CN1631117A - Hydrobiontic algae chlorophyll measuring method - Google Patents
Hydrobiontic algae chlorophyll measuring method Download PDFInfo
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- CN1631117A CN1631117A CN 200410073542 CN200410073542A CN1631117A CN 1631117 A CN1631117 A CN 1631117A CN 200410073542 CN200410073542 CN 200410073542 CN 200410073542 A CN200410073542 A CN 200410073542A CN 1631117 A CN1631117 A CN 1631117A
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Abstract
The invention discloses a hydrobiontic algae chlorophyll measuring method which consists of, using spectrophotometry method to measure the algae chlorophyll content in the water, filtering subaqueous algae with cellulose acetate microporous filtering film, carrying out interception to the algae cellulose acetate through immersion with 90% ethanol, extracting the chlorophyll in algae cell into ethanol, centrifuging the tobacco extract to obtain the clarified solution, using 90% ethanol as reference, determining absorbancy of the tobacco extract at 630, 647, 664 and 750 nm with a spectrophotometer, calculating content of chlorophyll a, b, c in the water sample by utilizing the measured data.
Description
Technical field
The present invention relates to a kind of Hydrobiontic algae chlorophyll measuring method, particularly, be suitable for measuring algae chlorophyll a, chlorophyll b, Chlorofucsin in the water with the method for spectrophotometry Hydrobiontic algae chlorophyll.
Background technology
Body eutrophication, algal bloom cause water quality deterioration.In order to estimate water body eutrophication degree, reply algae content is measured.Algae is green plankton, and body contains chlorophyll, is used to carry out photosynthesis.Algae chlorophyll comprises chlorophyll a, chlorophyll b and Chlorofucsin.All include chlorophyll a in all alginites, its content accounts for 1～2% of organism dry weight, and the part algae also contains chlorophyll b, c.Chlorophyll a accounts for the chlorophyllous overwhelming majority, is that the energy in the whole photosynthesis process transmits the center.Therefore, can reflect the algae bio amount more exactly, can be used for weighing the degree of body eutrophication with chlorophyll, especially chlorophyll a.Measuring the chlorophyllous method of algae has three classes, i.e. spectrophotometric method, fluorescence method, high performance liquid chromatography.The required instrument and equipment of spectrophotometric method is simple and easy, and is easy and simple to handle, is the method that generally adopts at present.U.S.'s water quality monitoring standard method provides the Unite States Standard with algae chlorophyll content in the spectrophotometry water.Some water quality monitoring departments of China also use the method and measure, but this method agents useful for same volatilization is strong, and have toxicity, pollute working environment, the tester is healthy in harm, and the required filter membrane dependence on import of this method, testing time is long, the testing cost height.China does not also measure the national standard method of algae chlorophyll content in the water at present, and the method that domestic recommendation is used exists shortcomings such as the measurement result deviation is big, measurement result is unstable, poor reproducibility, pollution working environment.
Summary of the invention
The invention provides a kind of Hydrobiontic algae chlorophyll measuring method, this method utilization spectrophotometry Hydrobiontic algae chlorophyll, this method has been selected avirulent reagent and low-cost filter membrane for use, required instrument and equipment is simple and easy, easy and simple to handle, the testing time is short, and measurement result is accurate, stable, favorable reproducibility, testing expense is low, safety non-pollution.
For solving the problems of the technologies described above, the present invention is achieved in that
The present invention adopts algae in the acetate fiber filtering with microporous membrane water, grinds the cellulose acetate film of having held back algae with 90% alcohol immersion, the chlorophyll in the alga cells is extracted in the ethanol, with extract
CentrifugalObtain clarified solution, make reference with 90% ethanol, with the spectrophotometric determination extract 630,647,664, the absorbance at 750nm place, adopt chlorophyll a, b, c content in the computing formula calculating water.
