CN108956501A - A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll - Google Patents

A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll Download PDF

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CN108956501A
CN108956501A CN201810983922.7A CN201810983922A CN108956501A CN 108956501 A CN108956501 A CN 108956501A CN 201810983922 A CN201810983922 A CN 201810983922A CN 108956501 A CN108956501 A CN 108956501A
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chlorophyll
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alga cells
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代堂刚
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising

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Abstract

The invention discloses a kind of spectrophotometric methods using ultrasonic disruption alga cells measurement chlorophyll, method includes the following steps: pretreatment, chlorophyll are extracted and spectrophotometry is measured and calculated.The invention has the following advantages: carrying out clasmatosis using the cavitation effect of ultrasonic wave and its mechanical oscillation, clasmatosis progress can be accelerated, be crushed more complete chlorophyll effectively in the most of alga cells of extraction.When correcting pheophytin a, the absorbance in the absorption cell of measurement acidification front and back, substitutes into formula and calculates respectively, and to correct interference of the de- U.S. chlorophyll a to chlorophyll a, this method makes the result for measuring chlorophyll more accurate.It does not need to be operated under freezing conditions, planktonic algae cell degeneration will not be made, can effectively shorten the broken time, improve broken recovery rate, can handle a large amount of cells, with good market promotion prospect.

Description

A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll
Technical field
The invention belongs to algological biology fields, and in particular to a kind of to measure chlorophyll using ultrasonic disruption alga cells Spectrophotometric method.
Background technique
The raised growth of algae is the principal phenomena of water eutrophication, and wherein chlorophyll a is all phytoplanktons The chlorophyll type that class all contains, serves not only as the important indicator of water nutrition state division, and can be used for characterizing and swim The standing crop of plant, therefore, chlorophyll a serve key in water eutrophication status evaluation.
Algae belongs to microphyte in water, to extract its chlorophyll a and mostly use chemical-agent technique, polishing, anti-greatly Multiple freeze-thaw method carries out clasmatosis processing, but chemical-agent technique, to the toxic effect of cell, the time is long, low efficiency；Polishing Time-consuming, labor intensive, grinding is insufficient, and it is low to be crushed recovery rate；Multigelation method is by the way that cell to be placed under extreme low temperature With expand with heat and contract with cold under room temperature being crushed, time-consuming, be crushed recovery rate it is low, under low temperature condition be easy so that cell degeneration.
Pheophytin a can interfere the measurement of chlorophyll a, and at present in the prior art, good method does not go correction de- The interference of magnesium pheophytin a, to will affect measurement result in measurement.
Therefore, it is necessary to study a kind of methods of efficient broken alga cells and Accurate Determining water quality chlorophyll.
Summary of the invention
The purpose of the present invention is to provide a kind of spectrophotometric sides using ultrasonic disruption alga cells measurement chlorophyll Method.
The object of the present invention is achieved like this, comprising the following steps:
1) pre-process: magnesium carbonate suspension is added in brown or dark bottles in acquisition earth's surface water sample, mixes, takes determined volume Mixing water sample, be put into the Vacuum filtration device with filter membrane and carry out suction filtration processing, obtain alga cells；
2) chlorophyll extracts: will use ultrasonic disruption by the alga cells filtered, adds 10mL acetone soln, be placed in It is spare that 4 ~ 12h of immersion is protected from light at a temperature of 4 DEG C, shake 3 ~ 4 times again, are centrifuged using centrifuge later, take supernatant during immersion；
3) spectrophotometry measurement and calculating: the supernatant obtained by centrifugation is poured into cuvette, using spectrophotometer It is measured under the wavelength of 750nm, 664nm, 647nm, 630nm, is calculated in surface water body according to three colour equation formula respectively Chlorophyll a, b, the c of planktonic algae cell, then pheophytin correction content is measured, and calculate de-magging leaf with monochromatic equation formulations Green element correction content.
Compared with prior art, the present invention has following technical effect that
1, clasmatosis is carried out using the cavitation effect of ultrasonic wave and its mechanical oscillation, clasmatosis progress can be accelerated, be crushed It is more complete, effectively extract the chlorophyll in most of alga cells.
2, when correcting pheophytin a, the absorbance in the absorption cell of measurement acidification front and back, substitutes into formula and falls into a trap respectively It calculates, to correct interference of the de- U.S. chlorophyll a to chlorophyll a, this method makes the result for measuring chlorophyll more accurate.
3, it does not need to be operated under freezing conditions, planktonic algae cell degeneration will not be made, can effectively shorten broken Time improves broken recovery rate, can handle a large amount of cells, has good market promotion prospect.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated, but the present invention is limited in any way, Based on present invention teach that it is made it is any transform or replace, all belong to the scope of protection of the present invention.
A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll, comprising the following steps:
1) pre-process: magnesium carbonate suspension is added in brown or dark bottles in acquisition earth's surface water sample, mixes, takes determined volume Mixing water sample, be put into the Vacuum filtration device with filter membrane and carry out suction filtration processing, obtain alga cells；
2) chlorophyll extracts: will use ultrasonic disruption by the alga cells filtered, adds 10mL acetone soln, be placed in It is spare that 4 ~ 12h of immersion is protected from light at a temperature of 4 DEG C, shake 3 ~ 4 times again, are centrifuged using centrifuge later, take supernatant during immersion；
3) spectrophotometry measurement and calculating: the supernatant obtained by centrifugation is poured into cuvette, using spectrophotometer It is measured under the wavelength of 750nm, 664nm, 647nm, 630nm, is calculated in surface water body according to three colour equation formula respectively Chlorophyll a, b, the c of planktonic algae cell, then pheophytin correction content is measured, and calculate de-magging leaf with monochromatic equation formulations Green element correction content.
Further, the resulting alga cells needs of filtering are protected from light and save at a temperature of -20 DEG C in the step 1), Low-temperature transport；Filtered alga cells need to wrap up one layer of aluminium foil in outside, save in -20 DEG C of following temperature, and in Analysis is completed in 25d.
Further, the magnesium carbonate suspension in the step 1) is the magnesium carbonate suspension of concentration 1%, and additional amount is 1mL is added in every liter of water sample, and the preparation method of the magnesium carbonate suspension is to take 1.0g magnesium carbonate powder, adds people's 100mL pure water, Suspension is stirred into, is sufficiently shaken up when using every time.
