CN115480019A - Method for determining chlorate in Chinese yam - Google Patents

Abstract

The invention relates to the field of analysis, and discloses a method for determining chlorate in Chinese yam, which comprises the following steps: (1) Mixing Chinese yam with water, performing ultrasonic treatment in ice water, performing first solid-liquid separation, mixing the obtained first clear liquid with a protein precipitator to precipitate protein in the first clear liquid, performing second solid-liquid separation, and purifying the obtained second clear liquid to obtain a purified liquid phase; (2) Preparing chlorate standard solutions with different concentrations, analyzing the content of chlorate ions by adopting ion chromatography, and drawing a standard curve to obtain a regression equation; (3) And carrying out ion chromatography analysis on the purified liquid phase, and substituting the obtained result into a regression equation to obtain the content of the chlorate. The method can accurately determine chlorate content in the yam sample, and the repeatability and stability of the analysis result are high.

Description

Method for determining chlorate in Chinese yam

Technical Field

The invention relates to the field of analysis, in particular to a method for determining chlorate in Chinese yam.

Background

The chlorate salt is a chlorate salt (ClO) ₃ ^- ) The salts of (2) mainly include sodium chlorate, potassium chlorate, magnesium chlorate, etc. Is an important chemical raw material, is used for preparing substances such as chlorine dioxide, sodium chlorite, perchlorate and the like, and is widely applied to industrial and agricultural production. Because of its strong oxidizing property, it has potential toxicity to organism, can cause hemoglobinemia, gastroenteritis, liver and kidney damage, etc., even suffocate, can not be used as food additive, also must not be added into food.

Bleaching water (sodium hypochlorite) is an inorganic chlorine-containing detergent and is widely applied to sterilization, deodorization, cleaning, environmental disinfection, water body disinfection and the like in the processes of medical treatment, health and epidemic prevention, industrial and agricultural food, beverage, fruit and vegetable processing; in addition, hypochlorous acid is extremely unstable and is easily hydrolyzed or reacts with CO ₂ Hypochlorous acid is generated, and strong oxidizing property and bleaching property are shown. The sodium hypochlorite solution can also perform disproportionation reaction at low temperature after disinfecting the surface of food to generate NaClO containing chlorate ₃ Chlorate is generally not present in the organism, and the presence of chlorate indirectly characterizes whether sodium hypochlorite is used in the food.

SN/T4049-2014 ion chromatography for determination of chlorate in export food is implemented by the State administration of quality supervision, inspection and quarantine in China in 2015, samples are extracted by pure water and purified by a solid-phase extraction column, and the State administration of food and drug supervision and administration in 2017 issues a food supplement inspection method BJS 201706 determination of chlorate and perchlorate in food in No. 64 notice in 2017. The pretreatment conditions of these standard methods are not very suitable for treating the substrate of yam, a plant-derived food containing a large amount of starch and mucin (a polysaccharide-protein mixture), and can not complete the determination of chlorate by simple water extraction, otherwise the blockage of a chromatographic sample feeding system and a chromatographic column is easily caused.

Disclosure of Invention

The invention aims to overcome the technical problems that the content of starch and mucin in a yam sample is high and the chlorate content in the yam sample cannot be directly analyzed and determined by sample injection in the prior art, and provides a method for determining chlorate in yam.

The inventor of the application finds that the chlorate ions in the yam sample are extracted by ultrasonic in an ice-water bath, so that the dissolution of starch in the yam can be effectively reduced, the supernatant is obtained after solid-liquid separation, the mucin in the yam is precipitated by utilizing the protein precipitation effect of potassium ferrocyanide and zinc acetate, the starch and the mucin in the yam sample can be removed, the chlorate content in the yam sample is accurately determined, and the repeatability and the stability of an analysis result are high.

Therefore, in order to achieve the above objects, the present invention provides a method for determining chlorate in yam, the method comprising:

(1) Mixing Chinese yam with water, performing ultrasonic treatment in ice water, performing first solid-liquid separation, mixing the obtained first clear liquid with a protein precipitator to precipitate protein in the first clear liquid, performing second solid-liquid separation, and purifying the obtained second clear liquid to obtain a purified liquid phase;

(2) Preparing chlorate standard solutions with different concentrations, analyzing the content of chlorate ions by adopting ion chromatography, and drawing a standard curve to obtain a regression equation;

(3) And carrying out ion chromatography analysis on the purified liquid phase, and substituting the obtained result into a regression equation to obtain the content of the chlorate.

The method for determining chlorate in Chinese yam provided by the invention can establish a new working curve and pretreatment conditions for chlorate ions in Chinese yam samples with large starch and mucin protein, so that the chlorate content in the Chinese yam samples can be accurately determined, and the repeatability and stability of analysis results are high.

