CN109444306A - A kind of method that efficient liquid phase fluorescence chromatography detects microcapsule algae toxin in water - Google Patents
A kind of method that efficient liquid phase fluorescence chromatography detects microcapsule algae toxin in water Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses the methods of microcapsule algae toxin in a kind of efficient liquid phase fluorescence chromatography detection water, and in particular to a kind of method that efficient liquid phase fluorescence chromatography detects microcapsule algae toxin in water belongs to monitoring water environment technology or agricultural monitoring technical field.The characteristics of fluorescence can be inspired under ultraviolet light the present invention is based on microcapsule algae toxin; by being filtered, being enriched with to sample, elute, constant volume is concentrated; using efficient liquid phase fluorescence chromatogram method realize Microcystins in Water-LR it is quick, accurately and efficiently detect, all there is directive significance to the monitoring of water pollution situation, improvement and protection.
Description
One, technical field
The invention belongs to monitoring water environment technology or agricultural monitoring technical fields, and in particular to Microcystins in Water-
A kind of efficient liquid phase fluorescence chromatogram detection method of LR.
Two, background technique
Microcystin (Microcystins, MCs) is by microcystic aeruginosa, Anabaena Flos-aquae, the algae that quivers etc. in Cyanophyta
The cyclic annular heptapeptide toxin metabolin of one kind caused by aquation occurs, has now been found that a isomers composition more than 90, has strong carcinogenic
Property, long-term drinking may induce liver cancer rich in the water of high concentration MCs, and MCs can also carry out biological concentration by food chain, to people
Body causes to damage, and has persistence biological pollution, and wherein microcapsule phycotoxin MC-LR (L represents leucine, and R represents arginine) is
A kind of configuration that toxicity is most strong, distribution is most wide.With the continuous aggravation of pollution situation, cyanobacterial bloom phenomenon is in the world
Lake, river frequently occur in reservoir, and the Microcystin generated in water body seriously threatens water quality and food safety, this is
Health and environmental problem as domestic and international common concern.MCs chemical property is stablized, at present the traditional treatment process in waterworks
(such as: precipitating, filtering, chlorination) cannot remove it, in view of the generality and stronger toxicity of MCs pollution, MC-LR in water body
Content is by multinational as the important indicator parameter for measuring water grade, the World Health Organization and China's " Drinking Water health
Standard " with defining earth surface water source, the concentration of Drinking Water MCs (using MC-LR as representative) must not be higher than 1.0 μ g/L.
Currently, the method for common detection MC-LR mainly has chemical measure, immunoassay and bioassary method.Chemistry
Detection method mainly includes thin-layered chromatography (TLC), gas chromatography (GC), Capillary Electrophoresis (CE), liquid chromatography/mass spectrometry analysis
Method (LC/MS) etc..These detection methods combine sensitive photoelectric technology to carry out analysis measurement using the physico-chemical property of MCs, though
Good measurement effect is so also achieved, but there are instrument and equipment operating cost height, complex pretreatment time-consuming, the rate of recovery be not high
It is insufficient.Immunoassay detects the specific recognition principle of antigen to various toxin using antibody, method sensitivity
High but specificity is not strong, and cross reaction easily occurs for antibody and toxin, it sometimes appear that false positive reaction, identifies not Type of toxin
It is good, it is of limited application.Bioassary method usually feeds with murine oral or is injected intraperitoneally the toxicity of evaluation MCs, this method behaviour
Make simply, intuitively, but cannot distinguish between the isomers of toxin and quantify not accurate enough, heavy workload.Since MC-LR belongs to monocycle seven
Peptide organic molecule, basic molecular structure is big ring and be spaced double bond has heat steady containing functional groups such as carbonyl, amino, amide groups
It is qualitative it is good, exciting light irradiation under can excite molecule issue fluorescence the characteristics of, itself have efficient liquid phase fluorescence chromatogram detection
Condition.MCs content is very low in natural water body, thus water sample should by after being enriched with, being concentrated could quantitative analysis, and efficient liquid phase
Chromatography (HPLC) has good separating capacity, as a result accurately, favorable reproducibility, and and fluorescence detector can be accurately sensitive right
The fluorescence inspired is detected.
