CN-122016691-A - Method for measuring total phosphorus content in cow urine

CN122016691ACN 122016691 ACN122016691 ACN 122016691ACN-122016691-A

Abstract

The invention discloses a method for measuring total phosphorus content in cow urine, which relates to the technical field of cow urine detection, and comprises the steps of collecting a cow urine sample; regulating pH value of cow urine sample, adding urease solution to react and deactivate urease, taking supernatant as standard cow urine sample, preparing phosphorus standard stock solution, adding vanadium ammonium molybdate color developing agent after gradient dilution to obtain standard working solutions with different concentrations, measuring absorbance of each standard working solution, drawing absorbance-phosphorus concentration standard curve, placing the standard cow urine sample in a microwave digestion tank, adding nitric acid and hydrogen peroxide, performing multi-stage heating program and constant-temperature acid-removing treatment to obtain digestion solution, absorbing the digestion solution, adding vanadium ammonium molybdate color developing agent, measuring absorbance of the digestion solution, and calculating total phosphorus content in the standard cow urine sample according to the absorbance-phosphorus concentration standard curve and absorbance of the digestion solution. The method is simple, convenient, quick and good in accuracy, and is suitable for measuring the total phosphorus content in a large quantity of cow urine.

Inventors

CHEN FANG
BU ZEMING
LIU ZUO
LI WENJING
ZOU CAIXIA
TANG QINGFENG
CHEN BAOYU
ZENG YUNFENG
Diao Peiyuan

Assignees

广西壮族自治区兽药监察所

Dates

Publication Date: 20260512
Application Date: 20260318

Claims (10)

