CN111257291A - Quantitative detection method for phosphate ions and application thereof - Google Patents

Quantitative detection method for phosphate ions and application thereof Download PDF

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CN111257291A
CN111257291A CN202010063875.1A CN202010063875A CN111257291A CN 111257291 A CN111257291 A CN 111257291A CN 202010063875 A CN202010063875 A CN 202010063875A CN 111257291 A CN111257291 A CN 111257291A
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CN111257291B (en
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韩颖超
张小君
廖丽璇
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Wuhan University of Technology WUT
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Abstract

The invention provides a quantitative detection method of phosphate radical ions and application thereof, and the method is based on a complex reaction between polyacrylic acid (PAA) and metal ions to prepare PAA-Ca2+(Eu3+) Complexing it with a specific concentration of phosphate ion (PO)4 3‑) Mixing the standard solutions in proportion, testing fluorescence intensity by using a fluorescence spectrophotometer, and establishing Eu3+Fluorescence intensity amplification ratio-PO4 3‑Linear relation of concentration, and finally, determining PO in the solution to be measured by using the linear relation4 3‑And (4) concentration. The method is simple and easy to operate, has low cost, can be used for detecting the concentration of phosphate radical in the fields of chemistry, biology, medicine, environment and the like, and is based on Eu3+Narrow emission band and high photochemical stability, so that the method of the invention is suitable for PO4 3‑The detection has higher sensitivity and stability.

Description

Quantitative detection method for phosphate ions and application thereof
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a quantitative detection method of phosphate ions and application thereof.
Background
Phosphorus plays an important role in biological, environmental and chemical recycling processes. The phosphorus pollution of the water body can be serious due to the over-standard inorganic phosphorus content in the water body; inorganic phosphorus in organisms participates in various metabolisms, and promotes synthesis, conversion and transport of carbohydrates; inorganic phosphorus is one of the indicators of kidney function, and discussion about phosphorus is common in patients with chronic kidney disease and dialysis. Therefore, quantitative detection of phosphate ions is of great significance.
Currently, inorganic phosphorus is detected by phosphomolybdate method, ICP method, and the like. The phosphomolybdate method involves reacting inorganic phosphorus with ammonium molybdate and then quantifying the color with a reducing agent, but the reducing agent is of a large variety and is easily oxidized, resulting in unstable color development. The ICP method can detect multiple elements simultaneously based on the unique emission spectrum of each element, but the test process has high requirements on samples and high instrument cost.
Disclosure of Invention
In view of this, the present invention aims to provide a method for quantitatively detecting phosphate ions, so as to solve the problems of unstable color development and high detection cost in the existing phosphate ion detection.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a quantitative detection method of phosphate ions comprises the following steps:
1) adding Ca2+Solution and Eu3+After the solutions are mixed, adding a polyacrylic acid solution at room temperature, stirring and mixing, then adjusting the pH to 6.0-8.0 by using ammonia water, and carrying out a complexing reaction to obtain a PAA-Ca (Eu) complex precursor;
2) to PO4 3-The solution is diluted into a series of PO with specific concentration4 3-Standard solution, and taking the series of PO with specific concentration4 3-Mixing a standard solution with the PAA-Ca (Eu) complex precursor, adjusting the pH to 9.0-10.0 by using ammonia water, testing the fluorescence intensity of the series of standard reaction solutions after full reaction, calculating the fluorescence intensity amplification rate of the series of standard reaction solutions, and establishing the fluorescence intensity amplification rate-PO4 3-A linear relationship of concentration;
3) mixing a solution to be detected with the PAA-Ca (Eu) complex precursor, adjusting the pH to 9.0-10.0 by using ammonia water, testing the fluorescence intensity of the reaction solution to be detected after full reaction, calculating the fluorescence intensity amplification rate of the reaction solution to be detected, and then, according to the fluorescence intensity amplification rateStrength amplification factor-PO4 3-Obtaining PO in the solution to be measured according to the concentration linear relation4 3-And (4) concentration.
