CN114235721A - Method for measuring total phosphorus concentration in fresh water body - Google Patents
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Abstract
The invention discloses a method for measuring the concentration of total phosphorus in a fresh water body, which comprises the following steps: preparing 5mL of each phosphorus working solution with different monopotassium phosphate concentrations, digesting each phosphorus working solution by adopting potassium persulfate or sodium persulfate, diluting the digested phosphorus working solution to 5mL by using ultrapure water, respectively adding ascorbic acid solution into each digested working solution, uniformly mixing, and reducing; adding molybdate solution, mixing, developing, referring to blank, and determining light absorption value and blank light absorption value of the working solution; drawing a standard working curve and solving a regression equation of the standard working curve; measuring 5mL of fresh water sample, and measuring and recording the absorbance of the water sample according to the same treatment steps as the phosphorus working solution; and (6) calculating a result. Compared with the national standard, the method has the advantages of wide measurement range of the total phosphorus concentration of the fresh water, high safety, simple operation, high working efficiency, low measurement cost, reduction of secondary pollution and the like.
Description
Technical Field
The invention particularly relates to a method for measuring the concentration of total phosphorus in a fresh water body.
Background
Phosphorus is an indispensable nutrient substance in various natural water bodies and is one of major elements necessary for biological growth, the growth of most algae in fresh water bodies is mainly limited by phosphorus, the change of the phosphorus concentration in the water bodies also has important influence on the total amount of phytoplankton and community structure, and when the concentration of soluble phosphorus in the water bodies is lower than 0.01 mg.L-1In time, part of the phytoplankton species are restricted from growing, the lower their concentration the more species are restricted, the phosphorus content controls the level of primary productivity in natural waters. In conclusion, the monitoring of phosphorus in water is the basic content of the research on zooplankton ecology, water quality management and water ecology.
Eutrophication of water is a phenomenon in which excessive proliferation of plankton such as algae, etc., due to the reception of excessive amounts of nutrients such as nitrogen, phosphorus, etc., causes deterioration of water quality and deterioration of water transparency. The problem of eutrophication of water bodies has become a ubiquitous environmental problem worldwide. The water eutrophication evolves very slowly under natural conditions, but with the rapid development of industrial and agricultural production, the development demand of human beings on environmental resources increases day by day, and a large amount of nutrient substances enter and accumulate in lakes, which causes the lake eutrophication to appear in a short period, so that human activities play an important role in accelerating the water eutrophication process. In addition, the input intensity, occurrence form, migration and transformation rule of phosphorus in the region of the outer river estuary are also one of the decisive factors influencing the eutrophication process of the lake. In conclusion, phosphorus is a key factor of water eutrophication, is one of important indexes for measuring the quality of water quality, analyzes long-term detection data of the water, pays attention to the content change of TP in the water, and becomes important detection and monitoring content of the eutrophication process and ecological change of natural water such as rivers, lakes and the like.
At present, a plurality of methods for measuring the TP in the fresh water comprise a spectrophotometric method, an atomic spectrometry method, a chromatography method and the like, and because of the advantages of low cost investment, simple operation, stable measurement result and the like of the ammonium molybdate spectrophotometry (hereinafter referred to as the national standard method) which is a determination method of total phosphorus in water quality in the national standard of the people's republic of China, the national standard method has wider application in actual water quality measurement. However, the national standard method has a narrow test range, and for water samples exceeding the upper test limit, the samples need to be subjected to gradient dilution, so that the operation process is not only complicated, but also the measurement precision is influenced; in addition, in the operation process of the method, an autoclave is required for digestion, in the process, the required reaction time is long, the determination efficiency is influenced, the requirement on the sealing property of the container is high, a large amount of gauze, strings and other sealing tools are required during sealing, certain difficulty is caused to the experiment operation, the phenomenon of untight sealing is easy to occur, and the solution rushes out of the container in the digestion process, so that the experiment result is influenced. In addition, still there is certain potential safety hazard at the in-process of clearing up, need have personnel's watch on to observe when using, also difficult control in the processing to pressure and temperature simultaneously, even intelligent pressure cooker, the precision promotes on the control of pressure and temperature, still will influence final result to some extent.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the method for measuring the total phosphorus concentration in the fresh water body, which has the advantages of improving the measurement range and the measurement precision of the total phosphorus concentration in the fresh water, along with high safety, simple operation, high working efficiency, low measurement cost and less secondary pollution.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for measuring the concentration of total phosphorus in a fresh water body comprises the following steps:
