CN101858857A - Analyzing and measuring method for chlorophyll a of phytoplankton in eutrophic water - Google Patents
Analyzing and measuring method for chlorophyll a of phytoplankton in eutrophic water Download PDFInfo
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Abstract
An analyzing and measuring method for chlorophyll a of phytoplankton in eutrophic water is a new method for extracting, analyzing and measuring the chlorophyll a of phytoplankton in eutrophic water and relates to the technical field of restoration of water environment in the environmental engineering. The method comprises the following steps of: filtering and freezing a collected water sample and then adding hot ethanol; after the water bathing and the ultrasonic treatment, extracting the water sample for a plurality of hours in dark, centrifuging the water sample, taking the supernatant and measuring the content of the chlorophyll in a water body by using the spectrophotometry. The invention has the advantages of simple and quick extraction and measurement of the sample, capability of extracting the chlorophyll in the phytoplankton to the most extent, high degree of extraction, low toxicity, good accuracy, low limit of detection and wide range of detection concentration; the problems that the traditional grinding method has high loss degree of chlorophyll and low extraction efficiency and the acetone extraction is toxic to human bodies to a certain extent are solved; and the invention is suitable for rapidly extracting and accurately measuring the chlorophyll of phytoplankton in the eutrophic water.
Description
Technical field
The invention belongs to environmental engineering water environment recovery technique field, relate in particular to the analysis determining method of phytoplankton chlorophyll a in a kind of eutrophication water, is a kind of new method of extracting, analyzing and measure the chlorophyll a of phytoplankton in the eutrophication water.
Background technology
Green plants utilizes chlorophyll to carry out photosynthesis, and chlorophyll mainly contains two kinds of chlorophyll a and chlorophyll bs, belongs to the synthesis of natural low-molecular-weight organic compound, and is water insoluble, and be dissolved in organic solvent, as ethanol, acetone, ether, chloroform etc.Wherein chlorophyll a can directly reflect algae grows situation in the water body, the content height, and then algae is many, and content is low, and then algae is few.Too much algae makes water transparency and dissolved oxygen DO reduce, and influence that biology carries out photosynthesis under the water surface, causes water quality deterioration, quicken water body and wear out, and be the major reason of impelling body eutrophication.Therefore, chlorophyll a is the important indicator of reflection body eutrophication, measures the concentration of chlorophyll a in the water body, to the control water body eutrophication degree, recovers the water environment effect and has important effect.When the concentration of chlorophyll a in the water body during, can be used as one of foundation of body eutrophication at 25～500ug/L.
Mensuration to algae in the water body and other phytoplankton chlorophyll a, method is numerous at present, generally use the more polishing that is, this method need with filter, plant tissue after freezing adds an amount of 90% acetone, then fully grind, in process of lapping, easily cause chlorophyllous loss in the tissue, and acetone there is under certain toxicity and the normal temperature extraction efficiency lower to human body, make that the chlorophyll a of measuring is on the low side, be unfavorable for evaluating and testing water body eutrophication degree.Study a kind of new extraction, the method for assay determination chlorophyll a, reduce traditional polishing extraction efficiency low, extract not exclusively, to the shortcoming of the toxic property of human body, to the monitoring water body eutrophication degree, administer body eutrophication, repair the impaired water environment and have great importance.
Summary of the invention
The technical problem to be solved in the present invention provides the analysis determining method of phytoplankton chlorophyll a in a kind of new eutrophication water, defectives such as the existing extraction of phytoplankton chlorophyll a detection method is incomplete, poisonous to remedy in traditional water body, complicated operating process, efficient to realize, quick, accurately, environmental protection ground measures the content of the contained chlorophyll a of phytoplankton in the water body.
The present invention is improved the master operation of traditional polishing, abandon grinding step, use hot ethanol to substitute propyl alcohol, introduce water-bath and ultrasonic operation, improve chlorophyll and extract and extraction efficiency, thereby reach the purpose that efficiently and accurately is measured chlorophyll a in the eutrophication water.
For solving the problems of the technologies described above, by the following technical solutions:
The analysis determining method of phytoplankton chlorophyll a in the eutrophication water (the following heat ethanol methods that also claims), concrete steps are as follows:
(1) pre-treatment of sample
Chlorophyllous sampling method according to phytoplankton in traditional detection water body is gathered water sample, with 200～500ml (V in the computing formula
2) water sample by the nutsch filter suction filtration.Filter membrane behind the suction filtration and phytoplankton attached to it are placed 10～25mL tool plug centrifuge tube, put into-18 ℃ of refrigerator and cooled then and froze 6～8 hours.