Therefore, a kind of high sensitivity is established, qualitative accurate, the rate of recovery and reproducibility are good, and easy, result swift to operate can
The method of the measurement Microcystins in Water-LR leaned on all has guidance meaning to the monitoring of water pollution situation, improvement and protection
Justice.
Three, summary of the invention
Technical problem to be solved by the invention is to provide microcystins in a kind of efficient liquid phase fluorescence chromatography detection water
The method of element-LR, this method is based on microcapsule algae toxin molecule can inspire specific fluorescent, heat surely under exciting light irradiation
Qualitative well equal physicochemical characteristics extract under the premise of finding and determining optimal excitation wavelength and wavelength of transmitted light in conjunction with solid phase
It follows the example of and the pre-treatments such as is filtered, is enriched with, elutes and is concentrated to water sample, and optimize high performance liquid chromatography detection condition in water body
Microcapsule algae toxin carries out quick, highly sensitive detection.Specific step is as follows for technical solution of the present invention:
(1) titer purchase microcapsule algae toxin standard solution is prepared, microcapsule algae toxin concentration standard value is 20 μ g/
mL。
It prepares Standard Stock solutions: accurately pipetting the microcapsule algae toxin standard solution (20 μ g/mL) of 1000 μ L in 10mL
In volumetric flask, with methanol dilution to graduation mark, the concentration containing microcapsule algae toxin is 2.0mg/L in the stock solution, is shaken up, 0 DEG C
~4 DEG C or less preservations, it is in one week effectively, spare.
Prepare standard working solution: by the Standard Reserving Solution methanol dilution of microcapsule algae toxin to 1.00,5.00,
10.00,6 concentration such as 50.00,100.00,250.00 μ g/L.This standard working solution faces the used time and now matches.
(2) the pretreatment water sampling 1000mL of water sample, and water sample is crossed to 0.45 μm of water system filter membrane, to Solid Phase Extraction.
(3) splitter 5~15mL of methanol, 2.0~5.0mL/min of flow velocity are activated, prewashing splitter, then with 5~15mL
Water, 2.0~5.0mL/min of flow velocity activate splitter, and ensure not allow splitter to drain off.
(4) enrichment of sample by pretreated 1000mL water sample with the flow velocity of 5~15mL/min after activated separation
Column carries out example enrichment.
(5) after dry splitter first uses 2~10mL water to elute splitter with 5~15mL/min flow velocity, then with 5~
15mL20% methanol elutes splitter with 2~10mL/min flow velocity again, finally with 10~30mL high pure nitrogen with 10~30mL/
Min flow velocity gas pushes away splitter, and nitrogen blows 10~50min of splitter, keeps splitter dry.
(6) sample elution and concentration: syringe pump uses 0.1% 1~5mL of trifluoroacetic acid methanol solution, 10~30mL/ of flow velocity
Min is cleaned, then elutes splitter with 0.1% 5~15mL of trifluoroacetic acid methanol, 1~5mL/min of flow velocity, then by eluent
It crosses anhydrous sodium sulfate column to be dehydrated, is finally pushed away splitter with 5~15mL of nitrogen, flow velocity 10~30mL/min gas, and received
Collect eluent.The eluent of collection is concentrated under the conditions of temperature is 35 DEG C~45 DEG C, nitrogen, which is blown, is concentrated into 1.0mL, and crosses 0.45 μ
Organic filter membrane of m is measured to high performance liquid chromatograph.
(7) chromatographic determination condition is measured are as follows: using SHIMPACKCLC-ODS (specification: 150mm × 6.0mm, 5.0 μm) color
Column is composed, excitation wavelength (λ ex) range of fluorescence detector is 249~254nm, and launch wavelength (λ em) range is 293~298nm.
Mobile phase is methanol (0.05% trifluoroacetic acid): water (0.05% trifluoroacetic acid)=(55~65): (35~45) (V/V), flow velocity
0.8~1.2mL/min, 30~50 DEG C of column temperature, 10 μ L of sample volume.
