CN103558242A - Methods for extracting and measuring granular organic phosphorus in water body - Google Patents

Abstract

The invention discloses a method for extracting granular organic phosphorus in water, which is characterized in that it comprises the following steps: sampling, filtering, freeze-drying, grinding, and extracting inorganic phosphorus and NaOH solution from EDTA-Na ₂ S ₂ O ₄ mixed solution The step of extracting organic phosphorus in the method; meanwhile, a nuclear magnetic resonance determination method based on the extraction method is also disclosed. The invention is beneficial in that it can detect the specific type of organic phosphorus in the sample; through on-site filtration, the change of the form of organic phosphorus in the sample transportation process is reduced; the amount of sample required for analysis is small, and the workload of on-site filtration is reduced; The analysis and extraction steps are few, which not only reduces the loss of phosphorus in the extraction process, but also saves the time for analyzing samples; it is found that the determination method of the present invention has higher accuracy of analysis and testing after calculation based on the relative recovery rate.

Description

Extraction and Determination Method of Granular Organic Phosphorus in Water Body

technical field

The invention relates to an extraction and determination method of organic phosphorus, in particular to an extraction and determination method of granular organic phosphorus in water, belonging to the field of chemistry.

Background technique

At present, the analysis methods of organic phosphorus in water mainly include subtraction method and step-by-step continuous chemical extraction method. Taking the step-by-step continuous chemical extraction method as an example, referring to Figure 1, it mainly includes sampling, filtration, freeze-drying, grinding, MgCl ₂ extraction of exchangeable phosphorus, NH ₄ F extraction of aluminum phosphorus, NaOH+Na ₂ CO ₃ extraction of iron Phosphorus, HAc+NaAc extraction of self-generated calcium phosphorus, hydrochloric acid extraction of detritus calcium phosphorus, and hydrochloric acid extraction of organic phosphorus after burning, etc., not only require many reagents, but also the operation process is cumbersome. The organic phosphorus content in the table is the content of total organic phosphorus, and the main form and content of granular organic phosphorus cannot be distinguished. The subtraction method has the same problem as the step-by-step continuous chemical extraction method.

Some kinds of particulate organic phosphorus are easily transformed and utilized by aquatic organisms, which is of great significance for understanding the biogeochemical cycle of aquatic ecosystems. However, the organic phosphorus extracted by the two existing methods is only particulate total organic phosphorus, which includes both easily transformed and difficult to be used biologically. Impact.

Contents of the invention

In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a method for extracting particulate organic phosphorus in water bodies with less sampling volume and quick and easy extraction, and a method for extracting particulate organic phosphorus in water bodies with fewer determination steps and higher test accuracy. Method for the determination of organophosphorus.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for extracting particulate organic phosphorus in water, characterized in that it comprises the following steps:

(1) Collect water samples, and use the vacuum filtration system to filter the collected water samples on site, collect the filter membranes, and store them in cold storage;

(2) Rinse the filter membrane with deionized water, keep the rinse solution L ₁ , then soak the filter membrane in deionized water, shake with an oscillator until all solid particles are separated into the water, and keep the extract solution L ₂ , combine L ₁ and L ₂ to obtain solution L _extraction ;

(3) _Extract the solution L and freeze-dry it at -80°C to obtain solid particles S ₀ ;

(4) Grinding solid particles S ₀ , numbering the ground solid particles S ₁ , and storing them sealed in a freezer at -80°C for later use;

(5) Under the condition of -20°C, put the solid particle S ₁ into the mixed solution of EDTA-Na ₂ S ₂ O ₄ with pH=4.5 for shaking pretreatment;

(6) Centrifuge the mixture at -4°C, add the precipitate obtained by centrifugation into 0.1M NaOH solution, and extract at -20°C;

(7) The extract is centrifuged at -4°C, and the supernatant is the final extract of granular organophosphorus.

The aforementioned method for extracting particulate organic phosphorus in water is characterized in that the aforementioned filter membrane is a 0.45 μm cellulose acetate filter membrane.

The method for extracting granular organic phosphorus in the aforementioned water body is characterized in that the aforementioned solid particles _S1 can all pass through a 100-mesh sieve.