s1: and (3) standard working curve formulation:
s1.1: concentrations of 0, 0.04, 0.08, 0.24, 0.4 and 0.8. mu.g.mL were prepared in six 18X 250mm round bottom screw cap digestion tubes, respectively, using phosphorus standard use solutions-15mL of each of the phosphorus standard solutions, wherein the concentration of potassium dihydrogen phosphate in the phosphorus standard solution is 2. mu.g.mL-1；
S1.2: adding 70-90 μ L of 0.05 g/mL into each phosphorus working solution-1The potassium persulfate or the sodium persulfate is placed in a digestion instrument for digestion for 30-40min, and is diluted to 5mL by ultrapure water after digestion;
s1.3: adding 20 μ L of working solution with concentration of 0.08-0.14 g/mL into each digested working solution-1Mixing the ascorbic acid solution uniformly, and reducing for 30 s; adding 40 mu L of molybdate solution, fully mixing, developing for 15min, wherein the molybdate solution is generated by reacting ammonium molybdate solution and antimony potassium tartrate solution, and the concentration of the ammonium molybdate solution and the antimony potassium tartrate solution is 15.50+0.12 mol/mL-1-38.69+0.29mol·mL-1；
S1.4: after the color development is finished, the absorbance A of the working solution is measured at a wavelength of 700nm by using a cuvette with an optical length of 10mm and taking a blank as a references；
S1.5: measuring 5mL of a blank water sample, and measuring and recording the absorbance A of the blank water sample according to the same treatment steps as the phosphorus working solutionb；
S1.6: with AsDeduction AbThe latter absorbance value AnAs ordinate, concentration C of phosphorus working solutionsDrawing a standard working curve for an abscissa, and solving an intercept a and a slope b of the standard working curve by using a linear regression method;
s2: water sample determination
Measuring 5mL of fresh water sample, and measuring and recording the absorbance of the water sample according to the same treatment steps as the phosphorus working solution;
s3: and (4) calculating a result:
the total phosphorus concentration in the fresh water sample is calculated according to the formula (1):
in the formula (I), the compound is shown in the specification,
c (TP) is the concentration of total phosphorus in the fresh water sample, and the unit is mu g.mL-1；
AwThe light absorption value of total phosphorus in the water sample;
Abblank light absorption values;
a is the intercept of a standard working curve;
b is the standard operating curve slope.
Optionally, the steps S1 and S2 further include:
digesting 5mL of fresh water sample by the method in the step S1.2, adding 60 mu L of turbidity-chromaticity compensation liquid, using a cuvette with an optical path of 10mm and taking a blank water sample as a reference under the wavelength of 700nm, and determining the absorbance A of the turbidity of the fresh water samplet，
The total phosphorus concentration in the fresh water sample is calculated according to the formula (2):
in the formula, AtThe absorbance value of turbidity in the water sample.
Optionally, in the step S1.2, the digestion pressure is 1.1kg/cm2The temperature was 120 ℃.
Optionally, the digestion time is 30 min.
Alternatively, the amount of potassium persulfate or sodium persulfate is 80 μ L.
Optionally, the concentration of the ascorbic acid solution is 0.1 g-mL-1。
Optionally, the molybdate solution is generated by reacting an ammonium molybdate solution and a potassium antimony tartrate solution, and the concentration of the ammonium molybdate solution and the potassium antimony tartrate solution is 31.00+0.32 mol/mL-1。
Alternatively, the molybdate solution is formulated as follows:
respectively dissolving 13g of ammonium molybdate and 0.35g of antimony potassium tartrate in 100mL of water, slowly adding the ammonium molybdate solution into 300mL of 50% sulfuric acid under continuous stirring, adding the antimony potassium tartrate solution, and fully and uniformly mixing.
Optionally, the turbidity-chromaticity compensation solution is prepared by the following method:
taking 50% sulfuric acid and a volume of 0.1 g.mL-1Ascorbic acid (b) was mixed well.
Compared with the prior art, the invention has the advantages that:
compared with a national standard method fresh water TP determination method, the method uses a digestion tube with a round bottom and a screw cover of 18-250 mm to replace a 50mL digestion tube as a reaction container, changes the effective reaction volume of TP determination from 25mL to 5mL, and optimizes five aspects of oxidant type, oxidant adding amount, digestion time, reducing agent ascorbic acid adding amount and color reagent molybdate adding amount. The new method for testing the fresh water TP is verified by adopting standard curve fitting and actually-measured environmental samples, and the result shows that: the small system for measuring the fresh water TP has good accuracy, precision, feasibility and applicability, and the feasibility of measuring the fresh water TP by the small system is proved. Furthermore, the detection range of the small-system rapid determination of TP concentration in fresh water determined by the research is 0-2 mug.mL-1Compared with the national standard method, the fresh water TP determination range is 0-0.6 mug/mL-1The measuring range of the experimental method is nearly four times enlarged. The research of the small-scale determination method obviously improves the upper limit of the fresh water TP determination concentration, can effectively determine water samples with higher TP concentration, saves a plurality of medicines and time consumed by gradient dilution, and greatly improves the efficiency of determining a large amount of water samples.
