CN102706827A - Novel extraction and determination method of content of chlorophyll by utilizing DMSO (dimethylsulfoxide) - Google Patents
Novel extraction and determination method of content of chlorophyll by utilizing DMSO (dimethylsulfoxide) Download PDFInfo
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 title claims abstract description 50
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 title abstract description 40
- 229930002875 chlorophyll Natural products 0.000 title abstract description 39
- 235000019804 chlorophyll Nutrition 0.000 title abstract description 39
- 238000000605 extraction Methods 0.000 title abstract description 20
- 238000000034 method Methods 0.000 title abstract description 14
- 210000002966 serum Anatomy 0.000 claims abstract description 15
- 239000004744 fabric Substances 0.000 claims abstract description 6
- 241001411320 Eriogonum inflatum Species 0.000 claims abstract description 3
- 230000003287 optical effect Effects 0.000 claims abstract description 3
- 208000003443 Unconsciousness Diseases 0.000 claims abstract 4
- 238000012360 testing method Methods 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims 1
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 abstract description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 12
- 241000196324 Embryophyta Species 0.000 abstract description 10
- 238000005070 sampling Methods 0.000 abstract description 3
- 239000007788 liquid Substances 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 238000005259 measurement Methods 0.000 description 10
- 239000012046 mixed solvent Substances 0.000 description 8
- 238000011160 research Methods 0.000 description 6
- 238000000354 decomposition reaction Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 241001464837 Viridiplantae Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000192700 Cyanobacteria Species 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 239000006004 Quartz sand Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229930002868 chlorophyll a Natural products 0.000 description 1
- 229930002869 chlorophyll b Natural products 0.000 description 1
- NSMUHPMZFPKNMZ-VBYMZDBQSA-M chlorophyll b Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C=O)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 NSMUHPMZFPKNMZ-VBYMZDBQSA-M 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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Abstract
植物叶绿素测定中应用丙酮和无水乙醇作为萃取液易挥发、且取样及样品处理、萃取时间长而造成叶绿素测定误差增大。本发明提出利用DMSO快速浸提、测定植物叶绿素的一种新方法。预先把洁净的血清瓶编号，向每个血清瓶中加入5mLDMSO后盖上瓶塞并准确称重，按编号顺序把血清瓶放入盖有遮光黑布的容器内带至田间，取待测部位样品后立即把样品放入装有DMSO的血清瓶中并盖好瓶塞和遮光黑布带回实验室，再次按编号称重，两次重量差即为待测样品重，把装有试验样品的血清瓶置于黑暗条件下浸提6-8小时，取浸提液1mL加入4mLDMSO混合均匀后，用分光光度计测定663nm和645nm波长下的光密度值。In the determination of plant chlorophyll, the use of acetone and absolute ethanol as the extraction liquid is volatile, and the sampling, sample processing, and extraction time are long, resulting in an increase in the error of chlorophyll determination. The invention proposes a new method for rapidly extracting and measuring plant chlorophyll by using DMSO. Number the clean serum bottles in advance, add 5mL DMSO to each serum bottle, cover the bottle and weigh it accurately, put the serum bottle in a container covered with a black-out cloth and take it to the field in the order of number, and take the part to be tested Immediately after taking the sample, put the sample into the serum bottle containing DMSO, cover the bottle stopper and blackout cloth, and bring it back to the laboratory, and weigh it again according to the number. The difference between the two weights is the weight of the sample to be tested. Place the serum bottle in the dark for 6-8 hours, take 1 mL of the extract and add 4 mL of DMSO to mix evenly, then use a spectrophotometer to measure the optical density values at 663 nm and 645 nm wavelengths.
Description
技术领域 technical field
本发明涉及一种植物叶绿素萃取、测定方法，尤其是利用DMSO快速萃取、测定植物叶绿素含量的新方法。 The invention relates to a method for extracting and measuring plant chlorophyll, in particular to a new method for rapidly extracting and measuring plant chlorophyll content by using DMSO.