Computing formula of the present invention is as follows:
Chla＝[12.12(D664-D750)-1.58(D647-D750)-0.08(D630-D750)]V
E/(Vsδ)
(1)
Chlb＝[-5.55(D664-D750)+21.51(D647-D750)-2.72(D630-D750)]V
E/(Vsδ)
(2)
Chlc＝[-1.71(D664-D750)-7.77(D647-D750)+25.08(D630-D750)]V
E/(Vsδ)
(3)
Chla, Chlb, Chlc in the formula---be respectively the concentration of chlorophyll a in the water sample, b, c, ug/L;
D630, D647, D664, D750---be respectively the absorbance of extract at wavelength 630,647,664,750nm place;
V
E-
CentrifugalThe constant volume of extract in the pipe, ml;
V
s-volume of water sample, L;
δ-cuvette light path, cm.
Algae is under vacuum condition in the described employing acetate fiber filtering with microporous membrane water.
Considering the MgCO3 suspension that drips at least one 1% on the film before the described acetate fiber filtering with microporous membrane.
Described acetate fiber miillpore filter adopts 0.8 μ m aperture.
The present invention is as follows compared with the prior art:
The present domestic generally method (being called for short domestic method) of employing that provides in " water and the effluent monitoring analytical method " book that has American National Standard method (abbreviation American process) and State Bureau of Environmental Protection to write with the similar method of the present invention shows the present invention and this two kinds of methods is compared.
1, compare with American process:
Aspect method of operating, distinguish and be: (1) extract difference.The American process extract is 90% acetone.(2) filter membrane difference.American process adopts glass fibre membrane.(3) the testing time difference of Xu Yaoing.The extract of American process need be placed 2～24 hours under low temperature (0～4 ℃) condition, and make chlorophyll in the alga cells be extracted liquid and fully extract, and then centrifugal mensuration.Extract extractability of the present invention is strong, can fully extract chlorophyll at short notice, does not need time-delay to extract, can direct centrifugal mensuration.(4) computing formula difference.Because 90% acetone is different with the specific absorption coefficient of 90% ethanol, thereby the computing formula difference.The computing formula of American process is:
Chla＝[11.85(D664-D750)-1.54(D647-D750)-0.08(D630-D750)]V
E/(Vsδ)???(4)
Chlb＝[-5.43(D664-D750)+21.03(D647-D750)-2.66(D630-D750)]V
E/(Vsδ)??(5)
Chlc＝[-1.67(D664-D750)-7.6(D647-D750)+24.52(D630-D750)]V
E/(Vsδ)???(6)
2, compare with domestic method:
Difference is: (1) extract difference, domestic method extract is 90% acetone.(2) operation sequence difference is placed on the interior low temperature drying of refrigerator 6～8 hours behind the membrane filtration of domestic method, take out to add the extract grinding again.(3) the testing time difference of Xu Yaoing, domestic method need increase by 6～8 hours low temperature drying time.(4) measure the wavelength difference.Domestic method measures that wavelength is 630,645,663,750nm.(5) computing formula difference, the computing formula of domestic method is:
Chla＝[11.64(D663-D750)-2.16(D645-D750)+0.1(D630-D750)]V
E/(Vsδ)????(7)
Chlb＝[-3.94(D663-D750)+20.97(D645-D750)-3.66(D630-D750)]V
E/(Vsδ)??(8)
Chlc＝[-5.53(D663-D750)-14.18(D645-D750)+54.22(D630-D750)]V
E/(Vsδ)?(9)
3, the comparison aspect the mensuration effect:
(1) measurement result of the present invention is stable, favorable reproducibility.To carrying out replication 10 times with three kinds of methods respectively with a water sample, measurement result sees Table 1.As can be seen from Table 1, the standard deviation and the American process of measurement result of the present invention are suitable, and be more much smaller than domestic method, and reappearance has had and increases substantially.