Compared with the method for measuring the fresh water TP by the national standard method, the method has the advantages that the consumption of medicines is obviously reduced, the waste amount of solution is greatly reduced, the pollution to the environment is reduced to the minimum while the measurement cost is saved, the measurement efficiency is improved, and the upper limit of measuring the fresh water TP is improved. The method provides a simple, convenient and rapid determination method for large-scale environmental sample determination and TP concentration higher sample determination, is favorable for realizing rapid detection and real-time dynamic monitoring of the TP content of the fresh water quality, and provides powerful technical support for exploring the eutrophication process and ecological change of natural water bodies such as lakes, rivers and the like.
Drawings
FIG. 1 is a graph showing the influence of the type of oxidizing agent on the fresh water TP measurement method of the present invention.
FIG. 2 is a graph showing the orthogonal validation of the concentration of TP in water by potassium persulfate and sodium persulfate as oxidants.
FIG. 3 is a graph showing the influence of the amount of potassium persulfate added as an oxidizing agent on the method for measuring fresh water TP of the present invention.
FIG. 4 is a graph showing the effect of digestion time on the fresh water TP assay method of the present invention.
FIG. 5 is a graph showing the effect of the amount of ascorbic acid as a reducing agent on the method for measuring fresh water TP of the present invention.
FIG. 6 is a graph showing the influence of the amount of the color-developing agent on the fresh water TP measurement method of the present invention.
FIG. 7 is a linear fitting graph of the standard curve for measuring the concentration of TP in fresh water by the small body system method and the national standard method.
FIG. 8 is an orthogonal verification curve diagram of the standard curve for measuring the concentration of TP in fresh water by the small body system method and the national standard method.
FIG. 9 is a diagram showing the detection range of the present invention for fresh water TP by the small body system method.
FIG. 10 is a graph of orthogonal validation of TP content of environmental samples measured by the small-system method and the national standard method according to the present invention.
Detailed Description
The invention is further described below with reference to specific preferred embodiments, without thereby limiting the scope of protection of the invention.
Example 1:
instrument and consumable
An ultraviolet spectrophotometer, an LX series pressure steam sterilization pot (self-control of a fastening type LX-B35L), a Lianhua digestion instrument (instrument model: LH-25A), a beaker, a digestion tube of 18 mm to 250mm, a 50mL graduated tube with a plug, a 10mm cuvette, a 25mL pipette, a 10mL pipette, a 5mL pipette, a 2mL pipette, a 1mL pipette, an ear washing ball, a 1000 muL pipette gun, a 200 muL pipette gun and a plurality of tips.
2.2 reagent preparation
The preparation of reagents for fresh water environment TP content determination is shown in Table 1.
TABLE 1TP assay reagent preparation
A method for measuring the concentration of total phosphorus in a fresh water body comprises the following steps:
s1: and (3) standard working curve formulation:
s1.1: concentrations of 0, 0.04, 0.08, 0.24, 0.4 and 0.8. mu.g.mL were prepared in six 18X 250mm round bottom screw cap digestion tubes, respectively, using phosphorus standard use solutions-15mL of each of the phosphorus standard solutions, wherein the concentration of potassium dihydrogen phosphate in the phosphorus standard solution is 2. mu.g.mL-1；
S1.2: 80 μ L of each phosphorus working solution was added to the solution at a concentration of 0.05 g/mL-1The potassium persulfate or the sodium persulfate is placed in a digestion instrument for digestion, and the digestion pressure is 1.1kg/cm2Digesting at 120 ℃ for 30min, and diluting to 5mL by using ultrapure water after digestion;
s1.3: to each digested working solution was added 20. mu.L of 0.1 g/mL-1Mixing the ascorbic acid solution uniformly, and reducing for 30 s; adding 40 mu L of molybdate solution, fully mixing, developing for 15min, wherein the molybdate solution is generated by reacting ammonium molybdate solution and antimony potassium tartrate solution, and the concentration of the ammonium molybdate solution and the antimony potassium tartrate solution is 31.00+0.32 mol/mL-1；
S1.4: after the color development is finished, the absorbance A of the working solution is measured at a wavelength of 700nm by using a cuvette with an optical length of 10mm and taking a blank as a references；
S1.5: measuring 5mL of a blank water sample, and measuring and recording the absorbance A of the blank water sample according to the same treatment steps as the phosphorus working solutionb；
S1.6: with AsDeduction AbThe latter absorbance value AnAs ordinate, concentration C of phosphorus working solutionsDrawing a standard working curve for an abscissa, and solving an intercept a and a slope b of the standard working curve by using a linear regression method;
s2: water sample determination
Measuring 5mL of fresh water sample, and measuring and recording the absorbance of the water sample according to the same treatment steps as the phosphorus working solution;
s3: turbidity measurement
Digesting 5mL of fresh water sample by the method in the step S1.2, adding 60 mu L of turbidity-chromaticity compensation liquid, using a cuvette with an optical path of 10mm and taking a blank water sample as a reference under the wavelength of 700nm, and determining the absorbance A of the turbidity of the fresh water samplet，
S3: and (4) calculating a result:
the total phosphorus concentration in the fresh water sample is calculated according to the formula (2):
in the formula (I), the compound is shown in the specification,
c (TP) is the concentration of total phosphorus in the fresh water sample, and the unit is mu g.mL-1；
AwThe light absorption value of total phosphorus in the water sample;
Abblank light absorption values;
Atthe light absorption value of turbidity in the water sample;
a is the intercept of a standard working curve;
b is the standard operating curve slope.