Further, the filter membrane in the step 1) is glass fiber filter, and diameter 47mm, aperture are 0.7 μm；Institute The negative pressure of suction filtration in the step 1) stated is less than 20kPa, gradually depressurizes, terminates to filter when water sample just passes through filter membrane completely, Filter membrane is taken out, there will be the one side doubling of sample, blot residual moisture with filter paper.
Further, the ultrasonic disruption in the step 2) is that ultrasound is carried out using ultrasonic cleaner, ultrasonic Frequency is 50 ~ 200kHz, and the processing time is 10 ~ 30min；Acetone soln concentration in the step 2) is 90%, preparation method To add 100mL pure water in 900mL acetone；The centrifugal speed of centrifuge in the step 2) is 3500 ~ 4200r/min, from The heart time is 10 ~ 30min.
Further, the spectrophotometry in the step 3) specifically uses light path for the cuvette of 1cm, 90% acetone Solutions as controls liquid carries out colorimetric, and spectrophotometer uses wavelength for the spectrophotometer of 380 ~ 780nm, during being measured Points for attention be measure 750nm wavelength at absorbance be greater than 0.005, need to aperture be 0.45 μm polytetrafluoroethylene (PTFE) it is organic Phase syringe filter is measuring after being filtered.
Further, three colour equation formula in the step 3) are as follows:
Ρchl-a=[11.85(A647-A750)-1.54(A664-A750)-0.08(A630-A750)] V1/( V2L),
Ρchl-b=[21.03(A647-A750)-5.43(A664-A750)-2.66(A630-A750)] V1/( V2L),
Ρchl-c=[24.52(A630-A750)-7.60(A647-A750)-1.67(A664-A750)] V1/( V2L);
Wherein, Pchl-a is the mass concentration of water sample Determination of Chlorophyll a, and unit is μ g/L, and Pchl-b is water sample Determination of Chlorophyll b's Mass concentration, unit are μ g/L, and Ρ chl-c is the mass concentration of water sample Determination of Chlorophyll c, and unit is μ g/L, A750For extracting solution Absorbance value at wavelength 750nm, A664For absorbance value of the extracting solution at wavelength 664nm, A647It is extracting solution in wavelength Absorbance value at 647nm, A630For absorbance value of the extracting solution at wavelength 630nm, V1For extracting liquid volume, unit mL, V2For volume of water sample, unit L；L is cuvette light path, unit cm.
Further, the monochromatic equation formulations in the step 3) calculate the concrete operations of pheophytin correction content Step is the hydrochloric acid solution that the 0.1mol/L of 40 μ L is added dropwise into the 1cm cuvette equipped with centrifuged supernatant, and the processing time is 10 Then ~ 30min measures absorbance at 750nm, 665nm.
Further, the monochromatic equation formulations in the step 3) are as follows:
Ρ,chl-a=26.7 [ (A664- A750)- (A665a-A750a)] V1/ (V2L),
Ρphe-a=26.7 [1.7 (A665a-A750a)- (A664-A750)] V1/ (V2L)；
Wherein, P,Chl-a is the mass concentration of chlorophyll a after correcting pheophytin a in water sample, and unit is μ g/L；Pphe-a For the mass concentration of pheophytin a in water sample, unit is μ g/L；A750For the suction before extracting solution acidification at wavelength 750nm Shading value；A664For the absorbance value before extracting solution acidification at wavelength 664nm；A750aIn wavelength 750nm after being acidified for extracting solution The absorbance value at place；A665aFor the absorbance value after extracting solution acidification at wavelength 665nm；V1For extracting liquid volume, unit is mL；V2For volume of water sample, unit L；L is cuvette light path, unit cm.
Embodiment 1
A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll, comprising the following steps:
1. the scope of application
This method provides the spectrophotometry of measurement water Determination of Chlorophyll.
This standard is suitable for the measurement of surface water Determination of Chlorophyll.
Volume of water sample is 300mL, and when using 1cm cuvette, the detection of chlorophyll a is limited to 0.11 μ g/L, and Determination Limit is 0. 5μg/L；The detection of chlorophyll b is limited to 0.25 μ g/L, and Determination Limit is 1.0 μ g/L；The detection of Chlorofucsin is limited to 0.25 μ g/ L. Determination Limit is 1.0 μ g/L.
2. Method And Principle
A certain amount of water sample is filtered with glass fibre membrane, algae is collected, alga cells is crushed using ultrasonic wave, with 90% Acetone soln extracts chlorophyll, according under chlorophyll spectral characteristic sequentially determining 750nm, 664nm, 647nm, 630nm wavelength Absorbance calculates the content of chlorophyll.
3 interference and elimination
Pheophytin a can interfere the measurement of chlorophyll a, should be while measuring chlorophyll a when containing pheophytin a The content of pheophytin a is measured, to correct interference.
When correcting pheophytin a, the absorbance in the absorption cell of measurement acidification front and back, substitutes into formula and calculates respectively, with Interference of the de- U.S. chlorophyll a of correction to chlorophyll a.
4. reagent and material
Unless otherwise indicated, analytical reagents that meet national standards and pure water are used when analysis.
4.1 magnesium carbonate suspension: w (MgCO3)=1%, people 100mL pure water, stirring are added in 1.0g magnesium carbonate powder At suspension.It should sufficiently be shaken up when using every time.
4.2 acetone solns: (C3H6O)=90%, 100mL pure water is added in 900mL acetone.
4.3 hydrochloric acid solutions: 8.5ml concentrated hydrochloric acid is added people into 500ml pure water, is cooled to room by c (HC1)=0.1mol/L 1000mL is diluted to after temperature.
5. instrument and equipment
A grades of volumetric glass that meet national standards is used when analysis.
5.1 visible spectrophotometer.
5.2 nutsch filter.
5.3 miillpore filters: the glass fiber filter that diameter 47mm, aperture are 0.7 μm.
5.4 ultrasonic cleaner.
5.5 centrifuges: revolving speed can reach 3500 ~ 4200r/min, use the centrifuge with temperature regulating device.
5.6 culture dishes: for holding, shifting filter membrane.