Each assay method reappearance of table 1 relatively
| The repeat sample numbering | ?????????????Chla(ug/L) | Total chlorophyll (ug/L) | ||||
| The present invention | American process | Domestic method | The present invention | American process | Domestic method | |
| ????1 | ????59.1 | ????56.7 | ????52.1 | ????67.5 | ????60.5 | ????63.4 |
| ????2 | ????58.3 | ????57.9 | ????55.5 | ????66.7 | ????62.1 | ????68.1 |
| ????3 | ????58.2 | ????58.6 | ????55.5 | ????65.7 | ????62.9 | ????68.1 |
| ????4 | ????60.6 | ????57.8 | ????51.0 | ????67.4 | ????61.7 | ????62.8 |
| ????5 | ????61.4 | ????57.1 | ????54.5 | ????69.6 | ????61.5 | ????70.0 |
| ????6 | ????61.9 | ????54.5 | ????50.3 | ????69.6 | ????59.2 | ????61.1 |
| ????7 | ????60.7 | ????59.1 | ????47.4 | ????68.9 | ????63.5 | ????58.3 |
| ????8 | ????59.8 | ????60.4 | ????58.4 | ????67.3 | ????65.2 | ????71.1 |
| ????9 | ????61.4 | ????57.7 | ????57.6 | ????70.4 | ????62.5 | ????69.7 |
| ????10 | ????57.5 | ????57.8 | ????54.1 | ????64.3 | ????61.4 | ????66.7 |
| On average | ????59.9 | ????57.8 | ????53.6 | ????67.8 | ????62.1 | ????65.9 |
| Standard deviation | ????1.47 | ????1.47 | ????3.25 | ????1.8 | ????1.6 | ????4.1 |
| Relative standard deviation (%) | ????2.4 | ????2.5 | ????6.1 | ????2.7 | ????2.6 | ????6.2 |
Annotate: total chlorophyll be Chla, Chlb, Chlc's and.
(2) measurement result of the present invention is accurate.Selected the water sample of a series of different chlorophyll concentrations, measured with three kinds of methods respectively, measurement result is listed in table 2.Can find out from table 2, the measurement result basically identical of the present invention and Unite States Standard method, the average relative error of the chlorophyll a of 12 samples only is 0.55%, total chlorophyllous average relative error also has only 2.5%.And the measurement result of domestic method and Unite States Standard method differs bigger, and the average relative error of the chlorophyll a of 6 samples is-19.7%, and total chlorophyllous average relative error is-3.45%.The order of accuarcy of measurement result of the present invention has been compared with domestic method largely and has been improved.
Each assay method measurement result of table 2 relatively
| Specimen coding | ?????????Chla(ug/L) | Total chlorophyll (ug/L) | Chla is with respect to the relative error (%) of American process | Total chlorophyll is with respect to the relative error (%) of American process | ||||||
| The present invention | American process | Domestic method | The present invention | American process | Domestic method | The present invention | Domestic method | The present invention | Domestic method | |
| ????1 | ??32.8 | ??35.1 | ??23.6 | ??36.3 | ??36.8 | ??32.6 | ??-6.6 | ??-32.8 | ??-1.4 | ??-11.4 |
| ????2 | ??45.6 | ??44.9 | ??34.9 | ??49.4 | ??46.6 | ??43.2 | ??1.6 | ??-22.3 | ??6 | ??-7.3 |
| ????3 | ??59.8 | ??57.7 | ??53.8 | ??66 | ??61.6 | ??66.5 | ??3.6 | ??-6.8 | ??7.1 | ??8 |
| ????4 | ??64.8 | ??60.8 | ??49.2 | ??69.3 | ??63.2 | ??67.1 | ??6.6 | ??-19.1 | ??9.7 | ??6.2 |
| ????5 | ??85.6 | ??89.6 | ??69.8 | ??93.7 | ??94.7 | ??85.8 | ??-4.5 | ??-22.1 | ??-1.1 | ??-9.4 |
| ????6 | ??125.3 | ??127.3 | ?108.1 | ??136.1 | ??135.3 | ??126.1 | ??-1.6 | ??-15.1 | ??0.6 | ??-6.8 |
| ????7 | ??27.1 | ??26 | ??31.3 | ??29.6 | ??4.2 | ??5.7 | ||||