1. A method for determining total phosphorus content in cow urine, the method comprising: S1, collecting all urine of each cow, uniformly mixing, sampling in parallel to obtain a cow urine sample, sealing the cow urine sample, and storing the cow urine sample at a temperature of-20 ℃ for later use; s2, regulating the pH value of the thawed cow urine sample, adding a urease solution to react until no bubbles are generated, inactivating urease after the reaction is finished, and taking supernatant as a standard cow urine sample; s3, preparing a phosphorus standard stock solution, adding an ammonium vanadium molybdate color developing agent after gradient dilution, fixing the volume, standing for color development to obtain standard working solutions with different concentrations; S4, placing a standard cow urine sample in a microwave digestion tank, adding nitric acid and hydrogen peroxide according to a preset proportion, performing a multi-stage heating program to perform initial digestion, performing constant-temperature acid-removing treatment after the initial digestion is completed, and cooling and fixing the volume to obtain a digestion solution; S5, absorbing the digestion liquid, adding an ammonium vanadium molybdate color developing agent, fixing the volume, standing for color development, and measuring the absorbance of the digestion liquid based on preset measurement conditions; s6, calculating the total phosphorus content in the standard cow urine sample according to the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution.
2. The method for determining total phosphorus content in cow urine according to claim 1, wherein the steps of adjusting the pH value of the thawed cow urine sample, adding a urease solution to react until no bubbles are generated, inactivating urease after the reaction is completed, and taking the supernatant as a standard cow urine sample comprise the following steps: S21, regulating the pH value of the thawed cow urine sample to 6.5-7.5 by using dilute hydrochloric acid or dilute sodium hydroxide solution; S22, adding 5-10U of active urease into each milliliter of cow urine sample with the pH value adjusted, and reacting at 35-40 ℃ for 20-30min until no bubbles are generated, so as to obtain a reacted sample; S23, placing the reacted cow urine sample in a boiling water bath with the temperature of 95-100 ℃ for heating for 5-10min until no bubbles are generated, and obtaining a cow urine sample with inactivated urease; s24, centrifuging the urease-inactivated cow urine sample at 4000-6000r/min for 5-10min, and taking the supernatant as a standard cow urine sample.
3. The method for measuring total phosphorus content in cow urine according to claim 1, wherein the preparation of phosphorus standard stock solution, adding vanadium ammonium molybdate color developing agent after gradient dilution, fixing volume and standing for color development to obtain standard working solutions with different concentrations, measuring the absorbance of each standard working solution based on preset measuring conditions, and drawing an absorbance-phosphorus concentration standard curve comprises the following steps: S31, transferring a phosphorus unit element standard solution, and diluting with water to prepare 50 mug/mL phosphorus standard stock solution; s32, carrying out gradient dilution on the phosphorus standard stock solution, respectively adding 10mL of ammonium vanadium molybdate color developing agent, fixing the volume, standing and developing to obtain standard working solutions with different concentrations; S33, measuring the absorbance of each standard working solution under the preset measurement condition, and drawing an absorbance-phosphorus concentration standard curve by taking the phosphorus mass concentration as an abscissa and the absorbance as an ordinate.
4. The method for determining total phosphorus content in cow urine according to claim 3, wherein the step of carrying out gradient dilution on the phosphorus standard stock solution, adding 10mL of ammonium vanadium molybdate color developing agent respectively, fixing the volume and standing for color development to obtain standard working solutions with different concentrations comprises the following steps: s321, respectively transferring the phosphorus standard stock solutions with different volumes into a plurality of 50mL volumetric flasks according to a preset gradient; S322, respectively adding 10mL of ammonium vanadium molybdate color reagent into each 50mL volumetric flask, fixing the volume to the scale with water, shaking uniformly, and standing for more than 10min to obtain standard working solutions with different concentrations.
5. A method for determining total phosphorus content in cow urine according to claim 3, wherein the predetermined determination conditions are: the absorbance was measured using an ultraviolet spectrophotometer at a wavelength of 400nm using a 1cm cuvette.
6. The method for determining total phosphorus content in cow urine according to claim 1, wherein the standard cow urine sample is placed in a microwave digestion tank, nitric acid and hydrogen peroxide are added according to a preset proportion, a multi-stage temperature raising program is executed for initial digestion, constant-temperature acid expelling treatment is carried out after the initial digestion is completed, and the volume is fixed after cooling, so that a digestion solution is obtained, and the method comprises the following steps: s41, transferring a standard cow urine sample, placing the standard cow urine sample in a microwave digestion tank, adding nitric acid and hydrogen peroxide according to a preset proportion, shaking uniformly, and sealing the microwave digestion tank; S42, placing the sealed microwave digestion tank in a microwave digestion instrument, and executing a multi-stage heating program to complete initial digestion to obtain an initial digestion sample; s43, carrying out constant-temperature acid-removing treatment on the initial digestion sample at the temperature of 140-160 ℃ until the acid is removed until about 1mL of the initial digestion sample remains; S44, transferring the initial digestion sample after acid removal to a 25mL volumetric flask, washing the inner wall of the microwave digestion tank for 2-3 times with a small amount of water, combining the washing liquid into the 25mL volumetric flask, and fixing the volume to the scale with water and shaking uniformly to obtain the digestion liquid.
7. The method for determining total phosphorus content in cow urine according to claim 6, wherein the predetermined ratio is: to each 1mL of standard cow urine sample, 5mL of nitric acid was added, and 2mL of hydrogen peroxide was added.
8. The method of claim 6, wherein the multi-stage temperature increasing program comprises a first stage, a second stage and a third stage; wherein, the power is controlled in the first stage to raise the temperature to 80 ℃ within 5-8min and keep for 5min; controlling the power in the second stage to raise the temperature to 120 ℃ within 5-8min, and keeping for 10min; The third stage controls the power to raise the temperature to 160 ℃ within 5-8min and hold for 20min.
9. The method for measuring total phosphorus content in cow urine according to claim 1, wherein the absorbing digestion solution, adding vanadium ammonium molybdate color developing agent, fixing volume and standing for color development, and measuring absorbance of the digestion solution based on preset measuring conditions comprises the following steps: S51, precisely transferring 5-10mL of digestion liquid into a 50mL volumetric flask; s52, adding 10mL of ammonium vanadium molybdate color reagent into a50 mL volumetric flask, fixing the volume to a scale with water, shaking uniformly, and standing for more than 10 min; S53, measuring absorbance of the digestion solution by using a 1cm cuvette and an ultraviolet spectrophotometer at a wavelength of 400 nm.
10. The method for determining total phosphorus content in cow urine according to claim 1, wherein the calculating the total phosphorus content in the cow urine sample based on the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution comprises the steps of: s61, obtaining the phosphorus concentration of the digestion solution according to an absorbance-phosphorus concentration standard curve; S62, multiplying the phosphorus concentration by the fixed volume of the digestion solution and the dilution multiple in measurement, dividing by the original volume of the standard cow urine sample participating in digestion, and calculating to obtain the total phosphorus content in the standard cow urine sample.