The aforementioned method for extracting particulate organic phosphorus in water is characterized in that the aforementioned EDTA-Na ₂ S ₂ O ₄ mixed solution is formed by mixing 0.1M EDTA and 2% (w/v) Na ₂ S ₂ O ₄ .

The aforementioned method for extracting particulate organic phosphorus in water is characterized in that in step (5), the mixed solution is oscillated every 20 minutes, the duration of each oscillating is 1 minute, and the total time of oscillating pretreatment is 2 hours.

The aforementioned method for extracting particulate organic phosphorus in water is characterized in that, in step (6), when the sediment starts to be extracted in NaOH solution, it is shaken every 30 minutes until all samples are frozen into ice cubes.

A method for assaying particulate organic phosphorus in water, characterized in that it comprises the following steps:

(1) Collect water samples, and use the vacuum filtration system to filter the collected water samples on site, collect the filter membrane, the aforementioned filter membrane is a 0.45 μm cellulose acetate filter membrane, and store it in cold storage;

(2) Rinse the filter membrane with deionized water, keep the rinse solution L ₁ , then soak the filter membrane in deionized water, shake with an oscillator until all solid particles are separated into the water, and keep the extract solution L ₂ , combine L ₁ and L ₂ to obtain solution L _extraction ;

(3) _Extract the solution L and freeze-dry it at -80°C to obtain solid particles S ₀ ;

(4) Grind solid particles S ₀ , number the ground solid particles S ₁ , all solid particles S ₁ can pass through a 100-mesh sieve, and store in a sealed freezer at -80°C for later use;

(5) At -20°C, put S ₁ into the EDTA-Na ₂ S ₂ O ₄ mixed solution with pH=4.5 for shaking pretreatment;

(6) Centrifuge the mixture at -4°C, add the precipitate obtained by centrifugation into 0.1M NaOH solution, and extract at -20°C;

(7) The extract was centrifuged at -4°C, and the type and content of organophosphorus in the supernatant was determined by nuclear magnetic resonance.

The aforementioned method for determining particulate organic phosphorus in water is characterized in that in step (5), the mixed solution is oscillated every 20 minutes, the duration of each oscillating is 1 minute, and the total time of oscillating pretreatment is 2 hours.

The aforementioned method for determining particulate organic phosphorus in water is characterized in that, in step (6), when the sediment is extracted in the NaOH solution, it is shaken every 30 minutes until all samples are frozen into ice cubes.

The aforementioned assay method for particulate organic phosphorus in water is characterized in that the conditions of nuclear magnetic resonance are: 90° pulse, 2s cycle delay time, 0.5s capture time, 19000 scan times, ^31P resonance frequency is 161.98MHz , chemical shifts are relative to the signal standard of 85% phosphoric acid.

The invention is beneficial in that: through on-site filtration, the change of organic phosphorus form in the sample transportation process is reduced; the amount of sample required for analysis is small, and the workload of on-site filtration is reduced; there are few analysis and extraction steps, which not only reduces the amount of organic phosphorus in the extraction process Phosphorus loss, and save the time of analyzing the sample; NMR method of the present invention can not only detect the type and content of specific organic phosphorus in the sample, but also can reflect the situation of organic phosphorus in the water body more accurately, the accuracy of its analysis and test higher degree.

Description of drawings

Fig. 1 is the flowchart of the major steps of existing step-by-step continuous chemical extraction method;

Fig. 2 is the flowchart of the main steps of extraction method of the present invention;

Fig. 3 is the NMR figure of granular organic phosphorus in the water body of Dianchi Lake sampling point 1;

Fig. 4 is the nuclear magnetic resonance figure of granular organic phosphorus in the water body of Dianchi Lake sampling point 2;

Fig. 5 is the nuclear magnetic resonance figure of the granular organic phosphorus in the water body of Hulun Lake sampling point;

Fig. 6 is the nuclear magnetic resonance figure of granular organic phosphorus in the water body of Taihu Lake sampling point 1;

Figure 7 is the nuclear magnetic resonance image of particulate organic phosphorus in the water body of Taihu Lake sampling point 2.

The meanings of reference signs in the figure: a—85% phosphate; b—phosphonate; c—DNA; d—pyrophosphate; e—ATPβ-phosphonate; f—unknown organic phosphorus; g—orthophosphate ; h—phosphonate monoester; i—phospholipid; j—polyphosphonate.