| ????8 | ??28.7 | ??30.1 | ??34 | ??34.3 | ??-4.7 | ??-0.8 | ||||
| ????9 | ??31.7 | ??32.4 | ??37.5 | ??36.4 | ??-2.2 | ??3 | ||||
| ????10 | ??53.7 | ??53 | ??59 | ??61.7 | ??1.3 | ??-1.8 | ||||
| ????11 | ??84.2 | ??84.2 | ??90.8 | ??87.6 | ??0 | ??3.7 | ||||
| ????12 | ??127.4 | ??133.1 | ??143.5 | ??145.1 | ??-4.3 | ??-1.1 | ||||
| On average | ??0.55 | ??-19.7 | ??2.5 | ??-3.45 | ||||||
Comprehensive above-mentioned comparative analysis can draw and the invention has the beneficial effects as follows: (1) measurement result is accurately stable, favorable reproducibility.Measurement result of the present invention and reappearance thereof have all reached the level of Unite States Standard method, and the method for using than domestic recommendation has had and increases substantially.(2) safety non-pollution.The present invention adopts 90% ethanol to make extract, has avoided in other two kinds of methods acetone extract to the injury of human body.(3) test is rapid, weak point consuming time.It is 0.5 hour that the present invention measures a required time of sample, and American process needs 2.5 hours at least, and domestic method needs 6.5 hours at least.(4) equipment needed thereby is few, and testing expense is low.Because the present invention does not need deepfreeze, therefore can save required refrigerating equipment in other two method.The present invention has adopted cellulose acetate film, and testing expense only is 1/5 of an American process.
Embodiment
The present invention includes following steps:
(1) algae concentrates.With the acetate fiber miillpore filter in 0.8um aperture, full glass vacuum filters device and vavuum pump filters the water sample that contains algae, and algae is trapped on the miillpore filter, drips some 1%MgCO on the film considering before filtering
3Suspension.
(2) pigment extracts.Ethanol extract with 90% soaks and grinds the filter membrane of having held back algae, chlorophyll is extracted mention in the ethanol, extract is poured in the centrifuge tube again, and constant volume is to several milliliters.
(3) centrifugation.It is centrifugal in the centrifuge that the centrifuge tube that fills the chlorophyll extract is packed into, makes the filter membrane paper scrap that is ground be deposited at the bottom of the centrifuge tube, obtains the extract of clarification.
(4) measure.The extract of clarification is poured in the cuvette, made reference with 90% ethanol, the mensuration extract is 630,647,664 and the absorbance at 750nm wavelength place on spectrophotometer.
(5) calculate.According to absorbance, volume of water sample, the extract volume at each the wavelength place that records, calculate the concentration of chlorophyll a, b, c in the water sample.Computing formula is:
Chla＝[12.12(D664-D750)-1.58(D647-D750)-0.08(D630-D750)]V
E/(Vsδ)
(1)
Chlb＝[-5.55(D664-D750)+21.51(D647-D750)-2.72(D630-D750)]V
E/(Vsδ)
(2)
Chlc＝[-1.71(D664-D750)-7.77(D647-D750)+25.08(D630-D750)]V
E/(Vsδ)
(3)
Chla, Chlb, Chlc in the formula---be respectively the concentration of chlorophyll a in the water sample, b, c, ug/L;
D630, D647, D664, D750---be respectively the absorbance of extract at wavelength 630,647,664,750nm place;
V
E---the constant volume of extract in the centrifuge tube, ml;
V
s---volume of water sample, L;
δ---cuvette light path, cm.