Compared with the method for measuring the TP in fresh water by the national standard method, the invention uses the round bottomA digestion tube with a screw cover of 18 mm and 250mm replaces a digestion tube with 50mL as a reaction container, the effective reaction volume of TP measurement is changed from 25mL to 5mL, and the five aspects of the type of an oxidant, the adding amount of the oxidant, the digestion time, the adding amount of a reducing agent ascorbic acid and the adding amount of a color developing agent molybdate are optimized. The new method for testing the fresh water TP is verified by adopting standard curve fitting and actually-measured environmental samples, and the result shows that: the small system for measuring the fresh water TP has good accuracy, precision, feasibility and applicability, and the feasibility of measuring the fresh water TP by the small system is proved. Furthermore, the detection range of the small-system rapid determination of TP concentration in fresh water determined by the research is 0-2 mug.mL-1Compared with the national standard method, the fresh water TP determination range is 0-0.6 mug/mL-1The measuring range of the experimental method is nearly four times enlarged. The research of the small-scale determination method obviously improves the upper limit of the fresh water TP determination concentration, can effectively determine water samples with higher TP concentration, saves a plurality of medicines and time consumed by gradient dilution, and greatly improves the efficiency of determining a large amount of water samples.
Compared with the method for measuring the fresh water TP by the national standard method, the method has the advantages that the consumption of medicines is obviously reduced, the waste amount of solution is greatly reduced, the pollution to the environment is reduced to the minimum while the measurement cost is saved, the measurement efficiency is improved, and the upper limit of measuring the fresh water TP is improved. The method provides a simple, convenient and rapid determination method for large-scale environmental sample determination and TP concentration higher sample determination, is favorable for realizing rapid detection and real-time dynamic monitoring of the TP content of the fresh water quality, and provides powerful technical support for exploring the eutrophication process and ecological change of natural water bodies such as lakes, rivers and the like.
Example 2:
this example specifically explains the inventive procedure of example 1:
1. design of experiments
1.1 fresh water TP determination influence factor optimization
In order to determine the optimal reaction conditions for the improved method, the present study designed a test for determining the influence of the type of the oxidizing agent, the amount of the oxidizing agent, the oxidation time, the amount of the reducing agent, and the amount of the color-developing agent on the fresh water TP determination.
(1) Selecting the oxidant category: 50mL graduated tubes and 18. mu.250 mm digestion tubes were used as reactors at room temperature, respectively, and concentration gradients of 0, 0.04, 0.08, 0.24, 0.4 and 0.8. mu.g. mL were set-1In order to further explore the precision difference of the detection results of the two oxidants, the results of the phosphorus standard use solution with different oxidants for measuring the same concentration gradient are subjected to linear fitting analysis of a standard working curve and cross validation of the two measurement results by adopting an orthogonal validation method, namely the results measured by a potassium persulfate system are taken as an abscissa and the results measured by a sodium persulfate system are taken as an ordinate, a standard working curve is established, linear cross fitting is performed, and the optimal oxidant type measured by the fresh water TP is determined by combining F test results and t test results and comprehensively measuring the cost;
(2) optimizing the dosage of the oxidant: on the premise of ensuring the consistency of the oxidant, the adding amount of the oxidant is changed at room temperature, 20, 40, 60, 80, 100 and 1200 mu L of room-temperature oxidant potassium persulfate is respectively added, and standard phosphorus use solutions (0, 0.04, 0.08, 0.24, 0.4 and 0.8 mu g/mL) with different concentrations are added-1) Oxidizing to research the influence of the adding amount of the potassium persulfate oxidant on the fresh water TP determination, setting six groups for each concentration sample, setting two parallel groups for each concentration sample, and performing graphical analysis on the final result to determine the optimal oxidant adding amount for the fresh water TP determination;
(3) digestion time optimization: the oxidation time was varied at room temperature to ensure consistency of oxidant and oxidant usage, and the standard use solutions (0, 0.04, 0.08, 0.24, 0.4, and 0.8 μ g/mL) for different concentrations of phosphorus were used-1) Performing oxidation, setting five groups for each concentration sample, setting two groups in parallel for each group, performing an experiment on influence of oxidation time (10 min, 20min, 30min, 40min and 50min) on fresh water TP determination, and performing mapping and partitioning on final resultsAnalyzing to determine the optimal digestion time of the fresh water TP determination;
(4) optimizing the dosage of the reducing agent: ensuring the consistency of the dosage of the oxidant, the oxidant and the oxidation time, changing the dosage of the reducing agent ascorbic acid at room temperature, and carrying out standard use solutions (0, 0.04, 0.08, 0.24, 0.4 and 0.8 mu g/mL) on phosphorus with different concentrations-1) Carrying out oxidation, setting five groups of concentration samples, setting two parallel groups in each group, and carrying out mapping analysis on the final result so as to determine the optimal reducing agent adding amount of the fresh water TP determination;
(5) optimizing the use amount of the color developing agent: to investigate the effect of the developer molybdate solution on fresh water TP assay, five sets of concentration gradients were set at 0, 0.04, 0.08, 0.24, 0.4 and 0.8. mu.g.mL-1The phosphorus standard use solution is provided with six groups of concentration samples, each group is provided with two parallel groups, an influence experiment of the color developing agent on the fresh water TP determination is carried out, and the final result is subjected to mapping analysis to determine the optimal color developing agent adding amount of the fresh water TP determination.