Optionally, in the step 3), the solution to be detected is taken and mixed with the PAA-Ca (Eu) complex precursor, the pH is adjusted to 9.0-10.0 by ammonia water, after full reaction, the fluorescence intensity of the reaction solution to be detected is tested, the fluorescence intensity amplification rate of the reaction solution to be detected is calculated, and then, the fluorescence intensity amplification rate-PO is calculated according to the fluorescence intensity amplification rate4 3-Obtaining PO in the solution to be measured according to the concentration linear relation4 3-Concentrations, including:
a) amplification ratio-PO according to the fluorescence intensity4 3-Linear relation of concentration, the amplification rate of fluorescence intensity corresponding to the lowest concentration in the linear relation is a0The maximum concentration corresponds to a fluorescence intensity amplification ratio of b0Determining the range a-b of reliable fluorescence intensity amplification rate, wherein a is a0+(b0-a0)×15％，b＝b0-(b0-a0)×15％；
b) Mixing a solution to be detected with the PAA-Ca (Eu) complex precursor, adjusting the pH to 9.0-10.0 by using ammonia water, testing the fluorescence intensity of the reaction solution to be detected after full reaction, and calculating the fluorescence intensity amplification rate of the reaction solution to be detected;
c) comparing the fluorescence intensity amplification rate of the reaction solution to be detected with the reliable fluorescence intensity amplification rate range a-b;
d) if the fluorescence intensity amplification rate of the reaction solution to be detected is within the reliable fluorescence intensity amplification rate range a-b, substituting the fluorescence intensity amplification rate of the reaction solution to be detected into the fluorescence intensity amplification rate-PO4 3-Obtaining PO in the solution to be measured according to the concentration linear relation4 3-Concentration;
e) if the fluorescence intensity amplification rate of the reaction solution to be detected is higher than the reliable fluorescence intensity amplification rate range a-b, diluting the solution to be detected until the fluorescence intensity amplification rate of the reaction solution to be detected is within the reliable fluorescence intensity amplification rate range a-b, and then diluting the reaction solution to be detected to obtain the diluted reaction solution to be detectedSubstituting the fluorescence intensity amplification rate of the solution into the fluorescence intensity amplification rate-PO4 3-Linear relation of concentration to obtain PO of the diluted reaction solution to be detected4 3-The concentration is obtained, and then the PO in the solution to be detected is obtained by dilution times4 3-Concentration;
f) if the fluorescence intensity amplification rate of the reaction solution to be detected is lower than the reliable fluorescence intensity amplification rate range a-b, increasing PO in the solution to be detected4 3-Until the fluorescence intensity amplification rate of the reaction solution to be detected is within the reliable fluorescence intensity amplification rate range a-b, and substituting the fluorescence intensity amplification rate of the reaction solution to be detected, which is obtained after the concentration is increased, into the fluorescence intensity amplification rate-PO4 3-Linear relationship of concentration to obtain PO of the reaction solution to be measured after concentration increase4 3-Concentration, and then by subtracting the added PO4 3-To obtain PO in the solution to be tested4 3-And (4) concentration.
Optionally, formulating the Ca in step 1)2+The calcium source of the solution is one of calcium chloride dihydrate and calcium nitrate tetrahydrate; preparing the Eu in step 1)3+The europium source of the solution is one of europium nitrate hexahydrate and europium chloride hexahydrate.
Optionally, Ca in said step 1)2+Solution and Eu3+The total molar concentration of Ca and Eu in the mixed solution after the solution is mixed is 0.07515M, and the ratio of the amount of Eu substances to the total amount of Ca and Eu substances is 5% -10%; the amount of carboxyl group in the polyacrylic acid solution and Ca in the step 1)2+Solution and Eu3+The ratio of the total amount of Ca and Eu in the mixed solution after the solution mixing is 2.0-4.0.
Optionally, the particle size of the PAA-Ca (Eu) complex precursor in the step 1) is 2-3 nm.
Optionally, formulating the PO in the step 2)4 3-The phosphorus source of the solution is one of diammonium hydrogen phosphate and disodium hydrogen phosphate dodecahydrate.
Alternatively, the series of specific concentrations of PO in the step 2)4 3-Standard solutionMiddle PO4 3-Amount of substance (a) and Ca2+Solution and Eu3+The total mass ratio of Ca and Eu in the mixed solution after the solution is mixed is less than or equal to 0.1.
Optionally, the PAA-Ca (Eu) complex precursor and the PO in the step 2)4 3-The reaction time of the standard solution is 6-8 h; the reaction time of the PAA-Ca (Eu) complex precursor and the solution to be detected in the step 3) is 6-8 h.