背景技术 Background technique
叶绿素是植物进行光合作用的主要色素，叶绿素吸收太阳光后经过光反应和暗反应两个连续过程，把CO2和H2O转变为糖类物质并储存在绿色植物营养器官中，从而为绿色植物生长发育和产量形成提供能源物质。叶绿素实际上存在于所有能营造光合作用的生物体中，包括绿色植物、原核的蓝绿藻（蓝菌）和真核的藻类。高等植物叶绿体中的叶绿素主要有叶绿素a 和叶绿素b 两种，可以说没有叶绿素，植物就不能把太阳光能转化成化学能储存在人类最终的食物中。因此，在提高农作物产量和品质的相关科学研究中常常需要测定叶绿素含量受栽培措施或作物生长环境影响的程度，以便正确评价人工调控作物生长发育及提高产量和品质的某项具体措施的可行性。 Chlorophyll is the main pigment for photosynthesis of plants. After absorbing sunlight, chlorophyll undergoes two continuous processes of light reaction and dark reaction, and converts CO 2 and H 2 O into sugar substances and stores them in the vegetative organs of green plants, thus turning green Plant growth and yield formation provide energy substances. Chlorophyll is present in virtually all photosynthetic organisms, including green plants, prokaryotic blue-green algae (cyanobacteria), and eukaryotic algae. The chlorophyll in the chloroplasts of higher plants mainly includes chlorophyll a and chlorophyll b. It can be said that without chlorophyll, plants cannot convert sunlight energy into chemical energy and store it in human final food. Therefore, in scientific research related to improving crop yield and quality, it is often necessary to determine the extent to which chlorophyll content is affected by cultivation measures or crop growth environments, in order to correctly evaluate the feasibility of a specific measure that artificially regulates crop growth and development and improves yield and quality. .
由于叶绿素不溶于水，因此目前在科学研究中对叶绿素进行测定时，常采用先配制丙酮:无水乙醇（V:V）=1:1的混合溶剂，把待测样品称重后放入盛有丙酮:无水乙醇（V:V）=1:1混合溶剂的容器内，并在黑暗中萃取24小时以上。采用丙酮:无水乙醇（V:V）=1:1混合溶剂萃取植物样品中叶绿素的方法，虽然改变了过去人们在叶绿素测定中使用丙酮或无水乙醇加入石英砂和待测样品进行研磨、过滤的测定方法，使测定技术变得省力，同时避免了研磨、过滤过程中叶绿素的损失，在叶绿素测定技术上前进了一步。然而，采用丙酮:无水乙醇（V:V）=1:1混合溶剂萃取叶绿素的方法仍然存在一些缺点，表现在：（1）丙酮和无水乙醇都是较易挥发的有机溶剂，萃取和测定过程中溶剂的挥发会造成叶绿素浓缩，使测定数值偏高而造成测定误差加大；（2）由于叶绿素不稳定，尤其在光照条件下很容易分解，因此要求叶绿素的萃取、测定时尽可能在黑暗条件下短时间内完成。然而，利用丙酮:无水乙醇（V:V）=1:1混合溶剂萃取样品中的叶绿素至少需要24小时，萃取效率低、时间长，很容易增加叶绿素的分解量而造成测定数据偏低。（3）叶绿素测定时需要把样品从田间取回到实验室，然后再进行取样、称重、萃取，试验样品从田间到实验室及室内样品处理、称重往往需要较长时间，植物离开赖以生存的土壤环境，叶绿素会加快分解而导致测定误差增大。 Since chlorophyll is insoluble in water, when measuring chlorophyll in scientific research, a mixed solvent of acetone: absolute ethanol (V:V) = 1:1 is often used first, and the sample to be tested is weighed and placed in a container. In a container with acetone: absolute ethanol (V:V) = 1:1 mixed solvent, and extract in the dark for more than 24 hours. Using acetone: absolute ethanol (V: V) = 1:1 mixed solvent to extract chlorophyll in plant samples, although it has changed the use of acetone or absolute ethanol to add quartz sand and the sample to be tested for grinding in the past. The measurement method of filtration makes the measurement technology labor-saving, and at the same time avoids the loss of chlorophyll in the process of grinding and filtering, which is a step forward in the chlorophyll measurement technology. However, the method of extracting chlorophyll with acetone: absolute ethanol (V: V) = 1:1 mixed solvent still has some shortcomings, which are shown in: (1) acetone and absolute ethanol are relatively volatile organic solvents, and the extraction and The volatilization of the solvent during the measurement process will cause the concentration of chlorophyll, which will cause the measurement value to be high and cause the measurement error to increase; (2) Since chlorophyll is unstable, especially under light conditions, it is easy to decompose, so it is required to extract and measure chlorophyll as much as possible. Complete in a short period of time in dark conditions. However, it takes at least 24 hours to extract the chlorophyll in the sample with acetone: absolute ethanol (V:V) = 1:1 mixed solvent. The extraction efficiency is low and the time is long. It is easy to increase the decomposition of chlorophyll and cause the measurement data to be low. (3) When measuring chlorophyll, it is necessary to take the sample from the field to the laboratory, and then carry out sampling, weighing, and extraction. It often takes a long time for the test sample to be processed and weighed from the field to the laboratory and indoors. In the living soil environment, chlorophyll will accelerate the decomposition and cause the measurement error to increase.