Drawings

Figure 1 typical chromatogram for chlorate standard;

figure 2 chlorate standard graph;

FIG. 3 chromatogram of test example yam sample A;

FIG. 4 chromatogram of yam sample B in test example;

FIG. 5 chromatogram of test example yam sample C.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and these ranges or values should be understood to encompass values close to these ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention provides a method for determining chlorate in Chinese yam, which comprises the following steps:

(1) Mixing Chinese yam with water, performing ultrasonic treatment in ice water, performing first solid-liquid separation, mixing the obtained first clear liquid with a protein precipitator to precipitate protein in the first clear liquid, performing second solid-liquid separation, and purifying the obtained second clear liquid to obtain a purified liquid phase;

(2) Preparing chlorate standard solutions with different concentrations, analyzing the content of chlorate ions by adopting ion chromatography, and drawing a standard curve to obtain a regression equation;

(3) And carrying out ion chromatography analysis on the purified liquid phase, and substituting the obtained result into a regression equation to obtain the content of the chlorate.

It will be appreciated that the temperature of the ice water is typically about 0 ℃.

In some embodiments of the present invention, in step (1), the solid-to-liquid ratio of yam to water is preferably (5-10) g: (50-100) mL.

In some embodiments of the present invention, to further ensure the extraction efficiency of the target object, the ultrasonic frequency is preferably 33 to 40Hz, and the time is preferably 20 to 30min.

In some embodiments of the present invention, in step (1), the present invention does not limit the first solid-liquid separation mode as long as the purpose of solid-liquid separation can be achieved, and for example, the first solid-liquid separation mode is centrifugation, and conditions of the centrifugation include: the temperature is preferably 4-8 ℃. The rotation speed is preferably 8000-10000rpm. The time is preferably 3-5min.

According to the invention, the protein precipitant is preferably used in an amount of 2 to 15g per liter of the first clear liquid.

In some embodiments of the invention, to further ensure removal of proteins contained in the sample, the protein precipitating agent comprises potassium ferrocyanide and zinc acetate.

In some embodiments of the invention, in step (1), the amount of potassium ferrocyanide is 2-4g and the amount of zinc acetate is 4.4-8.8g per liter of the first clear solution. For example, when potassium ferrocyanide and zinc acetate are used in the form of a solution, the concentration of potassium ferrocyanide is 100g/L, and the concentration of zinc acetate is 220g/L, the amount of potassium ferrocyanide and zinc acetate is 0.02-0.04mL per mL of the first clear liquid. Satisfying the above range, the introduction of metal ions can be further reduced, while further ensuring the protein removal effect.

In some embodiments of the present invention, in step (1), the present invention does not limit the first solid-liquid separation mode as long as the purpose of solid-liquid separation can be achieved, and for example, the first solid-liquid separation mode is centrifugation, and conditions of the centrifugation include: the temperature is preferably 4-8 ℃. The rotation speed is preferably 8000-10000rpm. The time is preferably 5-10min. The second solid-liquid separation mode is not limited in the present invention, and the purpose of solid-liquid separation can be achieved.

In some embodiments of the invention, in step (1), the purification treatment comprises: mixing the second clear solution with a pH adjusting agent. Preferably, the pH value of the mixed liquid obtained by mixing the second clear liquid and the pH regulator is 11-12. Further preferably, the pH adjusting agent is selected from sodium hydroxide and/or potassium hydroxide. When the above range is satisfied, excessive metal ions such as zinc ions and iron ions can be prevented from entering the ion chromatography and precipitating when encountering strong alkaline leacheate.

In some embodiments of the invention, the method further comprises: and carrying out third solid-liquid separation on the mixed solution obtained by mixing the second clear solution and the pH regulator, and filtering the third clear solution through a filter membrane after sequentially passing through an IC-Ag column and an IC-Na column. Preferably, the IC-Ag column and the IC-Na column are activated in advance before the mixed solution is sequentially passed through the IC-Ag column and the IC-Na column. Specifically, the activation treatment specifically comprises: rinsing with water, and keeping flat for 15-30min to equilibrate.

In some embodiments of the invention, in step (2), the concentration of the chlorate standard solution is in the range of 0.05-5 μ g/mL.

In the present invention, the ion chromatography conditions are:

a chromatographic column: a Dionex IonPacAS19 analytical column and an IonPacAS19 protective column;

mobile phase: DIONEXEG50 automatic drip washer generator, OH-type;

column temperature: 30 plus or minus 0.5 ℃;

flow rate: 1 plus or minus 0.1mL/min;

sample injection amount: 50 +/-0.5 mu L;

a suppressor: dionexassrs 4mm anion suppressor;

a detector: a conductivity detector.