Optionally, the fluorescence intensity amplification rate Δ I of the series of standard reaction solutions in step 2) and the fluorescence intensity amplification rate Δ I of the reaction solution to be detected in step 3) are calculated by the following formula:
ΔI＝(I-I0)/I0，
wherein I represents the fluorescence intensity of the series of standard reaction solutions and the reaction solution to be measured, I0Represents PAA-Ca (Eu) complex precursor and PO4 3-Fluorescence intensity after mixing of a standard solution with a concentration of 0.
The second purpose of the invention is to provide an application of the method for quantitatively detecting phosphate ions in organisms or environments.
The detection principle of the quantitative detection method of phosphate ions is as follows:
PAA－Ca2+(Eu3+) Complexes with PO4 3-After the reaction, Eu3+And PO4 3-O in (1)2-Formation of Eu-O bond based on Eu-O Charge Transfer Band (CTB) fluorescence mechanism, Eu at specific excitation wavelength (200-3+Can emit special fluorescence, the amplification rate of fluorescence intensity and the quantity of Eu-O bonds, namely PO4 3-The concentrations appeared to be positively correlated. Based on Eu3+Fluorescence intensity amplification and PO4 3-Linear relationship between concentrations, quantitative determination of PO by measuring fluorescence intensity4 3-And (4) concentration.
Compared with the prior art, the quantitative detection method for phosphate ions has the following advantages:
the invention is based on Eu-Fluorescence mechanism of O Charge Transfer Band (CTB) using phosphate ion concentration and Eu3+The quantitative relation between the fluorescence intensity amplification rates realizes the quantitative detection of the phosphate radical ions, the whole detection process is carried out in a water phase system, the method is simple and easy to operate, the cost is low, and the method can be used for detecting the concentration of the phosphate radical in the fields of chemistry, biology, medicine, environment and the like3+Narrow emission band and high photochemical stability, so that the method of the invention is suitable for PO4 3-The detection has higher sensitivity and stability.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph showing a particle size distribution of a PAA-Ca (Eu) complex precursor in example 1 of the present invention;
FIG. 2 shows fluorescence intensity amplification ratios-PO of examples 1 and 2 of the present invention4 3-A linear plot of concentration;
FIG. 3 is a graph showing fluorescence intensity amplification-PO of example 3 of the present invention4 3-And (4) a concentration linear relation graph.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The present invention will be described in detail with reference to examples.
Example 1
A quantitative detection method of phosphate ions specifically comprises the following steps:
1) 4.4103g of calcium chloride dihydrate was dissolved in 100ml of ultrapure water to prepare 0.3M Ca2+A solution;
2) 1.4898g of europium nitrate hexahydrate is dissolved in 100ml of ultrapure water to prepare 0.0334M Eu3+A solution;
3) dissolving 0.216g of polyacrylic acid in 20ml of ultrapure water, and continuously stirring to completely dissolve the polyacrylic acid to obtain a polyacrylic acid solution;
4) ca is added so that the ratio of Eu/(Ca + Eu) to the total of Ca and Eu becomes 10% (molar ratio), i.e., the ratio of the amount of Eu to the total of Ca and Eu becomes 10%2+Solution and Eu3+After the solution was mixed to obtain a mixed solution (20mL) having a total molar concentration of Ca and Eu of 0.07515M, the amount of the carboxyl group in the polyacrylic acid solution was determined in accordance with the amount of the substance and Ca at room temperature2+Solution and Eu3+The method comprises the following steps of (1) adding polyacrylic acid solution into a mixed solution obtained after mixing the solution, wherein the total content ratio of Ca and Eu in the mixed solution is 2.0, stirring to uniformly mix the solution, then adjusting the pH value to 6.0-8.0 by using ammonia water, and carrying out a complex reaction to obtain a PAA-Ca (Eu) complex precursor with the particle size of 2-3 nm, wherein a particle size distribution diagram is shown in figure 1;