5.7 tool plug glass centrifuge tubes: 10mL or 15mL.
5.8 aluminium foil.
5.9 tweezers.
5.10 syringe filters: the polytetrafluoroethylene (PTFE) organic phase syringe filter in 0.45 μm of aperture.
5.11 laboratory common instrument.
6. the acquisition and preservation of water sample
The acquisition of 6.1 water samples
According to different water bodys, 1000ml water sample is acquired in dark colored plastic bottle, adds the 1% magnesium carbonate suspension of people 1mL, to prevent Pigmentolysis caused by being only acidified.
The preservation of 6.2 water samples
Water sample should be kept in dark place, low-temperature transport.After sampling for 24 hours in glass fiber filter filter water sample, filtered filter membrane- It saves in 20 DEG C of refrigerators below, and is completed in analysis in 25d.
7. analytical procedure
7.1 filtering
Miillpore filter (see 5.3) is placed on the nutsch filter for being connected with vacuum pump, it is quasi- according to the concentration of water sample Determination of Chlorophyll The mixing water sample for really measuring determined volume is filtered, and negative pressure is 15kPa when suction filtration, is gradually depressurized, just completely logical in water sample Terminate to filter when filter membrane.Carefully filter membrane is taken out with tweezers, there will be the one side doubling of sample, blot residual moisture with filter paper.
If sample cannot extract in time, the filter membrane of suck dry moisture should be put in people's culture dish, one layer of aluminium foil is wrapped up in outside , put in -20 DEG C of people refrigerators below and save.
7.2 extracting
Filtered filter membrane is put people to have in plug glass centrifuge tube, covers tightly cock cap, is put into ultrasonic cleaner ultrasound, ultrasonic frequency Rate is 200kHz, and the processing time is 20min, and the concentration that 10mL is added into centrifuge tube is 90% acetone soln, and it is violent to cover tightly cock cap Shake a moment is placed in 4 DEG C of refrigerators to be protected from light and impregnates 8h, should shake 3 times again in soaking process.
7.3 centrifugation
Centrifuge tube is put in people's centrifuge, 20min is centrifuged with the speed of 4200r/min.
7.4 measurement
By in the supernatant 1cm of the falling people cuvette after centrifugation, reference is done with 90% acetone soln, respectively 750nm, 664nm, Absorbance value is measured at 647nm, 630nm wavelength.
When containing pheophytin a, the content of pheophytin a should be measured while measuring chlorophyll a.Specifically do Method is: be added dropwise 40 μ L hydrochloric acid solutions into the 1cm cuvette equipped with centrifuged supernatant, be acidified after 20min measure 750nm, Absorbance value at 665nm wavelength.
8 results calculate
8.1 chlorophyll calculation formula
The concentration of water body Determination of Chlorophyll is calculated according to the following formula:
Ρchl-a=[11.85(A647-A750)-1.54(A664-A750)-0.08(A630-A750)] V1L/( V2L),
Ρchl-b=[21.03(A647-A750)-5.43(A664-A750)-2.66(A630-A750)] V1L/( V2L),
Ρchl-c=[24.52(A630-A750)-7.60(A647-A750)-1.67(A664-A750)] V1L/( V2L);
Wherein, Pchl-a is the mass concentration of water sample Determination of Chlorophyll a, and unit μ g/L, Pchl-b are the matter of water sample Determination of Chlorophyll b Concentration is measured, unit μ g/L, Ρ chl-c is the mass concentration of water sample Determination of Chlorophyll c, unit μ g/L, A750It is extracting solution in wavelength Absorbance value at 750nm, A664For absorbance value of the extracting solution at wavelength 664nm, A647It is extracting solution at wavelength 647nm Absorbance value, A630For absorbance value of the extracting solution at wavelength 630nm, V1For extracting liquid volume, unit mL, V2For water sample Volume, unit L；L is cuvette light path, unit cm.
The calculation formula of 8.2 correction pheophytin a
The concentration of chlorophyll a and middle pheophytin a after correcting pheophytin a are calculated according to the following formula:
Ρ,chl-a=26.7 [ (A664- A750)- (A665a-A750a)] V1/ (V2L),
Ρphe-a=26.7 [1.7 (A665a-A750a)- (A664-A750)] V1/ (V2L)；
Wherein, P,Chl-a is the mass concentration of chlorophyll a after correcting pheophytin a in water sample, and unit is μ g/L；Pphe-a For the mass concentration of pheophytin a in water sample, unit is μ g/L；A750For the suction before extracting solution acidification at wavelength 750nm Shading value；A664For the absorbance value before extracting solution acidification at wavelength 664nm；A750aIn wavelength 750nm after being acidified for extracting solution The absorbance value at place；A665aFor the absorbance value after extracting solution acidification at wavelength 665nm；V1For extracting liquid volume, unit is mL；V2For volume of water sample, unit L；L is cuvette light path, unit cm.
The testing result of 1 chlorophyll a distinct methods of table compares unit: mg/L
Monitoring method summary Volume of water sample (mL) 1 2 3 4 It is average Remarks
SL88-2012 specification monitoring 300 0.0833 0.0846 0.0840 0.0839 0.0840 4 Duplicate Samples strictly are done by specification, acetone 10ml is added to place 8h, centrifugation 20min measurement
Method of the invention 300 0.0840 0.0833 0.0838 0.0839 0.0838 4 Duplicate Samples are done, are not frozen, is not added after acetone ultrasound 20min plus acetone 10ml, placement 8h, centrifugation 20min is measured
As can be seen from Table 1, the refrigerator freeze-thaw repeatedly of " the measurement spectrophotometry of SL88-2012 water quality chlorophyll " is used Method measured result (0.0840mg/L) is consistent with method testing result (0.0838mg/L) of the invention.As can be seen from Table 1 Two methods measured result has specification monitoring to be greater than supercritical ultrasonics technology monitoring, also has supercritical ultrasonics technology monitoring to be greater than specification monitoring, though The average value supercritical ultrasonics technology monitoring rate specification monitoring of right 4 data is small by 0.21%, but as can be seen from Table 1 may be to be supervised by specification The 2nd group surveyed causes greatly according to slightly volume.It can be seen that by the monitoring result comparison of two methods, supervised using ultrasonic fragmentation The chlorophyll method the data obtained surveyed in water body is reliable, and is greatly improved work efficiency, and is worthy of popularization.