The method of microcapsule algae toxin in a kind of efficient liquid phase fluorescence chromatography detection water, splitter is C18 points
From column, prewashing methanol volume is 10mL, flow velocity 3.0mL/min, and activation water volume is 10mL, flow velocity 3.0mL/min.
The method of microcapsule algae toxin, example enrichment are excessive in a kind of efficient liquid phase fluorescence chromatography detection water
Loading flow velocity from column is 10mL/min.
The method of microcapsule algae toxin, the dry leaching of splitter in a kind of efficient liquid phase fluorescence chromatography detection water
Wash water volume is 5mL, flow velocity 10mL/min, and used 20% methanol volume is 10mL, flow velocity 5mL/min, and it is dry that gas pushes away splitter
Dry nitrogen volume used is 20mL, flow velocity 20mL/min, continues nitrogen and blows the splitter time for 25min.
The method of microcapsule algae toxin, sample elution period in a kind of efficient liquid phase fluorescence chromatography detection water
It is 2mL that middle syringe pump, which is cleaned with 0.1% trifluoroacetic acid methanol solution volume, flow velocity 20mL/min, and splitter is eluted with 0.1% 3
Fluoroacetic acid methanol solution volume is 10mL, flow velocity 2mL/min；Gas pushes away N used in splitter2Volume is 10mL, flow velocity 20mL/
min。
The method of microcapsule algae toxin, the concentration of eluent in a kind of efficient liquid phase fluorescence chromatography detection water
Temperature is 40 DEG C.
The method of microcapsule algae toxin, sample continuous mode in a kind of efficient liquid phase fluorescence chromatography detection water
The excitation wavelength lambda ex=251nm of middle fluorescence detector, emission wavelength lambda em=296nm.
The method of microcapsule algae toxin, mobile phase volume ratio in a kind of efficient liquid phase fluorescence chromatography detection water
For 0.05% trifluoroacetic acid methanol solution: 0.05% trifluoroacetic acid aqueous solution=60:40, flow velocity 1.0mL/min, 40 DEG C of column temperature.
It is that the present invention reaches the utility model has the advantages that
(1) the present invention is based on the unique molecular structure feature of microcapsule algae toxin, it is explored under the conditions of fluorescence detector
Optimum detection excitation wave wavelength and transmitted wave wavelength, microcapsule algae toxin are stimulated the fluorescence of sending and the fluorescence of other substances
It is easily discriminated and comes, can be good at the problem of avoiding background interference, effectively preventing false positive, detection sensitivity is high.
(2) detection method provided by the invention is effectively rich to the microcapsule algae toxin in water sample using solid phase extraction techniques
Collection can greatly enhance the rate of recovery of microcapsule algae toxin, and analysis efficiency is high, cost is relatively low.
Four, Detailed description of the invention
Fig. 1 is λEx=238nm, the response schematic diagram under different Em wavelength；
Fig. 2 is λEm=296nm, the response schematic diagram under different Ex wavelength；
Fig. 3 is λEx=251nm, the response schematic diagram under different Em wavelength；
Fig. 4 is drinking water addition recycling chromatogram；
Fig. 5 is surface water addition recycling chromatogram；
The standard chromatogram of Fig. 6 microcapsule algae toxin；
Fig. 7 microcapsule algae toxin linear relationship chart.
Five, specific embodiment
1, titer purchase microcapsule algae toxin standard solution is prepared, microcapsule algae toxin concentration standard value is 20 μ g/
mL。
It prepares Standard Stock solutions: accurately pipetting the microcapsule algae toxin standard solution (20 μ g/mL) of 1000 μ L in 10mL
In volumetric flask, with methanol dilution to graduation mark, the concentration containing microcapsule algae toxin is 2.0mg/L in the stock solution, is shaken up, 0 DEG C
~4 DEG C or less preservations, it is in one week effectively, spare.
Prepare standard working solution: by the Standard Reserving Solution methanol dilution of microcapsule algae toxin to 1.00,5.00,
10.00,6 concentration such as 50.00,100.00,250.00 μ g/L.This standard working solution faces the used time and now matches.
2, the pretreatment water sampling 1000mL of water sample, and water sample is crossed to 0.45 μm of water system filter membrane, to Solid Phase Extraction.