Preferred filter membrane can adopt the glass fiber filter of diameter 47mm, aperture 0.22 μ m, or the composite fibre filter membrane of homemade diameter 60mm, aperture 0.45 μ m, or the glass fiber filter of diameter 25mm, aperture 0.65 μ m, wherein the effect of the glass fiber filter of diameter 47mm, aperture 0.22 μ m is best.
In this step operation, take volume of water sample also can adjust according to the concentration of the contained chlorophyll a of phytoplankton in the concrete water body environment, with reach handle the back testing sample solution concentration in the measurement range that spectrophotometer allows.
(2) extraction of sample
After the tool plug centrifuge tube of taking-up step (1) returns to room temperature state, in centrifuge tube, add the 10mL (V in the computing formula
1) 80 ℃ 90% ethanol, behind 80 ℃ of water-bath 2min, ultrasonic 10min (for example in supersonic wave cleaning machine, ultrasonic frequency is 40kHz, power is 150W), sonicated can make the phytoplankton cell pulverization more complete, then lucifuge leave standstill the extraction 3～5h after with the centrifugal 10min of the rotating speed of 4000r/min, it is to be measured to get supernatant.
If supernatant concentration exceeds the spectrophotometric determination scope, can carry out spectral photometry again to processing such as supernatant dilute, concentrates according to conventional method of analysis.
(3) concentration of phytoplankton chlorophyll a is calculated in the mensuration of supernatant and the water body
Get step (2) gained supernatant and on spectrophotometer, measure the absorbance of the chlorophyll a of supernatant.Make blank absorbency with 90% ethanol and measure, the supernatant absorbance is proofreaied and correct, then begin to measure the absorbance of supernatant.Survey supernatant absorptivity E at the 665nm wavelength earlier
665, survey supernatant absorptivity E at the 750nm wavelength again
750In the cuvette of supernatant is housed, add 0.1～0.2 milliliter of 1.0mol/L hydrochloric acid then and carry out acidifying, to eliminate the interference of phoeophytin to measurement result, obtain the absorption value of pure chlorophyll a, add a cover and shake up, leave standstill the absorptivity A that surveys the supernatant after acidification behind the 1min again at the 665nm wavelength
665, survey its absorptivity A at the 750nm wavelength again
750, be calculated as follows the concentration of phytoplankton chlorophyll a in the water body:
In the formula: Chla---chlorophyll-a concentration (ug/L);
V
1---the volume (L) behind the alcohol extraction liquid constant volume;
V
2---the volume (L) of water sample;
E
665, A
665---the absorptivity at 665nm wavelength place;
E
750, A
750---the absorptivity at 750nm wavelength place.
Compare with existing assay method (polishing), advantage of the present invention is as follows:
1. the relatively heat ethanol methods (being the inventive method) and the polishing operating process of chlorophyll a assay, the mensuration process of polishing is more numerous and diverse, wherein sample grinds needs cost plenty of time and energy, and be not easy the phytoplankton cell is ground fully, have influence on the extraction efficiency of chlorophyll a, and heat ethanol methods is because the sample process is freezing and the Rapid Thermal water-bath is extracted, use cold and hot difference with phytoplankton clasmatosis, add hyperacoustic pulverization, and the effect of extracting of hot solution is higher than cold soln, extraction to chlorophyll a is more complete, and time saving and energy saving.
2. the volatility of the used extraction solvent acetone of polishing is extremely strong, much larger than ethanol, long-term uses greatlyyer to operator's murder by poisoning to the murder by poisoning of human body, replaces polishing with heat ethanol methods and can make the operator be in relatively healthy experimental situation.Add the acetone and the ethanol of same amount simultaneously, the latter is cheap than the former, can reduce the sample analysis testing cost.
3. the chlorophyll-a concentration of polishing mensuration is on the low side, and its result is unfavorable for as the foundation of identifying eutrophication water, and hot heat ethanol methods detectability is low, the detectable concentration scope is wide, and the result is accurate, can correctly show the eutrophication degree of water body.
Description of drawings
Fig. 1 is a schematic flow sheet of the present invention.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.
Embodiment 1: Shanghai city river phytoplankton chlorophyll a is measured
After gathering the water sample of Shanghai city river according to the chlorophyllous sampling method of phytoplankton in traditional detection water body, use heat ethanol methods of the present invention and traditional polishing chlorophyll a respectively according to phytoplankton in the following step measurements water body.