1.2, drawing of Standard working Curve
According to a method for determining TP in a fresh water body by a national standard method, a standard working curve of an ammonium molybdate spectrophotometry under a small system is drawn, and the method comprises the following specific steps:
(1) putting 4mL of phosphorus standard stock solution into a 100mL volumetric flask, and adding ultrapure water to dilute the stock solution to a marked line;
(2) respectively adding 0mL, 0.1 mL, 0.2 mL, 0.6 mL, 1mL and 2mL of standard phosphorus use solution into two groups of digestion tubes with six round bottom screw caps of 18 mm and 250mm, and diluting to 5mL by using ultrapure water;
(3) adding 80 mu L of potassium persulfate solution as an oxidant, and reacting at 120 ℃ for 30 min;
(4) adding 20 μ L ascorbic acid solution as reducing agent, mixing, and standing for 30 s;
(5) adding 40 μ L molybdate solution as color developing agent, mixing, and standing for 15 min;
note: a, if the solution to be detected contains a turbidity or chromaticity influence measurement result, a blank reagent (diluted to 5mL by ultrapure water after digestion) needs to be prepared, 60 mu L of turbidity-chromaticity compensation solution is added, but ascorbic acid and molybdate solution are not added, and then the absorbance of the blank reagent is subtracted from the absorbance of the solution to be detected.
b arsenic is more than 0.002 mg/mL-1The assay was interfered with and removed with sodium thiosulfate. Sulfide is more than 0.002 mg/mL-1And (5) interfering the measurement, and introducing nitrogen to remove. Chromium is greater than 0.05 mg/mL-1Interference measurement, removal with sodium sulfite.
(6) After full reaction, taking a proper amount of solution to be detected in a cuvette with the thickness of 10mm, measuring the light absorption value of each sample at 700nm and recording data;
(7) and drawing a standard working curve and solving a linear regression equation of the standard working curve.
2. TP determination new method accuracy verification
2.1, the fitting verification of the standard working curve of the new TP determination method is to verify the accuracy and reliability of the standard curve of the new method for determining the fresh water TP, the fresh water TP is determined by using a small system and a national standard method, meanwhile, the standard working curve of the fresh water TP is established by using the determination result, the standard working curve established by the two methods is subjected to linear fitting analysis and an orthogonal verification method is adopted, namely, phosphorus standard use solution with the same concentration gradient is simultaneously determined by using the two methods, the result determined by using the national standard method is used as an abscissa, the result determined by using the small system is used as an ordinate, the linear fitting is carried out, and meanwhile, the determination result is subjected to cross verification.
2.4.2 verification of accuracy of actual measurement environmental sample TP five fresh water aquaculture surface waters of a culture pond near the western city school district of Guangdong petrochemical industry institute No. 1, scientific way of Mingmenao and a west lake of the official transition school district of Guangdong petrochemical industry institute are collected and are respectively numbered as 1, 2, 3, 4 and 5. After the water body sample is collected, the water body sample is directly filled into a PETG material plastic bottle and is stored at the temperature of 4 ℃. After the sample is sent back to the laboratory, the TP content in the sample is simultaneously measured by a small-body system measuring method and a national standard method respectively. And performing linear fitting analysis on the measurement result by using a standard working curve, and performing cross validation on the two results by using an orthogonal validation method, namely performing linear fitting by using the measurement result of the small body system as an abscissa and the measurement result of the national standard method as an ordinate. And the feasibility, the accuracy and the reliability of the small system for measuring the TP of the water body are further verified by utilizing F test and t test.