3, activation splitter first uses 10mL methanol with the flow velocity prewashing C18 column of 3.0mL/min, keeps solvent stream net, then use
10mL water not allow splitter to drain off with the flow velocity activation C18 column of 3.0mL/min in activation process.
4, the enrichment of sample carries out pretreated 1000mL water sample with the flow velocity of 10mL/min after activated C18 column
Example enrichment.
5, after dry splitter first uses 5mL water to elute C18 column with 10mL/min flow velocity, then with 10mL20% methanol with 5mL/
Min flow velocity elutes C18 column again, is finally pushed away with 20mL high pure nitrogen with 20mL/min flow velocity gas and blows C18 column, and nitrogen blows C18 column
25min keeps column dry.
6, sample elution and concentration first clean note with the methanol that 2mL contains 0.1% trifluoroacetic acid with the flow velocity of 20mL/min
Pump is penetrated, then contains the methanol of 0.1% trifluoroacetic acid with 10mL and C18 column is eluted with the flow velocity of 2mL/min, eluent is after anhydrous sulphur
Sour sodium column collects eluent, finally pushes away C18 column with 10mL nitrogen with the flow velocity gas of 20mL/min, and collect eluent.It will collect
Eluent under the conditions of 40 DEG C, nitrogen, which is blown, is concentrated into 1.0mL, and crosses 0.45 μm of organic filter membrane, surveys to high performance liquid chromatograph
It is fixed.
7, measurement chromatographic determination condition is
Chromatographic column: SHIMPACKCLC-ODS, (150mm × 6.0mm, 5.0 μm)；Or the suitable person of performance.Detector: fluorescence
Detector；λ ex=251nm, λ em=296nm.
Mobile phase: methanol (0.05% trifluoroacetic acid): water (0.05% trifluoroacetic acid)=60:40 (V/V).
Flow velocity: 1.0mL/min.Column temperature: 40 ± 1 DEG C.Sample volume: 10 μ L.
The method of microcapsule algae toxin passes through microcystin in a kind of efficient liquid phase fluorescence chromatography detection water of the invention
The excitation/emission wavelength of element-LR, and accuracy, the inspection of precision and this method are carried out to two kinds of drinking water, surface water samples
The determination carry out method confirmation of rising limit, lower limit of quantitation.
One, the determination of fluorescence detection wavelength
Using the spectral scan technology of liquid chromatogram fluorescence detector, by swashing to microcapsule algae toxin standard solution
Luminous spectrum and emission spectrum are scanned, so that it is determined that going out to detect the optimum excitation wave wavelength and transmitted wave of microcapsule algae toxin
Wavelength.The UV absorption wave 238nm recommended first by GB/T5750.8-2006.13 Microcystin (high pressure lipuid chromatography (HPLC))
As excitation wavelength (Ex=238nm), full scan detection, Microcystis aeruginosa are carried out to different emission Em (Em=263-400nm)
Launch wavelength Em when toxin response maximum is 296nm (Fig. 1)；
Then using the 296nm sought as launch wavelength (Em=296nm), to different excitation wavelength Ex (Ex=220-
Full scan detection 300nm) is carried out, excitation wavelength Ex when microcapsule algae toxin response maximum is 251nm (Fig. 2)；
Finally again using the 251nm sought as maximum excitation wavelength (Ex=251nm), again to different emission Em
(Em=276-400nm) carry out full scan detection, it is confirmatory seek microcapsule algae toxin response maximum when launch wavelength Em
For 296nm (Fig. 3).By analyzing spectrogram, the final excitation/emission wavelength for determining microcapsule algae toxin is λ ex=
251nm/ λ em=296nm.
Two, accuracy and precision
1,24 parts of each 1000mL of drinking water water sample are measured respectively, wherein 6 parts are used as sample blank, in remaining 18 parts of sample
Microcapsule algae toxin standard solution is added, is 3 pitch-based spheres, 0.10,0.50,1.00 μ g/L respectively.
(1) the pretreatment water sampling 1000mL of water sample, and water sample is crossed to 0.45 μm of water system filter membrane, to Solid Phase Extraction.