发明内容 Contents of the invention
由于目前叶绿素萃取和测定方法中存在明显的缺点，因此在我们的科学研究中，经过大量的研究找到了一种性质稳定、不易挥发并对叶绿素具有快速萃取作用的溶剂：二甲基亚砜（DMSO），叶绿素萃取过程中可以把萃取溶剂DMSO带至田间，植株活体条件下取待测样品的同时把样品放入DMSO萃取液中萃取，然后带回实验室并进行叶绿素含量的测定。 Due to the obvious shortcomings in the current chlorophyll extraction and determination methods, in our scientific research, after a lot of research, we found a solvent that is stable, non-volatile and has a rapid extraction effect on chlorophyll: dimethyl sulfoxide ( DMSO), the extraction solvent DMSO can be brought to the field during the chlorophyll extraction process, and the sample to be tested is taken under the living conditions of the plant, and the sample is extracted in the DMSO extraction solution, and then brought back to the laboratory for the determination of the chlorophyll content.
具体实施方式：预先把洁净的血清瓶编号，向每个血清瓶中加入5mL DMSO后盖上瓶塞并准确称重，按编号顺序把血清瓶放入盖有遮光黑布的容器内带至田间，取待测部位样品后立即把样品放入装有DMSO的血清瓶中并盖好瓶塞及遮光黑布带回实验室，再次按编号称重，两次重量差即为待测样品重，把装有试验样品的血清瓶置于黑暗条件下萃取6-8小时，取萃取液1mL加入4mL DMSO混合均匀后，用分光光度计测定663nm和645nm波长下的光密度值。 Specific implementation method: Number the clean serum bottles in advance, add 5mL DMSO to each serum bottle, cover the bottle and weigh it accurately, put the serum bottles in a container covered with a blackout cloth in the order of number and take them to the field After taking the sample of the part to be tested, immediately put the sample into a serum bottle filled with DMSO, cover the bottle stopper and blackout cloth and bring it back to the laboratory, and weigh again according to the number. The difference between the two weights is the weight of the sample to be tested. Place the serum bottle containing the test sample under dark conditions for extraction for 6-8 hours, take 1mL of the extract and add 4mL DMSO to mix evenly, then use a spectrophotometer to measure the optical density values at 663nm and 645nm wavelengths.
该方法避免了应用丙酮和无水乙醇萃取样品和测定过程中溶剂挥发造成的测定误差，样品萃取时间短、效率高、减少了叶绿素的分解量，样品叶绿素萃取可在田间进行操作，减少了样品经较长时间的运输和室内处理时间长而导致叶绿素分解和测定误差增大的不利影响。经过多年的研究和在科研中的应用表明：该方法6-8小时内即可完成待测样品中叶绿素的全部萃取，田间植物活体取样的同时进行叶绿素萃取，方法更简便、准确、快速。2010年-2011年，在大豆苗期、开花期和结荚期利用DMSO进行田间实际测定叶片叶绿素含量见表1。 This method avoids the measurement error caused by the application of acetone and absolute ethanol to extract samples and solvent volatilization during the determination process. The sample extraction time is short, the efficiency is high, and the decomposition of chlorophyll is reduced. The sample chlorophyll extraction can be performed in the field, reducing the sample The adverse effects of chlorophyll decomposition and increased measurement error are caused by long-term transportation and long indoor processing time. Years of research and application in scientific research have shown that this method can complete the extraction of all chlorophyll in the sample to be tested within 6-8 hours, and extract chlorophyll while sampling live plants in the field. The method is simpler, more accurate and faster. From 2010 to 2011, DMSO was used to measure the chlorophyll content of soybean leaves at the seedling stage, flowering stage and pod stage. See Table 1.
表1 不同萃取方法对叶绿素含量测定的影响 Table 1 Effect of different extraction methods on the determination of chlorophyll content
结果表明：利用DMSO萃取、测定叶绿素含量为：1.24 mg/g、1.28 mg/g和1.33 mg/g，利用丙酮:无水乙醇（V:V）=1:1混合溶剂萃取样品并测定叶绿素含量分别为：1.15 mg/g、1.19 mg/g和1.25 mg/g。统计分析表明利用丙酮:无水乙醇（V:V）=1:1混合溶剂萃取测定与利用DMSO萃取、测定叶绿素含量相比结果偏低，且差异达显著水平，这与丙酮:无水乙醇（V:V）=1:1混合溶剂萃取样品时萃取、测定时间较长和萃取液易挥发有关。 The results show that: using DMSO extraction, the measured chlorophyll content is: 1.24 mg/g, 1.28 mg/g and 1.33 mg/g, using acetone: absolute ethanol (V:V) = 1:1 mixed solvent to extract the sample and determine the chlorophyll content They are: 1.15 mg/g, 1.19 mg/g and 1.25 mg/g. Statistical analysis shows that using acetone: absolute ethanol (V:V) = 1:1 mixed solvent extraction to measure the chlorophyll content compared with the use of DMSO extraction to measure the result is on the low side, and the difference reaches a significant level, which is different from acetone: absolute ethanol ( V:V) = 1:1 mixed solvent extraction sample extraction, measurement time is longer and the extraction liquid is volatile.
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Application publication date: 20121003 |