Detailed ways

The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

1. Extraction of particulate organic phosphorus in water

Use the water collector to collect 30L of natural water 50cm below the surface of the eutrophic water body (the general collection volume is 20-50L, depending on the content of SS in the water body), and use the vacuum filtration system to filter the collected water samples on site and collect the filter membrane , refrigerated. The filter membrane is Whatman acetate filter membrane (OE67) with a pore size of 0.45 μm.

Collect 200mL water sample L ₀ on site, use molybdenum blue colorimetric method to measure the content C _TP and C _IDP of total phosphorus (TP) and dissolved total phosphoric acid (TDP) in the water body, then the content of particulate total phosphorus (TPP) C _IPP It can be expressed as:

C _IPP =C _TP -C _IDP .

Rinse the filter membrane with 30 mL of deionized water, and keep the rinse solution L ₁ .

Take the rinsed filter membrane with tweezers, soak it in 20mL of deionized water, shake it with an oscillator for 2-3min, until all the solid particles are separated into the water, and keep the extract L ₂ .

Combining _L1 with _L2 is called solution L _extraction . The solution L _{was extracted} and freeze-dried at -80°C to obtain solid particles S ₀ .

Grind the solid particles S ₀ until all the solid particles pass through a 100-mesh sieve, number the ground solid particles S ₁ , and store them sealed in a freezer at -80°C for later use.

Shake S 1 with 25 mL of EDTA-Na ₂ S ₂ O ₄ mixed solution (mixed with 0.1 MEDTA and 2% (w/v) Na ₂ S ₂ O ₄ ) at _-20 °C For pretreatment, shake the mixed solution once every 20 minutes, the duration of each shaking is 1 minute, and the total time of shaking pretreatment is 2 hours.

After 2 hours, the mixture was taken, centrifuged at 10,000 r·min ^-1 for 10 minutes at -4°C, and the supernatant and precipitate were separated. in,

The concentration of TP and phosphate ion (DIP) contained in the supernatant was measured by the molybdenum blue colorimetric method, and they were counted as C' _TP and C' _DIP respectively, then the corresponding content of TP and DIP in the natural water body C ₀ ' _TP and C ₀ ' _DIP can be converted, and the content percentage W ₁ of inorganic phosphorus in the mixed extract of EDTA-Na ₂ S ₂ O ₄ can also be calculated:

${\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; =</span>\frac{{{\mathrm{<span\; class="notranslate">\; C</span>}}^{<span\; class="notranslate">\; \&prime;</span>}}_{\mathrm{<span\; class="notranslate">\; DIP</span>}}}{{{\mathrm{<span\; class="notranslate">\; C</span>}}^{<span\; class="notranslate">\; \&prime;</span>}}_{\mathrm{<span\; class="notranslate">\; TP</span>}}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; \%</span><span\; class="notranslate">\; .</span>$

The precipitate obtained by centrifugation was added to 10mL of 0.1M NaOH solution, and extracted at -20°C for 16 hours. In the first 5-6 hours, shake once every 30 minutes to facilitate extraction until all samples were frozen into ice cubes. Then centrifuge at -4°C and 10000r min ^-1 for 10min, and the supernatant is the final extract of granular organophosphate.

2. Determination of particulate organic phosphorus in water

The final extract of granular organophosphorus was taken, and the type and content of organophosphorus in it were tested by nuclear magnetic resonance method.

NMR method: the instrument is Bruker AV400 NMR spectrometer with 5mm BBO probe, 90° pulse, cycle delay time 2s, capture time 0.5s, scan times 19000 times, the resonance frequency of ³¹ P is 161.98 MHz, and the chemical shift is Relative to the signal standard of 85% phosphoric acid.

The test results are shown in Figure 3 to Figure 7.

The total content of organic phosphorus is calculated as W ₂ '.