Embodiment 2
A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll, comprising the following steps:
1) pre-process: acquisition 1000ml earth's surface water sample is added 1% magnesium carbonate suspension, is then with diameter in brown bottle 47mm, the glass fiber filter that aperture is 0.7 μm are filtered, and obtain alga cells, then be placed on vacuum pump and filtered Processing, negative pressure is 20kPa when suction filtration, gradually depressurizes, terminates to filter when water sample just passes through filter membrane completely, filter membrane is taken out, There to be the one side doubling of sample, blots residual moisture with filter paper；
Wherein, the suspended liquid making method of magnesium carbonate is to take 1.0g magnesium carbonate powder, adds people's 100mL pure water, stirs into suspension, often It is sufficiently shaken up when secondary use.
Wherein, resulting alga cells needs are filtered to be protected from light and save at a temperature of -20 DEG C, low-temperature transport；It is filtered Alga cells need to wrap up one layer of aluminium foil in outside, save in -20 DEG C of following temperature.
2) chlorophyll extracts: will be crushed by the alga cells filtered using ultrasonic cleaner, ultrasonic frequency is 50kHz, the processing time be 30min, later be added 10mL acetone soln, at a temperature of being placed in 4 DEG C in be protected from light impregnate 12h, soaking Bubble during shake 4 times again, be centrifuged later using centrifuge, centrifugal speed be 4000 r/min, centrifugation time 30min, from Supernatant is taken after the heart；
Wherein, acetone soln concentration is 90%, and preparation method is to add 100mL pure water in 900mL acetone.
3) spectrophotometry measurement and calculating: the supernatant obtained by centrifugation is poured into cuvette, using light splitting light Degree meter is measured under the wavelength of 750nm, 664nm, 647nm, 630nm respectively, calculates surface water according to three colour equation formula The chlorophyll a, b of planktonic algae cell, c in body, then pheophytin correction content is measured, and calculated and taken off with monochromatic equation formulations Magnesium pheophytin corrects content.
Wherein, spectrophotometry specifically uses light path for the cuvette of 1cm, and 90% acetone soln makees reference liquid and carries out colorimetric, Spectrophotometer uses wavelength for the spectrophotometer of 380 ~ 780nm, and measurement points for attention are the extinction measured at 750nm wavelength Degree is greater than 0.005, need to be to measure after 0.45 μm of polytetrafluoroethylene (PTFE) organic phase syringe filter filters with aperture.
Wherein, three colour equation formula are as follows:
Ρchl-a=[11.85(A647-A750)-1.54(A664-A750)-0.08(A630-A750)] V1/( V2L),
Ρchl-b=[21.03(A647-A750)-5.43(A664-A750)-2.66(A630-A750)] V1/( V2L),
Ρchl-c=[24.52(A630-A750)-7.60(A647-A750)-1.67(A664-A750)] V1/( V2L);
Pchl-a is the mass concentration of water sample Determination of Chlorophyll a, and unit μ g/L, Pchl-b are that the quality of water sample Determination of Chlorophyll b is dense Degree, unit μ g/L, Ρ chl-c are the mass concentration of water sample Determination of Chlorophyll c, unit μ g/L, A750-It is extracting solution in wavelength Absorbance value at 750nm, A664For absorbance value of the extracting solution at wavelength 664nm, A647It is extracting solution at wavelength 647nm Absorbance value, A630For absorbance value of the extracting solution at wavelength 630nm, V1For extracting liquid volume, unit mL, V2For water sample Volume, unit L；L is cuvette light path, unit cm.
Wherein, monochromatic equation measurement pheophytin corrects content, and specific measuring process is to equipped with centrifuged supernatant 40 μ L hydrochloric acid solutions are added dropwise in 1cm cuvette and carry out acidification, the processing time is 30min, then at 750nm, 665nm Measure absorbance.
Wherein, hydrochloric acid solution is c (HC1)=0.1mol/L, and preparation method is to add people pure to 500ml 8.5ml concentrated hydrochloric acid In water, 1000mL is diluted to after being cooled to room temperature.
Wherein, monochromatic equation formulations are as follows:
Ρ,chl-a=26.7 [ (A664- A750)- (A665a-A750a)] V1/ (V2L),
Ρphe-a=26.7 [1.7 (A665a-A750a)- (A664-A750)] V1/ (V2L)；
P,Chl-a is the mass concentration of chlorophyll a after correcting pheophytin a in water sample, and unit is μ g/L；Pphe-a is water The mass concentration of pheophytin a in sample, unit are μ g/L；A750For the absorbance before extracting solution acidification at wavelength 750nm Value；A664For the absorbance value before extracting solution acidification at wavelength 664nm；A750aAfter being acidified for extracting solution at wavelength 750nm Absorbance value；A665aFor the absorbance value after extracting solution acidification at wavelength 665nm；V1For extracting liquid volume, unit mL；V2 For volume of water sample, unit L；L is cuvette light path, unit cm.
Embodiment 3
A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll, comprising the following steps:
1) pre-process: acquisition 1000ml earth's surface water sample is added 1% magnesium carbonate suspension, is then with diameter in brown bottle 47mm, the glass fiber filter that aperture is 0.7 μm are filtered, and obtain alga cells, then be placed on vacuum pump and filtered Processing, negative pressure is 20kPa when suction filtration, gradually depressurizes, terminates to filter when water sample just passes through filter membrane completely, filter membrane is taken out, There to be the one side doubling of sample, blots residual moisture with filter paper；
Wherein, the suspended liquid making method of magnesium carbonate is to take 1.0g magnesium carbonate powder, adds people's 100mL pure water, stirs into suspension, often It is sufficiently shaken up when secondary use.
Wherein, resulting alga cells needs are filtered to be protected from light and save at a temperature of -20 DEG C, low-temperature transport；It is filtered Alga cells need to wrap up one layer of aluminium foil in outside, save in -20 DEG C of following temperature.