5) 3.5814g of disodium hydrogen phosphate dodecahydrate was dissolved in 100ml of ultrapure water to prepare 0.1M PO4 3-Solution and mixing the prepared PO of 0.1M4 3-The solution is diluted into a series of PO with specific concentration4 3-Standard solutions (0, 0.25mM, 0.75mM, 1.5mM, 2mM, 2.5mM, 3.75mM, 5mM, 7.5mM, 10mM, 12.5mM) were then prepared by mixing PAA-Ca (Eu) complex precursors with 20ml of a series of specific concentrations of PO4 3-Mixing the standard solutions, respectively adjusting the pH to 9.0-10.0 by using ammonia water, fully reacting for 6 hours, measuring the fluorescence intensity of the series of standard reaction solutions by using a fluorescence spectrophotometer, calculating the fluorescence intensity amplification rate delta I of the series of standard reaction solutions, and establishing the fluorescence intensity amplification rate-PO4 3-A linear relationship of concentration, wherein the fluorescence intensity amplification rate Δ I of the series of standard reaction solutions is calculated by the following formula:
ΔI＝(I-I0)/I0，
wherein I represents the fluorescence intensity of a series of standard reaction solutions, I0Represents PAA-Ca (Eu) complex precursor and PO4 3-Fluorescence intensity of the mixed standard solution with the concentration of 0;
and obtaining the fluorescence intensity amplification rate (y) -PO by linear fitting4 3-The linear relationship of concentration (x) is shown in fig. 2, wherein y is 0.601x-0.098, and R is2＝0.99；
6) According to the above-mentioned fluorescenceLight intensity amplification-PO4 3-Obtaining PO by a concentration linear relation curve4 3-The linear range of the ion concentration was 0.25mM to 7.5mM, and the fluorescence intensity amplification factor a corresponding to the lowest concentration was obtained by bringing the linear ranges of 0.25mM and 7.5mM into the range of 0.601x-0.09800.052, the maximum concentration corresponds to the fluorescence intensity amplification rate b0Is 4.410, from a0And b0The range a-b for obtaining the reliable fluorescence intensity amplification rate is 0.71-3.76, wherein a is a0+(b0-a0)×15％，b＝b0-(b0-a0)×15％；
7) 0.0287g disodium hydrogenphosphate dodecahydrate was dissolved in 20ml ultrapure water to prepare 4mM PO4 3-Ionizing the solution to be detected, mixing the solution to be detected with the PAA-Ca (Eu) complex precursor in the step 4), adjusting the pH value to 9.0-10.0 by using ammonia water, fully reacting for 6 hours, measuring the fluorescence intensity of a reaction solution to be detected (the solution after the reaction of the solution to be detected and the PAA-Ca (Eu) complex precursor is finished) by using a fluorescence spectrophotometer, and calculating the fluorescence intensity amplification rate delta I of the reaction solution to be detected to be 2.355 within the reliable fluorescence intensity amplification rate range a-b (0.71-3.76), wherein the calculation method of the fluorescence intensity amplification rate delta I of the reaction solution to be detected is the same as the step 5);
8) substituting the fluorescence intensity amplification rate (2.355) of the reaction solution to be detected into the fluorescence intensity amplification rate-PO4 3-Obtaining PO in the solution to be measured according to a linear concentration relation (y is 0.601x to 0.098)4 3-At a concentration of 4.08mM, with the formulated PO4 3-The ionic solution concentration deviation rate was 2%.
Example 2
A quantitative detection method of phosphate ions specifically comprises the following steps:
1) 4.4103g of calcium chloride dihydrate was dissolved in 100ml of ultrapure water to prepare 0.3M Ca2+A solution;
2) 1.4898g of europium nitrate hexahydrate is dissolved in 100ml of ultrapure water to prepare 0.0334M Eu3+A solution;
3) dissolving 0.216g of polyacrylic acid in 20ml of ultrapure water, and continuously stirring to completely dissolve the polyacrylic acid to obtain a polyacrylic acid solution;
4) ca is added so that the ratio of Eu/(Ca + Eu) to the total of Ca and Eu becomes 10% (molar ratio), i.e., the ratio of the amount of Eu to the total of Ca and Eu becomes 10%2+Solution and Eu3+After the solution was mixed to obtain a mixed solution (20mL) having a total molar concentration of Ca and Eu of 0.07515M, the amount of the carboxyl group in the polyacrylic acid solution was determined in accordance with the amount of the substance and Ca at room temperature2+Solution and Eu3+The method comprises the following steps of (1) adding polyacrylic acid solution into a mixed solution obtained after mixing the solution, wherein the total content ratio of Ca and Eu in the mixed solution is 2.0, stirring to uniformly mix the solution, then adjusting the pH value to 6.0-8.0 by using ammonia water, and carrying out a complex reaction to obtain a PAA-Ca (Eu) complex precursor with the particle size of 2-3 nm;