Description

Method for measuring total phosphorus content in cow urine Technical Field The invention relates to the technical field of cow urine detection, in particular to a method for measuring total phosphorus content in cow urine. Background In recent years, with the continuous improvement of the national living standard and the quality requirements of people on beef cattle, dairy cows and dairy products (Shang Wenjie and the like, 2023), a large-scale intensive breeding mode of the beef cattle has become a development trend. Urine produced by a cattle farm mainly contains nutrient elements such as nitrogen, phosphorus and potassium and organic matters, and total phosphorus in the cattle urine mainly contains orthophosphate, metaphosphate, organic phosphorus compounds and the like, for example, surface water, underground water, soil and environmental air can be seriously polluted due to improper treatment, and the human health and ecological environment can be directly endangered. The total phosphorus content of the cow urine of the large-scale cow farm is monitored regularly, so that the ingestion and metabolism condition of phosphorus in cow daily ration can be reflected, the cow daily ration formula can be adjusted, the cultivation benefit is improved, the harmless treatment mode of the cow farm manure can be selected according to the total phosphorus content of the cow urine, the total phosphorus content of the cow manure organic fertilizer is reduced, the fertilizer utilization efficiency is improved, and the environmental problems such as water eutrophication and the like are avoided. Therefore, it is important to establish a method for determining the total phosphorus content in cow urine, which is simple to operate and accurate and reliable in result. At present, a method for detecting the total phosphorus content in cow urine and related national or industry standards are reported in the past, and only one document report in 1989 adopts a far infrared digestion method combined with an ultraviolet spectrophotometry to detect the total phosphorus content in human urine, and the digestion time of the method is long, the color development process is complex, and the working efficiency is low. The reported methods for detecting total phosphorus content mainly include inductively coupled plasma mass spectrometry (toffee et al, 2025), ion chromatography (Zhu Gongxia et al, 2022), polarography (Wang Zhonghui et al, 2000), enzyme-labeled method (Liu Yan et al, 2023), ultraviolet spectrophotometry (Jing, 2018) and the like. The first 4 methods have the advantages of high price, high experimental cost and more severe requirements on operators, compared with the conventional ultraviolet spectrophotometry, which is still the most classical and widely applied method due to the advantages of low experimental cost, mature technology, stable color development and the like. For the problems in the related art, no effective solution has been proposed at present. Disclosure of Invention Aiming at the problems in the related art, the invention provides a method for measuring the total phosphorus content in cow urine, which aims to overcome the technical problems in the prior art. For this purpose, the invention adopts the following specific technical scheme: a method for determining total phosphorus content in cow urine, the method comprising: S1, collecting all urine of each cow, uniformly mixing, sampling in parallel to obtain a cow urine sample, sealing the cow urine sample, and storing the cow urine sample at a temperature of-20 ℃ for later use; s2, regulating the pH value of the thawed cow urine sample, adding a urease solution to react until no bubbles are generated, inactivating urease after the reaction is finished, and taking supernatant as a standard cow urine sample; s3, preparing a phosphorus standard stock solution, adding an ammonium vanadium molybdate color developing agent after gradient dilution, fixing the volume, standing for color development to obtain standard working solutions with different concentrations; S4, placing a standard cow urine sample in a microwave digestion tank, adding nitric acid and hydrogen peroxide according to a preset proportion, performing a multi-stage heating program to perform initial digestion, performing constant-temperature acid-removing treatment after the initial digestion is completed, and cooling and fixing the volume to obtain a digestion solution; S5, absorbing the digestion liquid, adding an ammonium vanadium molybdate color developing agent, fixing the volume, standing for color development, and measuring the absorbance of the digestion liquid based on preset measurement conditions; s6, calculating the total phosphorus content in the standard cow urine sample according to the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution. Further, adjusting the pH value of the thawed cow urine sample, adding a urease solution to react un