2) chlorophyll extracts: will be crushed by the alga cells filtered using ultrasonic cleaner, ultrasonic frequency is 200kHz, the processing time be 30min, later be added 10mL acetone soln, at a temperature of being placed in 4 DEG C in be protected from light impregnate 10h, soaking Bubble during shake 4 times again, be centrifuged later using centrifuge, centrifugal speed be 4200 r/min, centrifugation time 30min, from Supernatant is taken after the heart；
Wherein, acetone soln concentration is 90%, and preparation method is to add 100mL pure water in 900mL acetone.
3) spectrophotometry measurement and calculating: the supernatant obtained by centrifugation is poured into cuvette, using light splitting light Degree meter is measured under the wavelength of 750nm, 664nm, 647nm, 630nm respectively, calculates surface water according to three colour equation formula The chlorophyll a, b of planktonic algae cell, c in body, then pheophytin correction content is measured, and calculated and taken off with monochromatic equation formulations Magnesium pheophytin corrects content.
Wherein, spectrophotometry specifically uses light path for the cuvette of 1cm, and 90% acetone soln makees reference liquid and carries out colorimetric, Spectrophotometer uses wavelength for the spectrophotometer of 380 ~ 780nm, and measurement points for attention are the extinction measured at 750nm wavelength Degree is greater than 0.005, need to be to measure after 0.45 μm of polytetrafluoroethylene (PTFE) organic phase syringe filter filters with aperture.
Wherein, three colour equation formula are as follows:
Ρchl-a=[11.85(A647-A750)-1.54(A664-A750)-0.08(A630-A750)] V1/( V2L),
Ρchl-b=[21.03(A647-A750)-5.43(A664-A750)-2.66(A630-A750)] V1/( V2L),
Ρchl-c=[24.52(A630-A750)-7.60(A647-A750)-1.67(A664-A750)] V1/( V2L);
Pchl-a is the mass concentration of water sample Determination of Chlorophyll a, and unit μ g/L, Pchl-b are that the quality of water sample Determination of Chlorophyll b is dense Degree, unit μ g/L, Ρ chl-c are the mass concentration of water sample Determination of Chlorophyll c, unit μ g/L, A750-It is extracting solution in wavelength Absorbance value at 750nm, A664For absorbance value of the extracting solution at wavelength 664nm, A647It is extracting solution at wavelength 647nm Absorbance value, A630For absorbance value of the extracting solution at wavelength 630nm, V1For extracting liquid volume, unit mL, V2For water sample Volume, unit L；L is cuvette light path, unit cm.
Wherein, monochromatic equation measurement pheophytin corrects content, and specific measuring process is to equipped with centrifuged supernatant 40 μ L hydrochloric acid solutions are added dropwise in 1cm cuvette and carry out acidification, the processing time is 30min, then at 750nm, 665nm Measure absorbance.
Wherein, hydrochloric acid solution is c (HC1)=0.1mol/L, and preparation method is to add people pure to 500ml 8.5ml concentrated hydrochloric acid In water, 1000mL is diluted to after being cooled to room temperature.
Wherein, monochromatic equation formulations are as follows:
Ρ,chl-a=26.7 [ (A664- A750)- (A665a-A750a)] V1/ (V2L),
Ρphe-a=26.7 [1.7 (A665a-A750a)- (A664-A750)] V1/ (V2L)；
P,Chl-a is the mass concentration of chlorophyll a after correcting pheophytin a in water sample, and unit is μ g/L；Pphe-a is water The mass concentration of pheophytin a in sample, unit are μ g/L；A750For the absorbance before extracting solution acidification at wavelength 750nm Value；A664For the absorbance value before extracting solution acidification at wavelength 664nm；A750aAfter being acidified for extracting solution at wavelength 750nm Absorbance value；A665aFor the absorbance value after extracting solution acidification at wavelength 665nm；V1For extracting liquid volume, unit mL；V2 For volume of water sample, unit L；L is cuvette light path, unit cm.
Embodiment 4
A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll, comprising the following steps:
1) pre-process: for acquisition 1000ml earth's surface water sample in brown bottle, addition concentration is 1% magnesium carbonate suspension, then with straight The glass fiber filter that diameter is 47mm, aperture is 0.7 μm is filtered, and obtains alga cells, then be placed on vacuum pump and carry out Suction filtration processing, negative pressure is 10kPa when suction filtration, gradually depressurizes, terminates to filter when water sample just passes through filter membrane completely, filter membrane is taken Out, the one side doubling that will have sample, blots residual moisture with filter paper；
Wherein, the suspended liquid making method of magnesium carbonate is to take 1.0g magnesium carbonate powder, adds people's 100mL pure water, stirs into suspension, often It is sufficiently shaken up when secondary use.
Wherein, resulting alga cells needs are filtered to be protected from light and save at a temperature of -20 DEG C, low-temperature transport；It is filtered Alga cells need to wrap up one layer of aluminium foil in outside, save in -20 DEG C of following temperature.
2) chlorophyll extracts: will be crushed by the alga cells filtered using ultrasonic cleaner, ultrasonic frequency is 75kHz, the processing time be 10min, later be added 10mL acetone soln, at a temperature of being placed in 4 DEG C in be protected from light impregnate 8h, impregnating Shake 3 times again in the process, are centrifuged using centrifuge later, and centrifugal speed is 3800 r/min, centrifugation time 10min, centrifugation After take supernatant；
Wherein, acetone soln concentration is 90%, and preparation method is to add 100mL pure water in 900mL acetone.
3) spectrophotometry measurement and calculating: the supernatant obtained by centrifugation is poured into cuvette, using light splitting light Degree meter is measured under the wavelength of 750nm, 664nm, 647nm, 630nm respectively, calculates surface water according to three colour equation formula The chlorophyll a, b of planktonic algae cell, c in body, then pheophytin correction content is measured, and calculated and taken off with monochromatic equation formulations Magnesium pheophytin corrects content.
Wherein, spectrophotometry specifically uses light path for the cuvette of 1cm, and 90% acetone soln makees reference liquid and carries out colorimetric, Spectrophotometer uses wavelength for the spectrophotometer of 380 ~ 780nm, and measurement points for attention are the extinction measured at 750nm wavelength Degree is greater than 0.005, need to be to measure after 0.45 μm of polytetrafluoroethylene (PTFE) organic phase syringe filter filters with aperture.