Embodiment:
1, equipment and reagent
(1) vavuum pump, vacuum is not more than 0.05MPa;
(2) full glass vacuum filtration device;
(3) magnetic substance or agate mortar;
(4) medical centrifuge, rotating speed 3500～4000r/min, load capacity 500～675g;
(5) centrifuge tube, 15mL, band scale division and nut;
(6) spectrophotometer, minimum scale 0.5～2nm;
(7) cuvette, light path 1～3cm;
(8) cellulose acetate film, diameter 50mm, aperture 0.8～1.0 μ m;
(9) 90% ethanol waters (volume ratio);
(10) 1%MgCO
3Suspension: in 100ml distilled water, add 1.0g MgCO
3Powder;
2, mensuration program
(1) sample is prepared.At underwater certain depth water sampling 500mL, if algae content height, the water yield can be cut down according to the circumstance.Because of algae is bigger in different depth of waters place content difference, answer the accurate recording sample point depth of water during sampling.Should carry out next step mensuration program as early as possible after the sampling,, can under lucifuge and low temperature (0～4 ℃) condition, preserve 24h if water sample needs to preserve.
(2) algae is filtered.With the cellulose acetate film full glass vacuum filtration device of packing into, the wet look of filter membrane up.Connect vavuum pump, on filter membrane, evenly drip several MgCO
3Suspension is measured 500ml water sample (algae content Gao Shike cuts down according to the circumstance) with graduated cylinder, pours in the suction filtration device.Treat that water sample is all by suction filtration 1 minute again behind the filter membrane, to filter the moisture on the dried filter membrane.Stop suction filtration, take off filter membrane, filter membrane is folding to a side of having held back algae, place it in the folding filter paper, to blot the moisture on the filter membrane.
(3) chlorophyll extracts.With scissors filter membrane is shredded, put into mortar, add 2～3mL90% ethanol, ground 2～3 minutes, help the chlorophyll in the extract extraction frustule, will extract chlorophyllous extract and pour in the centrifuge tube.In mortar, add 2～3mL90% ethanol again, ground 1～2 minute, bleach, illustrate that chlorophyll all is dissolved in the extract, pours extract in the centrifuge tube into together with filter membrane up to filter membrane.Rinse with 90% ethanol and to wash mortar 1～2 time, incorporate centrifuge tube into.With 90% ethanol with extract constant volume to 8～10mL in the centrifuge tube.
(4) centrifugal.Build centrifuge tube with nut, put into centrifuge, in the 500g centrifuge centrifugal 20～25 minutes, or in the 675g centrifuge centrifugal 15～20 minutes.
(5) measure.The careful centrifuge tube that takes out is poured supernatant in the cuvette into, makes reference with 90% ethanol, 630,647,664,750nm wavelength place measures absorbance.The reflection of the absorbance at 750nm place be the muddy degree of extract, this value should not surpass 0.005, if surpass this value, should measure centrifugal again back.
(6) calculate.According to absorbance, cuvette light path, volume of water sample, the extract constant volume of extract, utilize formula (1), (2), (3) to calculate the content of chlorophyll a, b, c in the water at each wavelength place.
Claims (5)
1, a kind of Hydrobiontic algae chlorophyll measuring method, it is characterized in that: adopt algae in the acetate fiber filtering with microporous membrane water, grind the cellulose acetate film of having held back algae with 90% alcohol immersion, chlorophyll in the alga cells is extracted in the ethanol, with the centrifugal acquisition clarified solution of extract, make reference with 90% ethanol, with the spectrophotometric determination extract 630,647,664, the absorbance at 750nm place, adopt chlorophyll a, b, c content in the computing formula calculating water.
2, Hydrobiontic algae chlorophyll measuring method according to claim 1, it is characterized in that: computing formula is as follows:
Chla＝[12.12(D664-D750)-1.58(D647-D750)-0.08(D630-D750)]V
E/(Vsδ)????(1)
Chlb＝[-5.55(D664-D750)+21.51(D647-D750)-2.72(D630-D750)]V
E/(Vsδ)???(2)
Chlc＝[-1.71(D664-D750)-7.77(D647-D750)+25.08(D630-D750)]V
E/(Vsδ)???(3)
Chla, Chlb, Chlc in the formula---be respectively the concentration of chlorophyll a in the water sample, b, c, ug/L;
D630, D647, D664, D750---be respectively the absorbance of extract at wavelength 630,647,664,750nm place;
V
E---
CentrifugalThe constant volume of extract in the pipe, ml;
V
s---volume of water sample, L;
δ---cuvette light path, cm.