5) 3.5814g of disodium hydrogen phosphate dodecahydrate was dissolved in 100ml of ultrapure water to prepare 0.1M PO4 3-Solution and mixing the prepared PO of 0.1M4 3-The solution is diluted into a series of PO with specific concentration4 3-Standard solutions (0, 0.25mM, 0.75mM, 1.5mM, 2mM, 2.5mM, 3.75mM, 5mM, 7.5mM, 10mM, 12.5mM) were then prepared by mixing PAA-Ca (Eu) complex precursors with 20ml of a series of specific concentrations of PO4 3-Mixing the standard solutions, respectively adjusting the pH to 9.0-10.0 by using ammonia water, fully reacting for 6 hours, measuring the fluorescence intensity of the series of standard reaction solutions by using a fluorescence spectrophotometer, calculating the fluorescence intensity amplification rate delta I of the series of standard reaction solutions, and establishing the fluorescence intensity amplification rate-PO4 3-A linear relationship of concentration, wherein the fluorescence intensity amplification rate Δ I of the series of standard reaction solutions is calculated by the following formula:
ΔI＝(I-I0)/I0，
wherein I represents the fluorescence intensity of a series of standard reaction solutions, I0Represents PAA-Ca (Eu) complex precursor and PO4 3-Fluorescence intensity of the mixed standard solution with the concentration of 0;
and obtaining the fluorescence intensity amplification rate (y) -PO by linear fitting4 3-The linear relationship of concentration (x) is shown in fig. 2, wherein y is 0.601x-0.098, and R is2＝0.99；
6) Amplification ratio-PO based on the fluorescence intensity4 3-Obtaining PO by a concentration linear relation curve4 3-The linear range of the ion concentration was 0.25mM to 7.5mM, and the fluorescence intensity amplification factor a corresponding to the lowest concentration was obtained by bringing the linear ranges of 0.25mM and 7.5mM into the range of 0.601x-0.09800.052, the maximum concentration corresponds to the fluorescence intensity amplification rate b0Is 4.410, from a0And b0The range a-b for obtaining the reliable fluorescence intensity amplification rate is 0.71-3.76, wherein a is a0+(b0-a0)×15％，b＝b0-(b0-a0)×15％；
7) 0.2865g of disodium hydrogen phosphate dodecahydrate were dissolved in 40ml of ultrapure water to prepare 20mM PO4 3-Ionizing a solution to be detected, then, mixing 20ml of the solution to be detected with the PAA-Ca (Eu) complex precursor in the step 4), adjusting the pH value to 9.0-10.0 by using ammonia water, after fully reacting for 8 hours, measuring the fluorescence intensity of the reaction solution to be detected by using a fluorescence spectrophotometer, and calculating the fluorescence intensity amplification rate delta I of the reaction solution to be detected to be 5.089, wherein the fluorescence intensity amplification rate delta I is not within the reliable fluorescence intensity amplification rate range a-b (0.71-3.76) and is higher than the reliable fluorescence intensity amplification rate range, and the calculation method of the fluorescence intensity amplification rate delta I of the reaction solution to be detected is the same as the step 5);
8) taking 10ml of the solution to be detected, diluting the solution to be detected into 20ml, mixing the diluted solution to be detected with the PAA-Ca (Eu) complex precursor in the step 4), adjusting the pH value to 9.0-10.0 by ammonia water, fully reacting for 8h, measuring the fluorescence intensity of the diluted reaction solution to be detected (the diluted solution to be detected and the PAA-Ca (Eu) complex precursor after reaction is finished) by using a fluorescence spectrophotometer, and calculating the fluorescence intensity amplification rate delta I of the diluted reaction solution to be detected to be 4.905, wherein the fluorescence intensity amplification rate delta I is still within the reliable fluorescence intensity amplification rate range a-b (0.71-3.76) and is higher than the reliable fluorescence intensity amplification rate range; taking 4ml of the solution to be tested, diluting the solution to be tested into 20ml, repeating the test process, and calculating the fluorescence intensity amplification rate delta I of the reaction solution to be tested obtained after dilution to be 2.323 within the range of reliable fluorescence intensity amplification rates a-b (0.71-3.76), wherein the calculation method of the fluorescence intensity amplification rate delta I of the reaction solution to be tested obtained after dilution is the same as the step 5);
9) substituting the fluorescence intensity amplification rate (2.323) of the diluted reaction solution to be detected into the fluorescence intensity amplification rate-PO4 3-The linear concentration relationship (y is 0.601x-0.098) indicates that PO in the diluted test solution was present4 3-PO concentration of 4.03mM in the test solution4 3-Multiplying the concentration by the dilution times to obtain PO in the solution to be detected4 3-At a concentration of 20.15mM, i.e. 4.03X 5 ═ 20.15mM, with the formulated PO4 3-The ionic solution concentration deviation rate was 7.5%.