(2) activation splitter first uses 10mL methanol with the flow velocity prewashing C18 column of 3.0mL/min, keeps solvent stream net, then use
10mL water not allow splitter to drain off with the flow velocity activation C18 column of 3.0mL/min in activation process.
(3) enrichment of sample by pretreated 1000mL water sample with the flow velocity of 10mL/min after activated C18 column into
Row example enrichment.
(4) after dry splitter first uses 5mL water to elute C18 column with 10mL/min flow velocity, then with 10mL20% methanol with
5mL/min flow velocity elutes C18 column again, is finally pushed away with 20mL high pure nitrogen with 20mL/min flow velocity gas and blows C18 column, and nitrogen is blown
C18 column 25min keeps column dry.
(5) sample elution and concentration first clean note with the methanol that 2mL contains 0.1% trifluoroacetic acid with the flow velocity of 20mL/min
Pump is penetrated, then contains the methanol of 0.1% trifluoroacetic acid with 10mL and C18 column is eluted with the flow velocity of 2mL/min, eluent is after anhydrous sulphur
Sour sodium column collects eluent, finally pushes away C18 column with 10mL nitrogen with the flow velocity gas of 20mL/min, and collect eluent.It will collect
Eluent under the conditions of 40 DEG C, nitrogen, which is blown, is concentrated into 1.0mL, and crosses 0.45 μm of organic filter membrane, surveys to high performance liquid chromatograph
It is fixed.
(6) measurement chromatographic determination condition is
Chromatographic column: SHIMPACKCLC-ODS, (150mm × 6.0mm, 5.0 μm)；Or the suitable person of performance.
Detector: fluorescence detector；λ ex=251nm, λ em=296nm.
Mobile phase: methanol (0.05% trifluoroacetic acid): water (0.05% trifluoroacetic acid)=60:40 (V/V).
Flow velocity: 1.0mL/min.Column temperature: 40 ± 1 DEG C.Sample volume: 10 μ L.
Method extraction, purification according to the above analysis, and detected according to above-mentioned chromatographic condition, average recovery rate and precision
Degree such as table 1, drinking water addition recycling chromatogram such as Fig. 4.
The TIANZHU XINGNAO Capsul and precision result of 1 microcystin in drinking water-LR of table
2,24 parts of each 1000mL of surface water water sample are measured respectively, wherein 6 parts are used as sample blank, in remaining 18 parts of sample
Microcapsule algae toxin standard solution is added, is 3 pitch-based spheres, 0.10,0.50,1.00 μ g/L respectively.
(1) the pretreatment water sampling 1000mL of water sample, and water sample is crossed to 0.45 μm of water system filter membrane, to Solid Phase Extraction.
(2) activation splitter first uses 10mL methanol with the flow velocity prewashing C18 column of 3.0mL/min, keeps solvent stream net, then use
10mL water not allow splitter to drain off with the flow velocity activation C18 column of 3.0mL/min in activation process.
(3) enrichment of sample by pretreated 1000mL water sample with the flow velocity of 10mL/min after activated C18 column into
Row example enrichment.
(4) after dry splitter first uses 5mL water to elute C18 column with 10mL/min flow velocity, then with 10mL20% methanol with
5mL/min flow velocity elutes C18 column again, is finally pushed away with 20mL high pure nitrogen with 20mL/min flow velocity gas and blows C18 column, and nitrogen is blown
C18 column 25min keeps column dry.
(5) sample elution and concentration first clean note with the methanol that 2mL contains 0.1% trifluoroacetic acid with the flow velocity of 20mL/min
Pump is penetrated, then contains the methanol of 0.1% trifluoroacetic acid with 10mL and C18 column is eluted with the flow velocity of 2mL/min, eluent is after anhydrous sulphur
Sour sodium column collects eluent, finally pushes away C18 column with 10mL nitrogen with the flow velocity gas of 20mL/min, and collect eluent.It will collect
Eluent under the conditions of 40 DEG C, nitrogen, which is blown, is concentrated into 1.0mL, and crosses 0.45 μm of organic filter membrane, surveys to high performance liquid chromatograph
It is fixed.