3, Hydrobiontic algae chlorophyll measuring method according to claim 1 is characterized in that: algae is under vacuum condition in the described employing acetate fiber filtering with microporous membrane water.
4, Hydrobiontic algae chlorophyll measuring method according to claim 1 is characterized in that: considering the MgCO that drips at least one 1% on the film before the described acetate fiber filtering with microporous membrane
3Suspension.
5, Hydrobiontic algae chlorophyll measuring method according to claim 1 is characterized in that: described acetate fiber miillpore filter adopts 0.8 μ m aperture.
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| CN 200410073542 CN1631117A (en) | 2004-12-30 | 2004-12-30 | Hydrobiontic algae chlorophyll measuring method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101303337A (en) * | 2008-07-09 | 2008-11-12 | 刘静玲 | Method for evaluating water nutrition state |
| CN102532146A (en) * | 2010-12-13 | 2012-07-04 | 依诺特生物能量控股公司 | Methods for extracting chlorophyll and detecting chlorophyll concentration |
| CN102564967A (en) * | 2010-12-13 | 2012-07-11 | 依诺特生物能量控股公司 | Chlorophyll fluorescent spectroscopy analyzer and method for measuring chlorophyll concentration |
| 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) |
| CN104629488A (en) * | 2014-12-29 | 2015-05-20 | 浙江海洋学院 | Method for extracting cytochrome from microalgae liquid |
| CN105021789A (en) * | 2015-07-29 | 2015-11-04 | 苏州翠浔水生态科技有限公司 | Method for quickly detecting standing stock of blue-green algae |
| CN106908395A (en) * | 2015-12-22 | 2017-06-30 | 南开大学 | The assay method of planktonic algae chlorophyll in water |
| 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 |
| CN108387535A (en) * | 2018-01-24 | 2018-08-10 | 河海大学 | A kind of computational methods of lake sediment Chlorophyll-a Content |
| CN109612945A (en) * | 2019-01-10 | 2019-04-12 | 深圳市周行环保科技有限公司 | A kind of method of algae content in quick detection water |
| CN113607649A (en) * | 2020-09-18 | 2021-11-05 | 中国科学院植物研究所 | Method for improving algae P700 detection signal |
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2004
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101303337A (en) * | 2008-07-09 | 2008-11-12 | 刘静玲 | Method for evaluating water nutrition state |
| CN102532146A (en) * | 2010-12-13 | 2012-07-04 | 依诺特生物能量控股公司 | Methods for extracting chlorophyll and detecting chlorophyll concentration |
| CN102564967A (en) * | 2010-12-13 | 2012-07-11 | 依诺特生物能量控股公司 | Chlorophyll fluorescent spectroscopy analyzer and method for measuring chlorophyll concentration |
| 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) |
| CN104629488A (en) * | 2014-12-29 | 2015-05-20 | 浙江海洋学院 | Method for extracting cytochrome from microalgae liquid |
| CN105021789A (en) * | 2015-07-29 | 2015-11-04 | 苏州翠浔水生态科技有限公司 | Method for quickly detecting standing stock of blue-green algae |
| CN106908395A (en) * | 2015-12-22 | 2017-06-30 | 南开大学 | The assay method of planktonic algae chlorophyll in water |
| 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 |
| CN108387535A (en) * | 2018-01-24 | 2018-08-10 | 河海大学 | A kind of computational methods of lake sediment Chlorophyll-a Content |
| CN109612945A (en) * | 2019-01-10 | 2019-04-12 | 深圳市周行环保科技有限公司 | A kind of method of algae content in quick detection water |
| CN113607649A (en) * | 2020-09-18 | 2021-11-05 | 中国科学院植物研究所 | Method for improving algae P700 detection signal |
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