3. Results and discussion
3.1 Effect of oxidant classes on fresh Water TP determination
The oxidizing agent is an important factor influencing the total oxidation of phosphorus in the water body into orthophosphate, in order to optimize the process of converting phosphorus in the water body into orthophosphate, the experiment researches the influence of different oxidizing agents on the oxidation process, and the result of the influence of two oxidizing agents, namely potassium persulfate and sodium persulfate, on the TP standard working curve is shown in figure 1. To further illustrate the high agreement between the results of the two oxidant measurements, the results of the two oxidant measurements on TP were cross-validated orthogonally, as shown in fig. 2: the linear equation for measuring the TP content of the fresh water by the two oxidants is Y-0.9819X-0.0014 (Y is the absorbance of sodium persulfate, and X is the absorbance of potassium persulfate), and the linear correlation coefficient R2The result of the two determination methods is highly consistent, the experimental results of TP determination by using two oxidants of potassium persulfate and sodium persulfate are basically the same, the F value of the test result of water sample by using potassium persulfate as the oxidant and sodium persulfate as the oxidant is 0.001 and is less than F table 5.318 through F detection with the confidence of 95%, and the precision of fresh water TP determination by using the two oxidants is not obviously different. The results of the two oxidant assays, sig 0.973 > 0.05, were tested with a confidence of 95% t, indicating that the two oxidants had no significant effect on the nitrogen TP assay. In summary, both of these oxidizing agents can be used as the oxidizing agent for measuring the total phosphorus TP content in fresh water by ammonium molybdate spectrophotometry, but from the aspect of experimental cost, the following experiment is continued by selecting potassium persulfate as the oxidizing agent in the present study.
3.2 influence of oxidant dose on fresh water TP assay TP standard use solutions were oxidized using different doses of potassium persulfate, the results are shown in FIG. 3: with the continuous increase of the adding amount of the potassium persulfate, the absorbance of the reaction substrate is not obviously changed, namely, the TP measuring results are not greatly influenced by different concentrations of the oxidant. Considering that the insufficient amount of the oxidizing agent causes the problem of insufficient oxidation of the substrate, the amount of the oxidizing agent used in the method is 80 muL according to the proportion of the oxidizing agent used in the national standard method.
3.3 Effect of digestion time on fresh water TP determination
The digestion time is an important influence factor for converting phosphorus in a water body into orthophosphate, different digestion times influence the determination of TP in fresh water, and the result is shown in figure 4: digestion time is 10-20min, absorbance of TP digestion substrates with various concentrations is reduced, absorbance is increased in 20-30min, and absorbance is slightly increased but tends to be stable in 30-50 min. When the digestion time is 10min, the digestion time is too short, so that the oxidation is insufficient, a certain intermediate product possibly exists in a reaction product, the absorbance at 700nm is higher, and the absorbance of a digestion substrate is unstable, so that the measurement result is influenced, and the intermediate product completely reacts and the absorbance is reduced along with the increase of the digestion time. After 20min, the phosphorus in the water body is gradually oxidized into orthophosphate, the absorbance is increased, the phosphorus in the water body is completely oxidized at 30min, and the absorbance is maintained in a certain range along with the increase of the digestion time. Therefore, the optimal digestion time for fresh water TP assay is within the range of 30-40 min. Digestion time of 30min was selected for this study.
3.4 influence of reducing agent dosage on fresh water TP determination
Different concentrations of TP standard use solutions were oxidized using different doses of ascorbic acid solution, and the results are shown in fig. 5: ascorbic acid solution concentration is from 0.006 g.mL-1Increased to 0.08 g.mL-1The absorbance of the reaction product decreases; the concentration of the ascorbic acid solution is from 0.08 g/mL-1Increased to 0.14 g.mL-1The absorbance of the reaction product was substantially leveled. When the concentration of the ascorbic acid solution is 0.006 g/mL-1In the process, the addition amount of the reducing agent is insufficient, so that the reduction is insufficient, certain intermediate products possibly exist in reaction products, the absorbance at 700nm is higher, and meanwhile, unreduced chloride ions possibly exist in the solution, so that the measurement result is influenced, the specific reaction mechanism needs to be further researched, and when the addition amount of the ascorbic acid solution is increased to 0.08 g/mL-1When the ascorbic acid solution is added, the reaction solution tends to be saturatedIn this state, the absorbance of the reaction product tends to be stable. In order to ensure that the reducing agent in the solution can fully react with the phosphomolybdic acid, the research selects to use the ascorbic acid with the concentration of 0.1 g/mL-1。
3.5 influence of color developing agent dosage on fresh water TP determination