3. Verification of measurement results

Take the same granular organophosphorus final extract, measure the content of TP and DIP in the final extract respectively by molybdenum blue colorimetry, count as C'' _TP and C'' _DIP respectively, then TP and DIP in natural water The corresponding content C ₀ '' _TP and C ₀ '' _DIP can be converted, and the content percentage W ₂ '' of organic phosphorus in the NaOH extract can also be calculated:

${{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}^{<span\; class="notranslate">\; \&prime;</span><span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; =</span>\frac{{{\mathrm{<span\; class="notranslate">\; C</span>}}^{<span\; class="notranslate">\; \&prime;</span><span\; class="notranslate">\; \&prime;</span>}}_{\mathrm{<span\; class="notranslate">\; TP</span>}}<span\; class="notranslate">\; -</span>{{\mathrm{<span\; class="notranslate">\; C</span>}}^{<span\; class="notranslate">\; \&prime;</span><span\; class="notranslate">\; \&prime;</span>}}_{\mathrm{<span\; class="notranslate">\; DIP</span>}}}{{{\mathrm{<span\; class="notranslate">\; C</span>}}^{<span\; class="notranslate">\; \&prime;</span><span\; class="notranslate">\; \&prime;</span>}}_{\mathrm{<span\; class="notranslate">\; TP</span>}}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; \%</span><span\; class="notranslate">\; .</span>$

Calculate the extraction efficiency η:

${\mathrm{<span\; class="notranslate">\; \&eta;</span>}}_{\mathrm{<span\; class="notranslate">\; TP</span>}}<span\; class="notranslate">\; =</span>\frac{{{{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="0"\; label="C"\; filenames="HDA0000413603560000022.png,HDA0000413603560000023.png"\; state="\{\{state\}\}">C</figure-callout></span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}}^{<span\; class="notranslate">\; \&prime;</span>}}_{\mathrm{<span\; class="notranslate">\; TP</span>}}<span\; class="notranslate">\; +</span>{{{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="0"\; label="C"\; filenames="HDA0000413603560000022.png,HDA0000413603560000023.png"\; state="\{\{state\}\}">C</figure-callout></span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}}^{<span\; class="notranslate">\; \&prime;</span><span\; class="notranslate">\; \&prime;</span>}}_{\mathrm{<span\; class="notranslate">\; TP</span>}}}{{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; TPP</span>}}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; \%</span><span\; class="notranslate">\; .</span>$

The verification results are shown in Table 1.

Table 1 Comparison table of measurement results

Method of the invention Molybdenum blue colorimetry W ₂ '/ W ₂ '' Dianchi Lake sampling point 1 W ₂ '=38.2μg·L ^-1 W ₂ ''=41.0μg·L ^-1 93.2% Dianchi Lake sampling point 2 W ₂ '=26.3μg·L ^-1 W ₂ ''=32.0μg·L ^-1 82.2% Hulun Lake sampling point W ₂ '=35.8μg·L ^-1 W ₂ ''=51.0μg·L ^-1 70.2% Taihu Lake Sampling Point 1 W ₂ '=126.3μg·L ^-1 W ₂ ''=199.0μg·L ^-1 63.5% Taihu Lake Sampling Point 2 W ₂ '=38.7μg·L ^-1 W ₂ ''=40.0μg·L ^-1 96.7%

In summary, 1, adopt the extraction method of the present invention, can extract 9 kinds of natural organic phosphorus, some kinds of organic phosphorus (such as DNA, polyphosphate and orthophosphate monoester) are easily converted and then bioutilized, It is of great significance to understand the biogeochemical cycle of phosphorus in water.

2. Compared with the molybdenum blue colorimetric method based on the NMR assay method of the extraction method of the present invention, the result measured by the NMR method is lower, that is, W ₂ '/W ₂ ''<1, because NMR measures Organic phosphorus is the main type of organic phosphorus in water bodies, and a large number of previous experiments have shown that W ₂ '/W ₂ ''>60%, which represents the main type of organic phosphorus in water bodies, so W ₂ ' can be used for Represents the situation of organic phosphorus in water.

3. Using this method to analyze and measure more than 20 lakes and reservoirs in the country, the extraction efficiency of organic phosphorus is greater than 82%, and the measurement effect is good. The proportion of the main organic phosphorus content in the water body to the total organic phosphorus in the nuclear magnetic resonance measurement is more than 60% %, and the higher the nutrient level of the water body, the better the extraction effect.

In addition, compared with the subtraction method, the extraction method of the present invention is a direct extraction method for organic phosphorus in the water body, and its result is more accurate; compared with the continuous chemical analysis method, the steps are greatly reduced, saving the time for analyzing samples. Therefore, the extraction method of the present invention and the determination method based on the extraction method have outstanding substantive features and significant progress.