According to a specific embodiment of the invention, the method for determining chlorate in yam comprises the following specific steps:

step one, pretreatment of yam samples and extraction of chlorate

Weighing a yam sample in a conical flask, adding water (the conductivity is more than or equal to 18.2M omega), and performing ultrasonic dissolution and extraction in an ice-water bath, wherein the solid-liquid ratio of yam to water is 5g:50-60mL, the ultrasonic frequency is 33-34Hz, and the time is 20-21min; then solid-liquid separation is carried out for 4-5min at 9000-10000rpm under the condition of 4-5 ℃, then supernatant fluid is taken, potassium ferrocyanide solution and zinc acetate solution are added, and the dosage of potassium ferrocyanide is 2.8-3.5g and the dosage of zinc acetate is 5-7g relative to each liter of first clear fluid.

Shaking, centrifuging at 9000-10000rpmm for 5-10min at 4-5 deg.C, transferring all supernatant, adjusting pH to 11.5-12, centrifuging again (centrifuging at 9500-10000rpm at 4-5 deg.C for 5-6 min), collecting supernatant, sequentially passing through serially connected IC-Ag and IC-Na columns, discarding the first 3mL, and purifying by introducing 0.22 μm water-based filter membrane to the end of the serially connected IC column to obtain purified liquid phase. Step two, solution preparation

Transferring the chlorate standard solution to prepare chlorate standard intermediate solution with the concentration of 100 mu g/mL; diluting the chlorate standard intermediate solution to prepare 0.05-5 mug/mL chlorate standard series working solution.

Step three, ion chromatography detection

Respectively injecting the chlorate standard series working solution and the purified solution of the sample into an ion chromatograph for chromatographic analysis. Performing linear regression by taking the concentration of the chlorate standard series working solution as a horizontal coordinate and the peak area as a vertical coordinate, and establishing a standard curve to obtain a regression equation; and calculating the chlorate content of the yam sample by comparing the retention time with a standard curve.

Wherein the ion chromatography conditions are as follows: and (3) chromatographic column: dionex IonPac AS19 analytical column (4 mm multiplied by 250 mm), ionPac AS19 protective column ((4 mm multiplied by 50 mm), mobile phase DIONEX EG50 automatic leaching solution generator, OH-type, column temperature: 30 ℃, flow rate: 1mL/min, sample injection amount: 50 muL, suppressor: DIONEX ASRS4mm anion suppressor, external water suppression mode, suppression current 100mA, detector: conductivity detector.

According to the method for determining chlorate content in Chinese yam, the adopted extraction method can be used for efficiently extracting chlorate in a Chinese yam sample; the adopted ion chromatography has good repeatability and stability, and the AS19 type anion exchange chromatographic column with high hydrophilicity and high selectivity to leacheate OH & lt- & gt and high flux is selected for analysis, so that the specificity is strong, and the accuracy is high. The ion chromatography is simple and convenient to operate, has high automation degree, and can realize batch processing, so that the detection working efficiency is improved, and the quality of analysis and test work is improved.

The present invention will be described in detail below by way of examples. In the following examples, the apparatus used is as follows: thermo Scientific ICS 5000+ system ion chromatograph (conductivity detector); analytical balance (Sartorius, germany); vortex mixer Vortex1 (IKA, germany); allegra 64R high speed refrigerated table centrifuge (Beckman corporation, usa); model Milli-Q Advantage A-10 ultrapure water purification system (Millipore, USA).

A chromatographic column: dionex IonPac AS19 analytical column (4 mm. Times.250 mm), ionPac AS19 guard column (4 mm. Times.50 mm); a small column (average particle size 40 μm, exchange capacity 2.0-2.2meq/1cc, tianjin Bonnert IC-Ag ion chromatography) is pretreated; cleanert IC-Na ion chromatography pretreatment of small column (average particle size 40 μm, exchange capacity 2.0-2.2meq/1cc, tianjin Bonner Aijiel Co.).

Medicine preparation: chlorate quasi-substance (1000 mug/mL, national center for analysis and test of nonferrous metals and electronic materials), sodium hydroxide solution (50%, germany Merck), zinc acetate (analytically pure, chemical reagents of national pharmaceutical group Co., ltd.), potassium ferrocyanide (analytically pure, chemical reagents of national pharmaceutical group Co., ltd.). The ultrapure water for the experiment was prepared by a Milli-Q ultrapure water machine.