(1) measure the concentration of the chlorophyll a of phytoplankton in the water body for heat ethanol methods:
Get 200ml water sample suction filtration and (use diameter 47mm respectively, the glass fiber filter of aperture 0.22 μ m and diameter 60mm, the composite fibre filter membrane of aperture 0.45 μ m carries out suction filtration), filter membrane and phytoplankton attached to it are placed 10mL tool plug centrifuge tube, putting into-18 ℃ of refrigerator and cooled then froze 6 hours, after taking-up tool plug centrifuge tube returns to room temperature state, 90% ethanol that in centrifuge tube, adds 80 ℃ of 10mL, and in 80 ℃ of water-bath 2min, (ultrasonic frequency is 40kHz to ultrasonic again 10min, power is 150W), extracted 5 hours and be placed on the dark place, centrifugal then 10min (4000r/min) gets supernatant and carry out colorimetric with 90% ethanol as reference liquid on spectrophotometer.The concrete way of measuring the supernatant absorbance is: the cuvette that adopts the 1cm light path, at first making blank absorbency with 90% ethanol measures, the supernatant absorbance is proofreaied and correct, then begin to measure the absorbance of supernatant, survey supernatant absorptivity E at the 665nm wavelength earlier
665, survey supernatant absorptivity E at the 750nm wavelength again
750, in the cuvette of supernatant is housed, add 0.1 milliliter of 1mol/L hydrochloric acid then and carry out acidifying, add a cover and shake up, leave standstill behind the 1min absorptivity A of the supernatant after the 665nm wavelength is measured acidified processing again
665, survey its absorptivity A at the 750nm wavelength again
750, by the concentration of phytoplankton chlorophyll a in formula (1.1) the calculating water body, identical water sample triplicate operation is averaged, and promptly gets measurement result.
(2) measure the concentration of the chlorophyll a of phytoplankton in this river water body for polishing:
Get 200ml water sample suction filtration (using the glass fiber filter of diameter 47mm, aperture 0.22 μ m and the composite fibre filter membrane of diameter 60mm, aperture 0.45 μ m to carry out suction filtration respectively), taking-up has the filter membrane of phytoplankton, freeze drying was put into mill after 8 hours under-18 ℃ of low temperature in refrigerator, add a small amount of carbonic acid magnesium dust and 5mL90% acetone, fully grinding (grinding approximately 15～20 minutes) is placed in the 10mL tool plug centrifuge tube, centrifugal 10min (4000r/min) pours supernatant in the 10mL volumetric flask into.In this centrifuge tube, add 5mL90% acetone again, place mill to continue milling and extracting then, centrifugal 10min, and supernatant is changed in the volumetric flask of aforementioned 10mL, with 90% acetone volumetric flask is settled to 10mL, shake up.Adopt the cuvette of 1cm light path, make blank absorbency with 90% acetone and measure, after the supernatant absorbance is proofreaied and correct, carry out the absorbance measurement of supernatant, read the absorbance D of 750nm, 663nm, 645nm, 630nm wavelength respectively
750, D
663, D
645, D
630Identical water sample triplicate operation is averaged, and promptly gets measurement result.
The concentration that polishing is measured the water phytoplankton chlorophyll a is calculated as follows:
In the formula: Chla---chlorophyll-a concentration (ug/L);
V---volume of water sample (L); V
1---the volume (mL) behind the extract constant volume;
D---absorbance; δ---cuvette light path (cm).
The result of two kinds of method mensuration is as shown in table 1.
Table 1 heat ethanol methods and polishing are measured phytoplankton chlorophyll-a concentration in the city river water body
Operating process by more above-mentioned two kinds of methods and measurement result adopt glass fiber filter to carry out the suction filtration effect and are better than the composite fibre filter membrane as can be known.The heat ethanol methods operating process is simple, and drug dosage is few, and extraction is complete, and identical water sample adopts same glass fibre to carry out suction filtration as filter membrane, and the concentration ratio polishing of the chlorophyll a that heat ethanol methods is measured exceeds 183.1%.Hence one can see that, heat ethanol methods the monitoring eutrophication water process in the gained measurement result than polishing more near actual conditions, will be with a wide range of applications.
Embodiment 2: Shanghai park water phytoplankton chlorophyll a is measured
Gather the water sample of Shanghai park water according to the chlorophyllous sampling method of phytoplankton in traditional detection water body, use heat ethanol methods of the present invention and traditional polishing to measure the concentration of the chlorophyll a of phytoplankton in the water body respectively.