Wherein, three colour equation formula are as follows:
Ρchl-a=[11.85(A647-A750)-1.54(A664-A750)-0.08(A630-A750)] V1/( V2L),
Ρchl-b=[21.03(A647-A750)-5.43(A664-A750)-2.66(A630-A750)] V1/( V2L),
Ρchl-c=[24.52(A630-A750)-7.60(A647-A750)-1.67(A664-A750)] V1/( V2L);
Pchl-a is the mass concentration of water sample Determination of Chlorophyll a, and unit μ g/L, Pchl-b are that the quality of water sample Determination of Chlorophyll b is dense Degree, unit μ g/L, Ρ chl-c are the mass concentration of water sample Determination of Chlorophyll c, unit μ g/L, A750-It is extracting solution in wavelength Absorbance value at 750nm, A664For absorbance value of the extracting solution at wavelength 664nm, A647It is extracting solution at wavelength 647nm Absorbance value, A630For absorbance value of the extracting solution at wavelength 630nm, V1For extracting liquid volume, unit mL, V2For water sample Volume, unit L；L is cuvette light path, unit cm.
Wherein, monochromatic equation measurement pheophytin corrects content, and specific measuring process is to equipped with centrifuged supernatant 40 μ L hydrochloric acid solutions are added dropwise in 1cm cuvette and carry out acidification, the processing time is 10min, then at 750nm, 665nm Measure absorbance.
Wherein, hydrochloric acid solution is c (HC1)=0.1mol/L, and preparation method is to add people pure to 500ml 8.5ml concentrated hydrochloric acid In water, 1000mL is diluted to after being cooled to room temperature.
Wherein, monochromatic equation formulations are as follows:
Ρ,chl-a=26.7 [ (A664- A750)- (A665a-A750a)] V1/ (V2L),
Ρphe-a=26.7 [1.7 (A665a-A750a)- (A664-A750)] V1/ (V2L)；
P,Chl-a is the mass concentration of chlorophyll a after correcting pheophytin a in water sample, and unit is μ g/L；Pphe-a is water The mass concentration of pheophytin a in sample, unit are μ g/L；A750For the absorbance before extracting solution acidification at wavelength 750nm Value；A664For the absorbance value before extracting solution acidification at wavelength 664nm；A750aAfter being acidified for extracting solution at wavelength 750nm Absorbance value；A665aFor the absorbance value after extracting solution acidification at wavelength 665nm；V1For extracting liquid volume, unit mL；V2 For volume of water sample, unit L；L is cuvette light path, unit cm.
Embodiment 5
A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll, comprising the following steps:
1) pre-process: acquisition 1000ml earth's surface water sample is added 1% magnesium carbonate suspension, is then with diameter in brown bottle 47mm, the glass fiber filter that aperture is 0.7 μm are filtered, and obtain alga cells, then be placed on vacuum pump and filtered Processing, negative pressure is 18kPa when suction filtration, gradually depressurizes, terminates to filter when water sample just passes through filter membrane completely, filter membrane is taken out, There to be the one side doubling of sample, blots residual moisture with filter paper；
Wherein, the suspended liquid making method of magnesium carbonate is to take 1.0g magnesium carbonate powder, adds people's 100mL pure water, stirs into suspension, often It is sufficiently shaken up when secondary use.
Wherein, resulting alga cells needs are filtered to be protected from light and save at a temperature of -20 DEG C, low-temperature transport；It is filtered Alga cells need to wrap up one layer of aluminium foil in outside, save in -20 DEG C of following temperature.
2) chlorophyll extracts: will be crushed by the alga cells filtered using ultrasonic cleaner, ultrasonic frequency is 100kHz, the processing time be 25min, later be added 10mL acetone soln, at a temperature of being placed in 4 DEG C in be protected from light impregnate 6h, soaking Bubble during shake 3 times again, be centrifuged later using centrifuge, centrifugal speed be 3600 r/min, centrifugation time 25min, from Supernatant is taken after the heart；
Wherein, acetone soln concentration is 90%, and preparation method is to add 100mL pure water in 900mL acetone.
3) spectrophotometry measurement and calculating: the supernatant obtained by centrifugation is poured into cuvette, using light splitting light Degree meter is measured under the wavelength of 750nm, 664nm, 647nm, 630nm respectively, calculates surface water according to three colour equation formula The chlorophyll a, b of planktonic algae cell, c in body, then pheophytin correction content is measured, and calculated and taken off with monochromatic equation formulations Magnesium pheophytin corrects content.
Wherein, spectrophotometry specifically uses light path for the cuvette of 1cm, and 90% acetone soln makees reference liquid and carries out colorimetric, Spectrophotometer uses wavelength for the spectrophotometer of 380 ~ 780nm, and measurement points for attention are the extinction measured at 750nm wavelength Degree is greater than 0.005, need to be to measure after 0.45 μm of polytetrafluoroethylene (PTFE) organic phase syringe filter filters with aperture.
Wherein, three colour equation formula are as follows:
Ρchl-a=[11.85(A647-A750)-1.54(A664-A750)-0.08(A630-A750)] V1/( V2L),
Ρchl-b=[21.03(A647-A750)-5.43(A664-A750)-2.66(A630-A750)] V1/( V2L),
Ρchl-c=[24.52(A630-A750)-7.60(A647-A750)-1.67(A664-A750)] V1/( V2L);
Pchl-a is the mass concentration of water sample Determination of Chlorophyll a, and unit μ g/L, Pchl-b are that the quality of water sample Determination of Chlorophyll b is dense Degree, unit μ g/L, Ρ chl-c are the mass concentration of water sample Determination of Chlorophyll c, unit μ g/L, A750-It is extracting solution in wavelength Absorbance value at 750nm, A664For absorbance value of the extracting solution at wavelength 664nm, A647It is extracting solution at wavelength 647nm Absorbance value, A630For absorbance value of the extracting solution at wavelength 630nm, V1For extracting liquid volume, unit mL, V2For water sample Volume, unit L；L is cuvette light path, unit cm.
Wherein, monochromatic equation measurement pheophytin corrects content, and specific measuring process is to equipped with centrifuged supernatant 40 μ L hydrochloric acid solutions are added dropwise in 1cm cuvette and carry out acidification, the processing time is 25min, then at 750nm, 665nm Measure absorbance.