Example 3
A quantitative detection method of phosphate ions specifically comprises the following steps:
1) 4.4103g of calcium chloride dihydrate was dissolved in 100ml of ultrapure water to prepare 0.3M Ca2+A solution;
2) 1.4898g of europium nitrate hexahydrate is dissolved in 100ml of ultrapure water to prepare 0.0334M Eu3+A solution;
3) dissolving 0.432g of polyacrylic acid in 20ml of ultrapure water, and continuously stirring to completely dissolve the polyacrylic acid to obtain a polyacrylic acid solution;
4) ca is added at a ratio of 5% (molar ratio) of Eu/(Ca + Eu), that is, 5% of the amount of Eu to the total amount of Ca and Eu2+Solution and Eu3+After the solution is mixed into a mixed solution (20mL) with the total molar concentration of Ca and Eu being 0.07515M, adding polyacrylic acid solution at room temperature, stirring to uniformly mix the polyacrylic acid solution, then adjusting the pH value to 6.0-8.0 by ammonia water, and carrying out a complexing reaction to obtain a PAA-Ca (Eu) complex precursor with the particle size of 2-3 nm;
5) 3.5814g of disodium hydrogen phosphate dodecahydrate was dissolved in 100ml of ultrapure water to prepare 0.1M PO4 3-Solution and mixing the prepared PO of 0.1M4 3-The solution is diluted into a series of PO with specific concentration4 3-Standard solution (0, 0.25mM, 0.75mM, 1.5mM, 2mM, 2.5mM, 3.75mM, 5mM, 7.5mM, 10mM, 12.5mM) was then takenPAA-Ca (Eu) complex precursor is mixed with 20ml series of PO with specific concentration4 3-Mixing the standard solutions, respectively adjusting the pH to 9.0-10.0 by using ammonia water, fully reacting for 6 hours, measuring the fluorescence intensity of the series of standard reaction solutions by using a fluorescence spectrophotometer, calculating the fluorescence intensity amplification rate delta I of the series of standard reaction solutions, and establishing the fluorescence intensity amplification rate-PO4 3-A linear relationship of concentration, wherein the fluorescence intensity amplification rate Δ I of the series of standard reaction solutions is calculated by the following formula:
ΔI＝(I-I0)/I0，
wherein I represents the fluorescence intensity of a series of standard reaction solutions, I0Represents PAA-Ca (Eu) complex precursor and PO4 3-Fluorescence intensity of the mixed standard solution with the concentration of 0;
and obtaining the fluorescence intensity amplification rate (y) -PO by linear fitting4 3-The linear relationship of concentration (x) is shown in fig. 3, where y is 0.897x-0.27, and R is2＝0.99；
6) Amplification ratio-PO based on the fluorescence intensity4 3-Obtaining PO by a concentration linear relation curve4 3-The linear range of the ion concentration was 0.25mM to 3.75mM, and the fluorescence intensity amplification factor a corresponding to the lowest concentration was obtained by bringing the linear ranges of 0.25mM and 3.75mM into the range of 0.897x-0.270The fluorescence intensity amplification rate b corresponding to the highest concentration is-0.0460Is 3.094, from a0And b0The range a-b for obtaining the reliable fluorescence intensity amplification rate is 0.43-2.62, wherein a is a0+(b0-a0)×15％，b＝b0-(b0-a0)×15％；
7) 0.0029g of disodium hydrogenphosphate dodecahydrate was dissolved in 40ml of ultrapure water to prepare 0.2mM PO4 3-Ionizing a solution to be detected, mixing 20ml of the solution to be detected with the PAA-Ca (Eu) complex precursor in the step 4), adjusting the pH value to 9.0-10.0 by ammonia water, fully reacting for 6h, measuring the fluorescence intensity of the reaction solution to be detected by using a fluorescence spectrophotometer, and calculating the fluorescence intensity amplification rate delta I of the reaction solution to be detected to be 0.020And the fluorescence intensity amplification rate does not fall into the range a-b (0.43-2.62) of the reliable fluorescence intensity amplification rate and is lower than the range a-b of the reliable fluorescence intensity amplification rate, wherein the calculation method of the fluorescence intensity amplification rate delta I of the reaction solution to be detected is the same as the step 5);