(6) measurement chromatographic determination condition is
Chromatographic column: SHIMPACKCLC-ODS, (150mm × 6.0mm, 5.0 μm)；Or the suitable person of performance.
Detector: fluorescence detector；λ ex=251nm, λ em=296nm.
Mobile phase: methanol (0.05% trifluoroacetic acid): water (0.05% trifluoroacetic acid)=60:40 (V/V).
Flow velocity: 1.0mL/min.Column temperature: 40 ± 1 DEG C.Sample volume: 10 μ L.
Method extraction, purification according to the above analysis, and detected according to above-mentioned chromatographic condition, average recovery rate and precision
Degree such as table 2, surface water addition recycling chromatogram such as Fig. 5.
The TIANZHU XINGNAO Capsul and precision of microcapsule algae toxin in 2 surface water of table
Three, detection limit and lower limit of quantitation
1, titer purchase microcapsule algae toxin standard solution is prepared, microcapsule algae toxin concentration standard value is 20 μ g/
mL。
It prepares Standard Stock solutions: accurately pipetting the microcapsule algae toxin standard solution (20 μ g/mL) of 1000 μ L in 10mL
In volumetric flask, with methanol dilution to graduation mark, the concentration containing microcapsule algae toxin is 2.0mg/L in the stock solution, is shaken up, 0 DEG C
~4 DEG C or less preservations, it is in one week effectively, spare.
Prepare standard working solution: by the Standard Reserving Solution methanol dilution of microcapsule algae toxin to 1.00,5.00,
10.00,6 concentration such as 50.00,100.00,250.00 μ g/L.This standard working solution faces the used time and now matches.
2, the microcapsule algae toxin standard solution for preparing 1.0 μ g/L, respectively with 3 times and 10 times of signal-to-noise ratio (S/N) calculating sides
The detection limit and lower limit of quantitation of method.
3, measurement chromatographic determination condition is
Chromatographic column: SHIMPACKCLC-ODS, (150mm × 6.0mm, 5.0 μm)；Or the suitable person of performance.
Detector: fluorescence detector；λ ex=251nm, λ em=296nm.
Mobile phase: methanol (0.05% trifluoroacetic acid): water (0.05% trifluoroacetic acid)=60:40 (V/V).
Flow velocity: 1.0mL/min.Column temperature: 40 ± 1 DEG C.Sample volume: 10 μ L.
The microcapsule algae toxin standard solution of various concentration is distinguished into sample introduction, Microcystin-first after instrument stabilizer
The standard chromatogram of LR such as Fig. 6 is abscissa mapping with concentration (x), i.e., then respectively with chromatographic peak area (y) for ordinate
For standard curve such as Fig. 7 of microcapsule algae toxin.Finally with the detection limit of 3 times and 10 times signal-to-noise ratio (S/N) calculation methods and calmly
Lower limit such as table 3 is measured, the method detection of microcapsule algae toxin is limited to 0.008 μ g/L, and lower limit of quantitation is 0.028 μ g/L, is below
1.0 μ g/L of standard value is controlled as defined in GB3838-2002.Therefore detection method provided by the invention exists to microcapsule algae toxin
The feature that detection in water has sample pretreatment process easy to operate, sensitive, quick, accuracy, sensitivity reach water quality
The requirement of research and application has important application value suitable for measuring the content of Study on Microcystins in Aquatic Environment-LR.
The method detection limit and lower limit of quantitation of 3 microcapsule algae toxin of table
Wherein: detection limit (3 times)=3*Nd*C*V/ (20*H)；Lower limit of quantitation (10 times)=10*Nd*C*V/ (20*H).