Wherein, hydrochloric acid solution is c (HC1)=0.1mol/L, and preparation method is to add people pure to 500ml 8.5ml concentrated hydrochloric acid In water, 1000mL is diluted to after being cooled to room temperature.
Wherein, monochromatic equation formulations are as follows:
Ρ,chl-a=26.7 [ (A664- A750)- (A665a-A750a)] V1/ (V2L),
Ρphe-a=26.7 [1.7 (A665a-A750a)- (A664-A750)] V1/ (V2L)；
P,Chl-a is the mass concentration of chlorophyll a after correcting pheophytin a in water sample, and unit is μ g/L；Pphe-a is water The mass concentration of pheophytin a in sample, unit are μ g/L；A750For the absorbance before extracting solution acidification at wavelength 750nm Value；A664For the absorbance value before extracting solution acidification at wavelength 664nm；A750aAfter being acidified for extracting solution at wavelength 750nm Absorbance value；A665aFor the absorbance value after extracting solution acidification at wavelength 665nm；V1For extracting liquid volume, unit mL；V2 For volume of water sample, unit L；L is cuvette light path, unit cm.
Embodiment 6
A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll, comprising the following steps:
1) pre-process: acquisition 500ml earth's surface water sample is added 1% magnesium carbonate suspension, is then with diameter in brown bottle 47mm, the glass fiber filter that aperture is 0.7 μm are filtered, and obtain alga cells, then be placed on vacuum pump and filtered Processing, negative pressure is 12kPa when suction filtration, gradually depressurizes, terminates to filter when water sample just passes through filter membrane completely, filter membrane is taken out, There to be the one side doubling of sample, blots residual moisture with filter paper；
Wherein, the suspended liquid making method of magnesium carbonate is to take 0.5g magnesium carbonate powder, adds people's 50mL pure water, stirs into suspension, often It is sufficiently shaken up when secondary use.
Wherein, resulting alga cells needs are filtered to be protected from light and save at a temperature of -20 DEG C, low-temperature transport；It is filtered Alga cells need to wrap up one layer of aluminium foil in outside, save in -20 DEG C of following temperature.
2) chlorophyll extracts: will be crushed by the alga cells filtered using ultrasonic cleaner, ultrasonic frequency is 150kHz, the processing time be 15min, later be added 10mL acetone soln, at a temperature of being placed in 4 DEG C in be protected from light impregnate 4h, soaking Bubble during shake 3 times again, be centrifuged later using centrifuge, centrifugal speed be 3500 r/min, centrifugation time 15min, from Supernatant is taken after the heart；
Wherein, acetone soln concentration is 90%, and preparation method is to add 100mL pure water in 900mL acetone.
3) spectrophotometry measurement and calculating: the supernatant obtained by centrifugation is poured into cuvette, using light splitting light Degree meter is measured under the wavelength of 750nm, 664nm, 647nm, 630nm respectively, calculates surface water according to three colour equation formula The chlorophyll a, b of planktonic algae cell, c in body, then pheophytin correction content is measured, and calculated and taken off with monochromatic equation formulations Magnesium pheophytin corrects content.
Wherein, spectrophotometry specifically uses light path for the cuvette of 1cm, and 90% acetone soln makees reference liquid and carries out colorimetric, Spectrophotometer uses wavelength for the spectrophotometer of 380 ~ 780nm, and measurement points for attention are the extinction measured at 750nm wavelength Degree is greater than 0.005, need to be to measure after 0.45 μm of polytetrafluoroethylene (PTFE) organic phase syringe filter filters with aperture.
Wherein, three colour equation formula are as follows:
Ρchl-a=[11.85(A647-A750)-1.54(A664-A750)-0.08(A630-A750)] V1/( V2L),
Ρchl-b=[21.03(A647-A750)-5.43(A664-A750)-2.66(A630-A750)] V1/( V2L),
Ρchl-c=[24.52(A630-A750)-7.60(A647-A750)-1.67(A664-A750)] V1/( V2L);
Pchl-a is the mass concentration of water sample Determination of Chlorophyll a, and unit μ g/L, Pchl-b are that the quality of water sample Determination of Chlorophyll b is dense Degree, unit μ g/L, Ρ chl-c are the mass concentration of water sample Determination of Chlorophyll c, unit μ g/L, A750It is extracting solution in wavelength 750nm The absorbance value at place, A664For absorbance value of the extracting solution at wavelength 664nm, A647For suction of the extracting solution at wavelength 647nm Shading value, A630For absorbance value of the extracting solution at wavelength 630nm, V1For extracting liquid volume, unit mL, V2For water sample body Product, unit L；L is cuvette light path, unit cm.
Wherein, monochromatic equation measurement pheophytin corrects content, and specific measuring process is to equipped with centrifuged supernatant 40 μ L hydrochloric acid solutions are added dropwise in 1cm cuvette and carry out acidification, the processing time is 15min, then at 750nm, 665nm Measure absorbance.
Wherein, hydrochloric acid solution is c (HC1)=0.1mol/L, and preparation method is to add people pure to 500ml 8.5ml concentrated hydrochloric acid In water, 1000mL is diluted to after being cooled to room temperature.
Wherein, monochromatic equation formulations are as follows:
Ρ,chl-a=26.7 [ (A664- A750)- (A665a-A750a)] V1/ (V2L),
Ρphe-a=26.7 [1.7 (A665a-A750a)- (A664-A750)] V1/ (V2L)；
P,Chl-a is the mass concentration of chlorophyll a after correcting pheophytin a in water sample, and unit is μ g/L；Pphe-a is water The mass concentration of pheophytin a in sample, unit are μ g/L；A750For the absorbance before extracting solution acidification at wavelength 750nm Value；A664For the absorbance value before extracting solution acidification at wavelength 664nm；A750aAfter being acidified for extracting solution at wavelength 750nm Absorbance value；A665aFor the absorbance value after extracting solution acidification at wavelength 665nm；V1For extracting liquid volume, unit mL；V2 For volume of water sample, unit L；L is cuvette light path, unit cm.

