscible liquids of DMSO and 80% acetone"

Figure 5

The time of extracting chlorophyll from spinach leaf with DMSO under different temperatures Different lowercase letters indicated significant difference at P<0.05."

Figure 6

The degradation of chlorophyll in DMSO solution under different temperatures Different lowercase letters indicated significant difference at P<0.05."

Table 3

Comparison of chlorophyll absorbance at absorption peak wavelength in two solvents"

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

Figure 7

The absorption spectrum of mixed chlorophyll (Ca/Cb=3:1) in 80% acetone or miscible solvent (DMSO:80% acetone=1:4, v/v)"

Table 4

The calculation formulas (A) and comparison of chlorophyll concentration (B) calculated by different formulas (means±SD)"

(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

Table 5

Comparison of chlorophyll concentration and Ca/Cb extracted and measured by three methods (means±SD)"

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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