Claims (8)
1. a kind of method of microcapsule algae toxin in efficient liquid phase fluorescence chromatography detection water, which is characterized in that be to utilize micro-capsule
The irradiation of microcystins-LR molecule stimulated luminescence can launch the physicochemical characteristics such as specific fluorescent, thermal stability be good, find and determination
Out under the premise of optimal excitation wavelength and wavelength of transmitted light, water sample is filtered in conjunction with solid phase extraction, is enriched with, is eluted and
Concentration etc. pre-treatments, and optimize high performance liquid chromatography detection condition Microcystins in Water-LR is carried out it is quick, highly sensitive
Detection；Specific step is as follows:
Step 1: the pretreatment of water sample
Water sampling 1000mL, and water sample is crossed to 0.45 μm of water system filter membrane, to Solid Phase Extraction；
Step 2: activation splitter
With 5~15mL of methanol, flow velocity 2.0~5.0mL/min prewashing splitter, then with 5~15mL water, 2.0~5.0mL/ of flow velocity
Min activates splitter, and ensures not allow splitter to drain off；
Step 3: example enrichment
Pretreated 1000mL water sample is subjected to example enrichment after activated splitter with the flow velocity of 5~15mL/min；
Step 4: dry splitter
After first eluting splitter with 2~10mL water with 5~15mL/min flow velocity, then with 5~15mL, 20% methanol with 2~10mL/
Min flow velocity elutes splitter again, finally pushes away separation splitter with 10~30mL high pure nitrogen with 10~30mL/min flow velocity gas,
And nitrogen blows 10~50min of splitter, keeps splitter dry；
Step 5: sample elution and concentration
Syringe pump is cleaned using 0.1% 1~5mL of trifluoroacetic acid methanol solution, 10~30mL/min of flow velocity, then with 0.1%
5~15mL of trifluoroacetic acid methanol, 1~5mL/min of flow velocity elute splitter, then eluent is crossed anhydrous sodium sulfate column and is dehydrated
It is dry, finally splitter is pushed away, and is collected eluent with 5~15mL of nitrogen, flow velocity 10~30mL/min gas；By the eluent of collection
It is concentrated under the conditions of temperature is 35 DEG C~45 DEG C, nitrogen, which is blown, is concentrated into 1.0mL, and crosses 0.45 μm of organic filter membrane, to efficient liquid phase
Chromatograph measurement；
Step 6: measurement
Chromatographic determination condition are as follows: use SHIM PACK CLC-ODS chromatographic column, the excitation wavelength range of fluorescence detector is 249
~254nm, launch wavelength range are 293~298nm；Mobile phase is methanol (0.05% trifluoroacetic acid): water (0.05% trifluoro second
Acid)=(55~65): (35~45) (V/V), 0.8~1.2mL/min of flow velocity, 30~50 DEG C of column temperature, 10 μ L of sample volume.
2. the method for microcapsule algae toxin in a kind of efficient liquid phase fluorescence chromatography detection water according to claim 1,
Be characterized in that, splitter be C18 splitter, prewashing methanol volume be 10mL, flow velocity 3.0mL/min, activation water volume be 10mL,
Flow velocity 3.0mL/min.
3. the method for microcapsule algae toxin in a kind of efficient liquid phase fluorescence chromatography detection water according to claim 1,
It is characterized in that, a kind of method that efficient liquid phase fluorescence chromatography detects microcapsule algae toxin in water described in claim 1, sample
The loading flow velocity that product were enriched with splitter is 10mL/min.
4. the method for microcapsule algae toxin in a kind of efficient liquid phase fluorescence chromatography detection water according to claim 1,
It is characterized in that, for the dry water volume that elutes of splitter for 5mL, flow velocity 10mL/min, used 20% methanol volume is 10mL, flow velocity
5mL/min, it is 20mL, flow velocity 20mL/min that gas, which pushes away the dry used nitrogen volume of splitter, continue nitrogen and blow the splitter time be
25min。
5. the method for microcapsule algae toxin in a kind of efficient liquid phase fluorescence chromatography detection water according to claim 1,
It is characterized in that, it is 2mL that syringe pump, which is cleaned with 0.1% trifluoroacetic acid methanol solution volume, in sample elution period, flow velocity 20mL/
Min, it is 10mL that splitter, which is eluted with 0.1% trifluoroacetic acid methanol solution volume, flow velocity 2mL/min；Gas pushes away used in splitter
N2Volume is 10mL, flow velocity 20mL/min.
6. the method for microcapsule algae toxin in a kind of efficient liquid phase fluorescence chromatography detection water according to claim 1,
It is characterized in that, the thickening temperature of eluent is 40 DEG C.