The color developing agent directly influences the reaction of the whole system, is a key factor for measuring the content of the fresh water TP by the ammonium molybdate spectrophotometry, is very important to research in the experiment, and influences of molybdate solutions with different dosages on the measurement of the fresh water TP are shown in the figure 6: when the concentration of molybdate (ammonium molybdate and antimony potassium tartrate) is 7.75+0.058 mol/mL-1To 15.50+0.12 mol/mL-1In the meantime, the absorbance of the reaction substrate is increased, and when the molybdate concentration exceeds 15.50+0.12 mol/mL-1Thereafter, the absorbance of the reaction substrate was substantially leveled. When the concentration of molybdate is 7.75+0.058 mol/mL-1When the concentration of the color developing agent is too low, the reaction with orthophosphate is not sufficient, and the absorbance of a reaction product is low; when the addition amount of the color developing agent is increased to 15.50+0.12 mol/mL-1During the reaction, orthophosphate and ammonium molybdate fully react to generate phosphomolybdic heteropoly acid in the presence of antimonate, the phosphomolybdic heteropoly acid is reduced by ascorbic acid to generate blue complex, the absorbance is increased, the solution reaches a saturated state, namely, the absorbance of a reaction product is kept unchanged after the concentration of molybdate is increased. To ensure that the developer in solution can react sufficiently with orthophosphate, the developer has a molybdate concentration of 31.00+0.32 mol/mL-1。
3.6 Standard Curve fitting verification of New method for fresh water TP determination
According to the experimental results, the optimal reaction conditions of the new fresh water TP determination method are determined as follows: the type of the oxidant is potassium persulfate, the adding amount of the oxidant is 80 μ L, the digestion time is 30min, and the concentration of the reducing agent ascorbic acid is 0.1 g/mL-1The concentration of the color developing agent molybdate solution is 31.00+0.32 mol/mL-1。
Standard using liquid of phosphorus is used, standard curves of a small system and a national standard system are drawn according to the conditions, and the linear fitting result is shown in figure 7: TP concentration and absorbance toolHas good linear regression relationship, the regression equation of small-system TP concentration and absorbance is that Y is 0.3991x-0.0018, and the linear correlation coefficient R20.9998; the regression equation of TP concentration and absorbance by the national standard method is Y-0.3965 x-0.0028, and the linear correlation coefficient R2＝0.9998。
To further illustrate the accuracy of the small system, the research adopts an orthogonal verification method to perform cross verification on fresh water TP determined by the small system and the national standard method, and the result is shown in fig. 8: the linear equation of the two measurement methods is that Y is 0.9934X-0.0011(Y is the absorbance of a small body system, and X is the absorbance of the national standard method), and the linear correlation coefficient R20.9999. Analysis of variance (F ═ 0.001 < F)Watch (A)5.318 with 95% confidence) further confirmed that there was no significant difference between the results of the small system and the national standard method for fresh water TP determination. The results show that the results of the small system and the method for measuring the fresh water TP by the national standard are highly consistent, namely the method for measuring the fresh water TP by the small system is accurate and reliable.
3.7 Linear detection Range of fresh Water TP Small body System determination
The optimized experimental conditions are adopted to carry out the linear detection range experiment of the fresh water TP small system, and the result is shown in figure 9: when the concentration of TP is 0-2. mu.g.mL-1Within the range, the total phosphorus concentration and the absorbance form a linear relation, and the correlation coefficient R20.9968; when the concentration of TP exceeds 2. mu.g/mL-1When the concentration of the solution exceeds 2 mu g/mL, the absorbance value is still increased, but the absorbance value exceeds 1, and the subsequent data are not recommended in consideration of the optimal measurement range of the ultraviolet-visible spectrophotometer-1When this is the case, it should be measured after dilution. In conclusion, the detection range of the small-body system rapid determination of TP concentration in fresh water determined by the research is 0-2 mug. multidot.mL-1Compared with the national standard method, the fresh water TP determination range is 0-0.6 mug/mL-1The measuring range of the experimental method is nearly four times enlarged. The research of the small-scale determination method obviously improves the upper limit of the fresh water TP determination concentration, can effectively determine water samples with higher TP concentration, saves a plurality of medicines and time consumed by gradient dilution, and greatly improves the efficiency of determining a large amount of water samples.
3.8, verifying the accuracy of the actual measurement environmental sample TP
The TP content in the collected fresh water sample is respectively and simultaneously measured by using a small system and a national standard method, the measurement results are compared by adopting an orthogonal verification method, and the results are shown in figure 10: fitting straight line correlation coefficient R of TP results in two kinds of determination water samples2The linear relationship was good at 0.9995. And F detection and t detection are carried out on two results of the TP of the determined sample, the F value of the TP result of the water sample is 0.001 and is smaller than 5.318 of an F table through F detection with the confidence coefficient of 95%, the Sig of the two methods is 0.981 and is larger than 0.05 through t detection with the confidence coefficient of 95% through small system and national standard method determination, and the results show that the precision and the accuracy of the TP of the fresh water determined by the small system and the national standard method are not obviously different. In conclusion, the small-system ammonium molybdate spectrophotometry method for determining the TP content in the fresh water body has strong feasibility and applicability.
The above description is only for the preferred embodiment of the present application and should not be taken as limiting the present application in any way, and although the present application has been disclosed in the preferred embodiment, it is not intended to limit the present application, and those skilled in the art should understand that they can make various changes and modifications within the technical scope of the present application without departing from the scope of the present application, and therefore all the changes and modifications can be made within the technical scope of the present application.