Claims (9)

1. a kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll, it is characterised in that including following step It is rapid:
1) pre-process: magnesium carbonate suspension is added in brown or dark bottles in acquisition earth's surface water sample, mixes, takes determined volume Mixing water sample, be put into the Vacuum filtration device with filter membrane and carry out suction filtration processing, obtain alga cells；
2) chlorophyll extracts: will use ultrasonic disruption by the alga cells filtered, adds 10mL acetone soln, be placed in It is spare that 4 ~ 12h of immersion is protected from light at a temperature of 4 DEG C, shake 3 ~ 4 times again, are centrifuged using centrifuge later, take supernatant during immersion；
3) spectrophotometry measurement and calculating: the supernatant obtained by centrifugation is poured into cuvette, using spectrophotometer It is measured under the wavelength of 750nm, 664nm, 647nm, 630nm, is calculated in surface water body according to three colour equation formula respectively Chlorophyll a, b, the c of planktonic algae cell, then pheophytin correction content is measured, and calculate de-magging leaf with monochromatic equation formulations Green element correction content.
2. the spectrophotometric method according to claim 1 using ultrasonic disruption alga cells measurement chlorophyll, special Sign is in the step 1) that the resulting alga cells needs of filtering are protected from light and save at a temperature of -20 DEG C, low-temperature transport；It takes out Alga cells after filter need to wrap up one layer of aluminium foil in outside, save in -20 DEG C of following temperature, and test in analyzing in 25d It finishes.
3. the spectrophotometric method according to claim 1 using ultrasonic disruption alga cells measurement chlorophyll, special Sign is that the magnesium carbonate suspension in the step 1) is the magnesium carbonate suspension of concentration 1%, and additional amount is that every liter of water sample is added 1mL, the preparation method of the magnesium carbonate suspension are to take 1.0g magnesium carbonate powder, add people's 100mL pure water, stir into suspended Liquid sufficiently shakes up when using every time.
4. the spectrophotometric method according to claim 1 using ultrasonic disruption alga cells measurement chlorophyll, special Sign is that the filter membrane in the step 1) is glass fiber filter, and diameter 47mm, aperture are 0.7 μm；The step 1) In suction filtration negative pressure be less than 20kPa, gradually depressurize, terminate to filter when water sample just passes through filter membrane completely, filter membrane is taken out, There to be the one side doubling of sample, blots residual moisture with filter paper.
5. the spectrophotometric method according to claim 1 using ultrasonic disruption alga cells measurement chlorophyll, special Sign is that the ultrasonic disruption in the step 2) is that ultrasound is carried out using ultrasonic cleaner, ultrasonic frequency is 50 ~ 200kHz, processing time are 10 ~ 30min；Acetone soln concentration in the step 2) is 90%, and preparation method is 900mL third Add 100mL pure water in ketone；The centrifugal speed of centrifuge in the step 2) is 3500 ~ 4200r/min, centrifugation time 10 ~30min。
6. the spectrophotometric method according to claim 1 using ultrasonic disruption alga cells measurement chlorophyll, special Sign is that the spectrophotometry in the step 3) specifically uses light path for the cuvette of 1cm, and 90% acetone soln makees reference liquid Colorimetric is carried out, spectrophotometer uses wavelength for the spectrophotometer of 380 ~ 780nm, and the points for attention during being measured are It measures the absorbance at 750nm wavelength and is greater than 0.005, the polytetrafluoroethylene (PTFE) organic phase syringe filter that need to be 0.45 μm with aperture It is measured again after being filtered.
7. the method according to claim 1 using ultrasonic disruption planktonic algae raji cell assay Raji water quality chlorophyll, special Sign is three colour equation formula in the step 3) are as follows:
Ρchl-a=[11.85(A647-A750)-1.54(A664-A750)-0.08(A630-A750)] V1/( V2L),
Ρchl-b=[21.03(A647-A750)-5.43(A664-A750)-2.66(A630-A750)] V1/( V2L),
Ρchl-c=[24.52(A630-A750)-7.60(A647-A750)-1.67(A664-A750)] V1/( V2L);
Wherein, Pchl-a is the mass concentration of water sample Determination of Chlorophyll a, and unit is μ g/L, and Pchl-b is water sample Determination of Chlorophyll b's Mass concentration, unit are μ g/L, and Ρ chl-c is the mass concentration of water sample Determination of Chlorophyll c, and unit is μ g/L, A750Exist for extracting solution Absorbance value at wavelength 750nm, A664For absorbance value of the extracting solution at wavelength 664nm, A647It is extracting solution in wavelength Absorbance value at 647nm, A630For absorbance value of the extracting solution at wavelength 630nm, V1For extracting liquid volume, unit mL, V2For volume of water sample, unit L；L is cuvette light path, unit cm.
8. the method according to claim 1 using ultrasonic disruption planktonic algae raji cell assay Raji water quality chlorophyll, special Sign is that the concrete operation step of the measurement pheophytin correction content in the step 3) is to equipped with centrifuged supernatant 1cm cuvette in be added dropwise 40 μ L 0.1mol/L hydrochloric acid solution, the processing time be 10 ~ 30min, then in 750nm, Absorbance is measured at 665nm.
9. the method according to claim 1 using ultrasonic disruption planktonic algae raji cell assay Raji water quality chlorophyll, special Sign is the monochromatic equation formulations in the step 3) are as follows:
Ρ,chl-a=26.7 [ (A664- A750)- (A665a-A750a)] V1/ (V2L),
Ρphe-a=26.7 [1.7 (A665a-A750a)- (A664-A750)] V1/ (V2L)；
Wherein, P,Chl-a is the mass concentration of chlorophyll a after correcting pheophytin a in water sample, and unit is μ g/L；Pphe-a For the mass concentration of pheophytin a in water sample, unit is μ g/L；A750For the extinction before extracting solution acidification at wavelength 750nm Angle value；A664For the absorbance value before extracting solution acidification at wavelength 664nm；A750aAfter being acidified for extracting solution at wavelength 750nm Absorbance value；A665aFor the absorbance value after extracting solution acidification at wavelength 665nm；V1For extracting liquid volume, unit mL； V2For volume of water sample, unit L；L is cuvette light path, unit cm.
CN201810983922.7A 2018-08-28 2018-08-28 A kind of spectrophotometric method using ultrasonic disruption alga cells measurement chlorophyll Pending CN108956501A (en)

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Application publication date: 20181207