8) taking 20ml of the solution to be detected, adding 0.0145g of disodium hydrogen phosphate dodecahydrate, mixing the solution to be detected with the increased concentration with the PAA-Ca (Eu) complex precursor in the step 4), adjusting the pH value to 9.0-10.0 with ammonia water, fully reacting for 6 hours, measuring the fluorescence intensity of the reaction solution to be detected (the solution after the reaction of the solution to be detected with the increased concentration and the PAA-Ca (Eu) complex precursor) obtained after the concentration is increased by using a fluorescence spectrophotometer, and calculating the fluorescence intensity amplification rate delta I of the reaction solution to be detected with the increased concentration to be 1.741, wherein the fluorescence intensity amplification rate delta I of the reaction solution to be detected with the increased concentration is within the reliable fluorescence intensity amplification rate range a-b (0.43-2.62), and the calculation method of the fluorescence intensity amplification rate delta I of the reaction solution to be detected with the increased concentration is the same as the step 5);
9) substituting the fluorescence intensity amplification rate (1.741) of the reaction solution to be detected obtained after the concentration is increased into the fluorescence intensity amplification rate-PO4 3-The concentration linear relationship (y is 0.897x-0.27) indicates that PO in the test solution obtained after the concentration is increased4 3-PO in the test solution obtained by increasing the concentration of 2.242mM4 3-Concentration minus added PO4 3-The amount of (a), wherein the PO was increased by adding 0.0145g of disodium hydrogen phosphate dodecahydrate4 3-The concentration is 2.024mM, and PO in the solution to be tested is obtained4 3-At a concentration of 0.218mM, i.e., 2.242mM-2.024mM ═ 0.218mM, with formulated PO4 3-The ionic solution concentration deviation rate was 7.9%.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for quantitatively detecting phosphate ions is characterized by comprising the following steps:
1) adding Ca2+Solution and Eu3+After the solutions are mixed, adding a polyacrylic acid solution at room temperature, stirring and mixing, then adjusting the pH to 6.0-8.0 by using ammonia water, and carrying out a complexing reaction to obtain a PAA-Ca (Eu) complex precursor;
2) to PO4 3-The solution is diluted into a series of PO with specific concentration4 3-Standard solution, and taking the series of PO with specific concentration4 3-Mixing a standard solution with the PAA-Ca (Eu) complex precursor, adjusting the pH to 9.0-10.0 by using ammonia water, testing the fluorescence intensity of the series of standard reaction solutions after full reaction, calculating the fluorescence intensity amplification rate of the series of standard reaction solutions, and establishing the fluorescence intensity amplification rate-PO4 3-A linear relationship of concentration;
3) mixing a solution to be detected with the PAA-Ca (Eu) complex precursor, adjusting the pH to 9.0-10.0 by using ammonia water, testing the fluorescence intensity of the reaction solution to be detected after full reaction, calculating the fluorescence intensity amplification rate of the reaction solution to be detected, and then, according to the fluorescence intensity amplification rate-PO4 3-Obtaining PO in the solution to be measured according to the concentration linear relation4 3-And (4) concentration.