(1) measure the concentration of the chlorophyll a of phytoplankton in the water body for heat ethanol methods:
Get 500ml water sample suction filtration and (use diameter 47mm, the glass fiber filter of aperture 0.22 μ m), filter membrane behind the suction filtration and phytoplankton attached to it are placed 10mL tool plug centrifuge tube, putting into-18 ℃ of refrigerator and cooled then froze 8 hours, take out tool plug centrifuge tube and return to 90% ethanol that adds 80 ℃ of 10mL behind the room temperature state, and in 80 ℃ of water-bath 2min, (ultrasonic frequency is 40kHz to ultrasonic again 10min, power is 150W), extracted 3 hours and be placed on the dark place, centrifugal then 10min (4000r/min) gets supernatant and carry out colorimetric with 90% ethanol as reference liquid on spectrophotometer.The concrete way of measuring the supernatant absorbance is: the cuvette that adopts the 1cm light path, at first making blank absorbency with 90% ethanol measures, the supernatant absorbance is proofreaied and correct, then begin to measure the absorbance of supernatant, survey supernatant absorptivity E at the 665nm wavelength earlier
665, survey supernatant absorptivity E at the 750nm wavelength again
750, in the cuvette of supernatant is housed, add 0.2 milliliter of 1mol/L hydrochloric acid then and carry out acidifying, add a cover and shake up, leave standstill behind the 1min absorptivity A of the supernatant after the 665nm wavelength is measured acidified processing again
665, survey its absorptivity A at the 750nm wavelength again
750, by the concentration of phytoplankton chlorophyll a in formula (1.1) the calculating water body, identical water sample triplicate operation is averaged, and promptly gets measurement result.
(2) measure the concentration of the chlorophyll a of phytoplankton in this river water body for polishing:
Get 200ml water sample suction filtration (using the glass fiber filter of diameter 47mm, aperture 0.22 μ m), taking-up has the filter membrane of phytoplankton, freeze drying was put into mill after 8 hours under-18 ℃ of low temperature in refrigerator, add a small amount of carbonic acid magnesium dust and 5mL90% acetone, fully grinding (grinding approximately 15～20 minutes) is placed in the 10mL tool plug centrifuge tube, centrifugal 10min (4000r/min) pours supernatant in the 10mL volumetric flask into.In this centrifuge tube, add 5mL90% acetone again, place mill to continue milling and extracting then, centrifugal 10min, and supernatant is changed in the volumetric flask of aforementioned 10mL, with 90% acetone volumetric flask is settled to 10mL, shake up.Adopt the cuvette of 1cm light path, make blank absorbency with 90% acetone and measure, after the supernatant absorbance is proofreaied and correct, carry out the absorbance measurement of supernatant, read the absorbance D of 750nm, 663nm, 645nm, 630nm wavelength respectively
750, D
663, D
645, D
630Identical water sample triplicate operation is averaged, and promptly gets measurement result.
The result of two kinds of method mensuration is as shown in table 2.
Table 2 heat ethanol methods and polishing are measured phytoplankton chlorophyll-a concentration in the park water
Operating process by above-mentioned two kinds of methods more as can be known, the heat ethanol methods operating process is simple, drug dosage is few, extraction fully, identical water sample, the concentration ratio polishing of the chlorophyll a that heat ethanol methods is measured exceeds 157.3%.
Above-mentioned two embodiment are compared as can be known, and chlorophyll-a concentration is high more, and the advantage of heat ethanol methods is obvious more, and this is because for the water sample of the chlorophyll a of higher concentration, when adopting polishing to measure, the turnover rate of chlorophyll a is higher in the process of lapping.In addition, 3 repetitive operations in embodiment 1 and embodiment 2, same water sample being carried out according to the inventive method, gained measurement result good reproducibility shows that this method possesses operational stability, can be widely used in research and the real work.
Above-mentioned description to embodiment is can understand and apply the invention for the ease of those skilled in the art.The person skilled in the art obviously can easily make various modifications to these embodiment, and needn't pass through performing creative labour being applied in the General Principle of this explanation among other embodiment.Therefore, the invention is not restricted to the embodiment here, those skilled in the art should be within protection scope of the present invention for improvement and modification that the present invention makes according to announcement of the present invention.