7. the method for microcapsule algae toxin in a kind of efficient liquid phase fluorescence chromatography detection water according to claim 1,
It is characterized in that, the excitation wavelength lambda of fluorescence detector in sample continuous modeex=251nm, emission wavelength lambdaem=296nm.
8. the method for microcapsule algae toxin in a kind of efficient liquid phase fluorescence chromatography detection water according to claim 1,
It is characterized in that, mobile phase volume ratio is 0.05% trifluoroacetic acid methanol solution: 0.05% trifluoroacetic acid aqueous solution=60:40, stream
Fast 1.0mL/min, 40 DEG C of column temperature.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110186886A (en) * | 2019-05-29 | 2019-08-30 | 扬州大学 | The inversion method of Microcystins in Water MC-LR concentration |
| CN113029994A (en) * | 2021-03-31 | 2021-06-25 | 扬州大学 | Microcystin concentration inversion method based on multi-source characteristic spectrum of extracellular organic matter |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435703A (en) * | 2011-11-25 | 2012-05-02 | 中国环境科学研究院 | Method for simultaneously detecting various microcystins in water |
| CN103048398A (en) * | 2012-12-05 | 2013-04-17 | 上海应用技术学院 | Method for determining microcystin MC-LR in water body |
| US20130196309A1 (en) * | 2012-01-31 | 2013-08-01 | Syracuse University | Woven hydrogel based biosensor |
| CN205003159U (en) * | 2015-08-17 | 2016-01-27 | 大连众信检测技术有限公司 | Microcystic toxins - LR fluorescence immunity quick quantitative detection card |
| CN107037155A (en) * | 2017-04-28 | 2017-08-11 | 哈尔滨工业大学 | Simultaneously in extracting and enriching gauge water typical four kinds of Taste and odor compounds and three kinds of Algae toxins method |
| CN108072763A (en) * | 2016-11-15 | 2018-05-25 | 杭州绿洁水务科技股份有限公司 | The micro-fluidic chip and detection method of Microcystin in a kind of detection water |
-
2018
- 2018-12-24 CN CN201811579440.1A patent/CN109444306A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435703A (en) * | 2011-11-25 | 2012-05-02 | 中国环境科学研究院 | Method for simultaneously detecting various microcystins in water |
| US20130196309A1 (en) * | 2012-01-31 | 2013-08-01 | Syracuse University | Woven hydrogel based biosensor |
| CN103048398A (en) * | 2012-12-05 | 2013-04-17 | 上海应用技术学院 | Method for determining microcystin MC-LR in water body |
| CN205003159U (en) * | 2015-08-17 | 2016-01-27 | 大连众信检测技术有限公司 | Microcystic toxins - LR fluorescence immunity quick quantitative detection card |
| CN108072763A (en) * | 2016-11-15 | 2018-05-25 | 杭州绿洁水务科技股份有限公司 | The micro-fluidic chip and detection method of Microcystin in a kind of detection water |
| CN107037155A (en) * | 2017-04-28 | 2017-08-11 | 哈尔滨工业大学 | Simultaneously in extracting and enriching gauge water typical four kinds of Taste and odor compounds and three kinds of Algae toxins method |
Non-Patent Citations (4)
| Title |
|---|
| MICHAEL J. BROPHY等: "Fluorescence Spectra Predict Microcystin-LR and Disinfection Byproduct Formation Potential in Lake Water", 《ENVIRON. SCI. TECHNOL.》 * |
| 中华人民共和国国家质量监督检验检疫总局: "《中华人民共和国国家标准GB/T 20466-2006》", 31 December 2006 * |
| 朱美洁: "水库型水源地微囊藻毒素污染现状及环境影响因素研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 李好枝: "《体内药物分析》", 31 August 2003 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110186886A (en) * | 2019-05-29 | 2019-08-30 | 扬州大学 | The inversion method of Microcystins in Water MC-LR concentration |
| CN110186886B (en) * | 2019-05-29 | 2022-02-11 | 扬州大学 | Inversion method of microcystin MC-LR concentration in water body |
| CN113029994A (en) * | 2021-03-31 | 2021-06-25 | 扬州大学 | Microcystin concentration inversion method based on multi-source characteristic spectrum of extracellular organic matter |
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