Chinese Bulletin of Botany ›› 2016, Vol. 51 ›› Issue (5): 667-678.DOI: 10.11983/CBB15190
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Nianwei Qiu1†, Xiushun Wang1†, Fabin Yang1, Xiaogang Yang1, Wen Yang1, Runjie Diao1, Xiu Wang1, Jing Cui1, Feng Zhou2*
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
Nianwei Qiu, Xiushun Wang, Fabin Yang, Xiaogang Yang, Wen Yang, Runjie Diao, Xiu Wang, Jing Cui, Feng Zhou. Fast Extraction and Precise Determination of Chlorophyll[J]. Chinese Bulletin of Botany, 2016, 51(5): 667-678.
Figure 1 The volatility of 80% acetone (A) and DMSO (B) under different temperatures Different lowercase letters indicated significant difference at P<0.05.
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 |
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 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.
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) |
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 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.
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 |
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 |
(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 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 |
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 |
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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