It should be noted that the above embodiments do not limit the present invention in any form, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims (10)

1. the extraction method of granular organic phosphorus in water body is characterized in that, comprises the following steps: (1) Collect water samples, and use the vacuum filtration system to filter the collected water samples on site, collect the filter membranes, and store them in cold storage; (2) Rinse the filter membrane with deionized water, keep the rinse solution L ₁ , then soak the filter membrane in deionized water, shake with an oscillator until all solid particles are separated into the water, and keep the extract solution L ₂ , combine L ₁ and L ₂ to obtain solution L _extraction ; (3) _Extract the solution L and freeze-dry it at -80°C to obtain solid particles S ₀ ; (4) Grinding solid particles S ₀ , numbering the ground solid particles S ₁ , and storing them sealed in a freezer at -80°C for later use; (5) Under the condition of -20°C, put the solid particle S ₁ into the mixed solution of EDTA-Na ₂ S ₂ O ₄ with pH=4.5 for shaking pretreatment; (6) Centrifuge the mixture at -4°C, add the precipitate obtained by centrifugation into 0.1M NaOH solution, and extract at -20°C; (7) The extract is centrifuged at -4°C, and the supernatant is the final extract of granular organophosphorus. 2. The method for extracting particulate organic phosphorus in water according to claim 1, wherein the filter membrane is a cellulose acetate filter membrane of 0.45 μm. 3. the extraction method of granular organic phosphorus in the water body according to claim 1, is characterized in that, solid particle _S All can pass through 100 mesh sieves. 4. The method for extracting particulate organic phosphorus in water according to claim 1, wherein the EDTA-Na ₂ S ₂ O ₄ mixed solution is composed of 0.1M EDTA and 2% (w/v) Na ₂ S ₂ O ₄ mixed. 5. The method for extracting particulate organic phosphorus in water according to any one of claims 1 to 4, characterized in that in step (5), the mixed solution is oscillated once every 20 minutes, and the duration of each oscillation is The total time of shaking pretreatment is 2 hours. 6. The method for extracting particulate organic phosphorus in water according to any one of claims 1 to 4, characterized in that, in step (6), when the sediment starts to be extracted in NaOH solution, it is shaken once every 30 minutes until all samples are frozen. 7. the assay method of granular organic phosphorus in water body, is characterized in that, comprises the following steps: (1) Collect water samples, and use the vacuum filtration system to filter the collected water samples on site, and collect the filter membrane, which is a 0.45 μm cellulose acetate filter membrane, and store it in cold storage; (2) Rinse the filter membrane with deionized water, keep the rinse solution L ₁ , then soak the filter membrane in deionized water, shake with an oscillator until all solid particles are separated into the water, and keep the extract solution L ₂ , combine L ₁ and L ₂ to obtain solution L _extraction ; (3) _Extract the solution L and freeze-dry it at -80°C to obtain solid particles S ₀ ; (4) Grind solid particles S ₀ , number the ground solid particles S ₁ , all solid particles S ₁ can pass through a 100-mesh sieve, and store in a sealed freezer at -80°C for later use; (5) At -20°C, put S ₁ into the EDTA-Na ₂ S ₂ O ₄ mixed solution with pH=4.5 for shaking pretreatment; (6) Centrifuge the mixture at -4°C, add the precipitate obtained by centrifugation into 0.1M NaOH solution, and extract at -20°C; (7) The extract was centrifuged at -4°C, and the type and content of organophosphorus in the supernatant was determined by nuclear magnetic resonance. 8. The method for measuring particulate organic phosphorus in water according to claim 7, characterized in that in step (5), the mixed solution is oscillated once every 20 min, and the duration of each oscillation is 1 min, and the oscillation pre- The total time for processing was 2h. 9. The method for measuring particulate organic phosphorus in water according to claim 7, characterized in that, in step (6), when the sediment starts to be extracted in the NaOH solution, shake once every 30 minutes until all samples are Freeze into ice cubes. 10. the assay method of granular organophosphorus in water according to claim 7, is characterized in that, the condition of nuclear magnetic resonance is: 90 ° of pulses, cycle delay time 2s, capture time 0.5s, number of scans is 19000 times, ³¹ P has a resonance frequency of 161.98 MHz and chemical shifts are relative to the signal standard of 85% phosphoric acid.

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