2. The method according to claim 1, wherein the solution to be detected is taken in the step 3), mixed with the PAA-Ca (Eu) complex precursor, and adjusted to pH 9.0-10.0 with ammonia water, after the reaction, the fluorescence intensity of the reaction solution to be detected is measured, the fluorescence intensity amplification rate of the reaction solution to be detected is calculated, and then, the fluorescence intensity amplification rate-PO is determined according to the fluorescence intensity amplification rate4 3-Obtaining PO in the solution to be measured according to the concentration linear relation4 3-Concentrations, including:
a) amplification ratio-PO according to the fluorescence intensity4 3-Linear relation of concentration, the amplification rate of fluorescence intensity corresponding to the lowest concentration in the linear relation is a0The maximum concentration corresponds to a fluorescence intensity amplification ratio of b0Determining the range a-b of reliable fluorescence intensity amplification rate, wherein a is a0+(b0-a0)×15％，b＝b0-(b0-a0)×15％；
b) Mixing the solution to be tested with the PAA-Ca (Eu) complex precursor, adjusting the pH to 9.0-10.0 by using ammonia water, fully reacting, testing the fluorescence intensity of the reaction solution to be tested, and calculating the fluorescence intensity amplification rate of the reaction solution to be tested;
c) comparing the fluorescence intensity amplification rate of the reaction solution to be detected with the reliable fluorescence intensity amplification rate range a-b;
d) if the fluorescence intensity amplification rate of the reaction solution to be detected is within the reliable fluorescence intensity amplification rate range a-b, substituting the fluorescence intensity amplification rate of the reaction solution to be detected into the fluorescence intensity amplification rate-PO4 3-Obtaining PO in the solution to be measured according to the concentration linear relation4 3-Concentration;
e) if the fluorescence intensity amplification rate of the reaction solution to be detected is higher than the reliable fluorescence intensity amplification rate range a-b, diluting the solution to be detected until the fluorescence intensity amplification rate of the reaction solution to be detected is within the reliable fluorescence intensity amplification rate range a-b, and substituting the fluorescence intensity amplification rate of the reaction solution to be detected obtained after dilution into the fluorescence intensity amplification rate-PO4 3-Linear relation of concentration to obtain PO of the diluted reaction solution to be detected4 3-The concentration is obtained, and then the PO in the solution to be detected is obtained by dilution times4 3-Concentration;
f) if the fluorescence intensity amplification rate of the reaction solution to be detected is lower than the reliable fluorescence intensity amplification rate range a-b, increasing PO in the solution to be detected4 3-Until the fluorescence intensity amplification rate of the reaction solution to be detected is within the reliable fluorescence intensity amplification rate range a-b, and substituting the fluorescence intensity amplification rate of the reaction solution to be detected, which is obtained after the concentration is increased, into the fluorescence intensity amplification rate-PO4 3-Linear relationship of concentration to obtain PO of the reaction solution to be measured after concentration increase4 3-Concentration, and then by subtracting the added PO4 3-Is obtained byPO in the solution to be tested4 3-And (4) concentration.
3. The method according to claim 1, wherein the Ca is prepared in the step 1)2+The calcium source of the solution is one of calcium chloride dihydrate and calcium nitrate tetrahydrate; preparing the Eu in step 1)3+The europium source of the solution is one of europium nitrate hexahydrate and europium chloride hexahydrate.
4. The method according to claim 1, wherein Ca is used in the step 1)2+Solution and Eu3+The total molar concentration of Ca and Eu in the mixed solution after the solution is mixed is 0.07515M, and the ratio of the amount of Eu substances to the total amount of Ca and Eu substances is 5% -10%; the amount of carboxyl group in the polyacrylic acid solution and Ca in the step 1)2+Solution and Eu3+The ratio of the total amount of Ca and Eu in the mixed solution after the solution mixing is 2.0-4.0.
5. The method for quantitatively detecting phosphate ions according to claim 1, wherein the particle size of the precursor of the PAA-Ca (Eu) complex in the step 1) is 2 to 3 nm.
6. The method according to claim 1, wherein the PO is prepared in the step 2)4 3-The phosphorus source of the solution is one of diammonium hydrogen phosphate and disodium hydrogen phosphate dodecahydrate.
7. The method for quantitatively detecting phosphate ions according to claim 1, wherein the series of specific concentrations of PO in the step 2)4 3-PO in standard solution4 3-Amount of substance (a) and Ca2+Solution and Eu3+The total mass ratio of Ca and Eu in the mixed solution after the solution is mixed is less than or equal to 0.1.
8. According to claim 1The method for quantitatively detecting phosphate ions, wherein the PAA-Ca (Eu) complex precursor and the PO are used in the step 2)4 3-The reaction time of the standard solution is 6-8 h; the reaction time of the PAA-Ca (Eu) complex precursor and the solution to be detected in the step 3) is 6-8 h.
9. The method for quantitatively detecting phosphate ions according to claim 1, wherein the fluorescence intensity amplification rate Δ I of the series of standard reaction solutions in the step 2) and the fluorescence intensity amplification rate Δ I of the reaction solution to be detected in the step 3) are calculated by the following formula:
ΔI＝(I-I0)/I0，
wherein I represents the fluorescence intensity of the series of standard reaction solutions and the reaction solution to be measured, I0Represents PAA-Ca (Eu) complex precursor and PO4 3-Fluorescence intensity after mixing of a standard solution with a concentration of 0.
10. The method for quantitatively detecting phosphate ions according to any one of claims 1 to 9, which is applied to quantitatively detecting phosphate in organisms or environments.
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