Claims (9)
1. A method for measuring the concentration of total phosphorus in a fresh water body is characterized by comprising the following steps:
s1: and (3) standard working curve formulation:
s1.1: concentrations of 0, 0.04, 0.08, 0.24, 0.4 and 0.8. mu.g.mL were prepared in six 18X 250mm round bottom screw cap digestion tubes, respectively, using phosphorus standard use solutions-15mL of each of the phosphorus standard solutions, wherein the concentration of potassium dihydrogen phosphate in the phosphorus standard solution is 2. mu.g.mL-1；
S1.2: adding 70-90 μ L of 0.05 g/mL into each phosphorus working solution-1The potassium persulfate or the sodium persulfate is placed in a digestion instrument for digestion for 30-40min, and is diluted to 5mL by ultrapure water after digestion;
s1.3: adding 20 μ L of working solution with concentration of 0.08-0.14 g/mL into each digested working solution-1Mixing the ascorbic acid solution uniformly, and reducing for 30 s; adding 40 mu L of molybdate solution, fully mixing, developing for 15min, wherein the molybdate solution is generated by reacting ammonium molybdate solution and antimony potassium tartrate solution, and the concentration of the ammonium molybdate solution and the antimony potassium tartrate solution is 15.50+0.12 mol/mL-1-38.69+0.29mol·mL-1；
S1.4: after the color development is finished, the absorbance A of the working solution is measured at a wavelength of 700nm by using a cuvette with an optical length of 10mm and taking a blank as a references；
S1.5: measuring 5mL of a blank water sample, and measuring and recording the absorbance A of the blank water sample according to the same treatment steps as the phosphorus working solutionb；
S1.6: with AsDeduction AbThe latter absorbance value AnAs ordinate, concentration C of phosphorus working solutionsDrawing a standard working curve for an abscissa, and solving an intercept a and a slope b of the standard working curve by using a linear regression method;
s2: water sample determination
Measuring 5mL of fresh water sample, and measuring and recording the absorbance of the water sample according to the same treatment steps as the phosphorus working solution;
s3: and (4) calculating a result:
the total phosphorus concentration in the fresh water sample is calculated according to the formula (1):
in the formula (I), the compound is shown in the specification,
c (TP) is the concentration of total phosphorus in the fresh water sample, and the unit is mu g.mL-1；
AwThe light absorption value of total phosphorus in the water sample;
Abblank light absorption values;
a is the intercept of a standard working curve;
b is the standard operating curve slope.
2. The method for determining the total phosphorus concentration in the fresh water body according to claim 1, wherein the steps between S1 and S2 further comprise:
digesting 5mL of fresh water sample by the method in the step S1.2, adding 60 mu L of turbidity-chromaticity compensation liquid, using a cuvette with an optical path of 10mm and taking a blank water sample as a reference under the wavelength of 700nm, and determining the absorbance A of the turbidity of the fresh water samplet，
The total phosphorus concentration in the fresh water sample is calculated according to the formula (2):
in the formula, AtThe absorbance value of turbidity in the water sample.
3. The method for determining the concentration of total phosphorus in the fresh water body according to claim 1, wherein in the step S1.2, the digestion pressure is 1.1kg/cm2The temperature was 120 ℃.
4. The method for determining the concentration of total phosphorus in the fresh water body according to claim 1, wherein the digestion time is 30 min.
5. The method according to claim 1, wherein the amount of potassium persulfate or sodium persulfate is 80 μ L.
6. The method according to claim 1, wherein the concentration of the ascorbic acid solution is 0.1 g-mL-1。
7. The method for determining the total phosphorus concentration in the fresh water body according to claim 1, wherein the molybdate solution is generated by reacting an ammonium molybdate solution and an antimony potassium tartrate solution, and the ammonium molybdate solution and the antimony potassium tartrate solution are mixed to form the molybdate solutionHas a concentration of 31.00+0.32 mol/mL-1。
8. The method according to claim 7, wherein the molybdate solution is prepared by the following steps:
respectively dissolving 13g of ammonium molybdate and 0.35g of antimony potassium tartrate in 100mL of water, slowly adding the ammonium molybdate solution into 300mL of 50% sulfuric acid under continuous stirring, adding the antimony potassium tartrate solution, and fully and uniformly mixing.
9. The method for determining the total phosphorus concentration in the fresh water body according to claim 1, wherein the turbidity-chromaticity compensation solution is prepared by the following method:
taking 50% sulfuric acid and a volume of 0.1 g.mL-1Ascorbic acid (b) was mixed well.
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| CN107367470A (en) * | 2017-05-22 | 2017-11-21 | 安徽宏远职业卫生技术服务有限公司 | A kind of method of Judgment of Determination of Total Phosphorus In Water With Ammonium Molybdate Spectrophotometry |
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| CN106841062A (en) * | 2016-12-29 | 2017-06-13 | 贵港市芭田生态有限公司 | The detection method of total phosphorus content in water quality |
| CN107367470A (en) * | 2017-05-22 | 2017-11-21 | 安徽宏远职业卫生技术服务有限公司 | A kind of method of Judgment of Determination of Total Phosphorus In Water With Ammonium Molybdate Spectrophotometry |
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