Claims (4)
1. the analysis determining method of phytoplankton chlorophyll a in the eutrophication water, concrete steps are as follows:
(1) pre-treatment of sample: the chlorophyllous sampling method according to phytoplankton in traditional detection water body is gathered water sample, the water sample of 200～500ml is passed through the nutsch filter suction filtration, filter membrane behind the suction filtration and phytoplankton attached to it are placed 10～25mL tool plug centrifuge tube, put into-18 ℃ of refrigerator and cooled then and froze 6～8 hours;
(2) extraction of sample: after the tool plug centrifuge tube that takes out step (1) from refrigerator returns to room temperature state, 90% ethanol that in centrifuge tube, adds 80 ℃ of 10mL, behind 80 ℃ of water-bath 2min, ultrasonic 10min in supersonic wave cleaning machine, then lucifuge leave standstill the extraction 3～5h after with the centrifugal 10min of the rotating speed of 4000r/min, it is to be measured to get supernatant;
(3) concentration of phytoplankton chlorophyll a is calculated in the mensuration of supernatant and the water body: get step (2) gained supernatant is measured the chlorophyll a of supernatant on spectrophotometer absorbance; At first make blank absorbency and measure, the supernatant absorbance is proofreaied and correct with 90% ethanol; Then begin to measure the absorbance of supernatant, survey supernatant absorptivity E at the 665nm wavelength earlier
665, survey supernatant absorptivity E at the 750nm wavelength again
750, in the cuvette of supernatant is housed, add 0.1～0.2 milliliter of 1.0mol/L hydrochloric acid then and carry out acidifying, add a cover and shake up, leave standstill behind the 1min absorptivity A of the supernatant after the 665nm wavelength is measured acidified processing again
665, survey its absorptivity A at the 750nm wavelength again
750, be calculated as follows the concentration of phytoplankton chlorophyll a in the water body:
In the formula: Chla---chlorophyll-a concentration (ug/L);
V
1---the volume (L) behind the alcohol extraction liquid constant volume;
V
2---the volume (L) of water sample;
E
665, A
665---the absorptivity at 665nm wavelength place;
E
750, A
750---the absorptivity at 750nm wavelength place.
2. analysis determining method according to claim 1, it is characterized in that: the filter membrane that adopts in the step (1) is the glass fiber filter of diameter 47mm, aperture 0.22 μ m, or the composite fibre filter membrane of homemade diameter 60mm, aperture 0.45 μ m, or the glass fiber filter of diameter 25mm, aperture 0.65 μ m.
3. analysis determining method according to claim 1 and 2 is characterized in that: the filter membrane that adopts in the step (1) is the glass fiber filter of diameter 47mm, aperture 0.22 μ m.
4. analysis determining method according to claim 1 and 2 is characterized in that: the ultrasonic frequency in the step (2) during sonicated is 40kHz, and power is 150W.
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| CN102590118A (en) * | 2012-02-27 | 2012-07-18 | 哈尔滨工业大学 | Method for measuring chlorophyll a in water |
| CN102706827A (en) * | 2012-07-02 | 2012-10-03 | 东北农业大学 | Novel extraction and determination method of content of chlorophyll by utilizing DMSO (dimethylsulfoxide) |
| CN103076297A (en) * | 2012-12-27 | 2013-05-01 | 河北科技大学 | Method for quickly and real-timely measuring water chlorophyll through replacing chlorophyll standard substance with microcystis aeruginosa |
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| CN102590118A (en) * | 2012-02-27 | 2012-07-18 | 哈尔滨工业大学 | Method for measuring chlorophyll a in water |
| CN102706827A (en) * | 2012-07-02 | 2012-10-03 | 东北农业大学 | Novel extraction and determination method of content of chlorophyll by utilizing DMSO (dimethylsulfoxide) |
| CN103076297A (en) * | 2012-12-27 | 2013-05-01 | 河北科技大学 | Method for quickly and real-timely measuring water chlorophyll through replacing chlorophyll standard substance with microcystis aeruginosa |
| CN104406922A (en) * | 2014-11-17 | 2015-03-11 | 临沂大学 | Method for determining content of trace elements in DunalieUa salina |
| CN104406922B (en) * | 2014-11-17 | 2017-05-24 | 临沂大学 | Method for determining content of trace elements in DunalieUa salina |
| CN106908395A (en) * | 2015-12-22 | 2017-06-30 | 南开大学 | The assay method of planktonic algae chlorophyll in water |
| CN106769903A (en) * | 2017-01-12 | 2017-05-31 | 南京渔管家物联网科技有限公司 | A kind of breeding water body algae concentration detection method |
| CN106769903B (en) * | 2017-01-12 | 2020-05-19 | 南京渔管家物联网科技有限公司 | Method for detecting concentration of algae in aquaculture water |
| KR101965979B1 (en) * | 2017-11-02 | 2019-04-04 | 서원대학교산학협력단 | Method for Extracting chlorophylls from Spirulina |
| CN107907494A (en) * | 2017-11-23 | 2018-04-13 | 上海国齐检测技术有限公司 | The assay method of leaf green a in a kind of water |
| CN108872107A (en) * | 2018-07-02 | 2018-11-23 | 湖北省农业科学院中药材研究所 | Plant maturation leaf pigment measuring method |
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