The yam samples used in the examples were chlorate free. Before use, the IC-Ag and IC-Na small column needs to be subjected to activation treatment, and the activation treatment specifically comprises the following steps: rinsed with 10mL of ultrapure water and then kept flat for 20min.

Example 1

Taking a blank yam sample (namely the yam sample which is considered to contain no chlorate), adding a certain amount of chlorate standard substance (so as to know the real chlorate content N in the yam sample), and preparing low, medium and high concentration standard samples (N) _{Is low in} 、N _In 、N _{Height of} ) 6 parts of each. Respectively detecting the standard-added samples according to the following steps from one step to three, respectively measuring and calculating the sulfate radical content X in each standard-added sample ₁ The recovery rate W was calculated as follows:

taking a low-concentration standard sample as an example, calculating the recovery rates corresponding to 6 standard samples respectively, and averaging the six recovery rate values to obtain an average recovery rate. The average recovery rates for the low, medium and high concentration spiked samples are shown in Table 2.

Taking a low-concentration standard sample as an example, the Relative Standard Deviation (RSD) is calculated as follows:

wherein X represents chlorate content in each spiked sample,

refers to the average chlorate content and n refers to the total number of samples.

The relative standard deviations for the low, medium and high concentrations of spiked samples are shown in Table 2.

Firstly, pretreatment of yam samples and extraction of chlorate

Precisely weighing 5g of the above prepared rhizoma Dioscoreae sample in 250mL conical flask, adding 50mL of ultrapure water, ultrasonic dissolving in ice water bath (frequency 33Hz; time 20 min), and centrifuging at 4 deg.C and 10000rpm for 5min. Taking 25mL of supernatant, adding 0.75mL each of 100g/L potassium ferrocyanide solution and 220g/L zinc acetate solution, shaking up, and centrifuging at 10000rpm at 4 ℃ for 5min. The whole supernatant was transferred, the pH was adjusted to 12 with 100g/L potassium hydroxide, and the mixture was centrifuged again (4 ℃ centrifugation at 10000rpm for 5 min). And (3) taking 5mL of supernatant, passing through the serially connected IC-Ag and IC-Na columns, discarding the front 3mL, and then introducing a 0.22 mu m water system filter membrane to the tail end of the serially connected IC column for purification to obtain the sample purification solution.

Step two, solution preparation

Precisely transferring the chlorate standard solution to prepare chlorate standard intermediate solution with the concentration of 100 mu g/mL; the chlorate standard intermediate solution was diluted to prepare 6 concentrations of chlorate standard working solutions, such as 0.05. Mu.g/mL, 0.1. Mu.g/mL, 0.5. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 5. Mu.g/mL, etc.

Step three, ion chromatography detection

Respectively injecting the chlorate standard series working solution and the sample purifying solution into an ion chromatograph for chromatographic analysis. The ion chromatography operating conditions were: and (3) chromatographic column: dionex IonPac AS19 analytical column (4 mmx250 mm), ionPacAS19 guard column ((4 mm x 50 mm), mobile phase DIONEX EG50 automatic eluent generator, OH-type, column temperature: 30 ℃, flow rate: 1.0mL/min, sample introduction: 50. Mu.L, suppressor: DIONEX ASRS4mm anion suppressor, water addition suppression mode, suppression current 75mA, detector: conductivity detector.

1) And performing linear regression by taking the concentration of the working solution of the chlorate standard series as a horizontal coordinate and the peak area as a vertical coordinate, and establishing a standard curve to obtain a regression equation shown in table 1.

TABLE 1

Test object	Linear Range, μ g/mL	Regression equation	Coefficient of correlation, r
				Chlorate (in chlorate)	0.05-5	Y＝0.2388X-0.009	0.9998

2) Substituting the peak area of the ion chromatogram corresponding to the sample purification solution into the regression equation to obtain the concentration c of the component (chlorate) to be detected in the sample purification solution, calculating the chlorate content in the marked yam sample adopted in the step one, and calculating by adopting the following formula:

x: chlorate content (in chlorate) mg/kg;

c: the concentration of the solution of the component to be detected in the sample solution obtained from the standard working curve is mu g/mL;

c ₀ : the concentration of the component solution to be detected in the blank solution obtained from the standard working curve is mu g/mL;

v: volume of extract, mL

m: weight of sample, g

f: and (4) dilution times.

Example 2

The chlorate content in yam was determined according to the method of example 1, except that in the first step, 8g of yam sample was precisely weighed into a 250mL conical flask, 80mL of ultrapure water was added to the flask for ultrasonic dissolution extraction in an ice-water bath (frequency 40Hz, time 28 min), and centrifugation was carried out at 8000rpm for 3min at 6 ℃. Taking 40mL of supernatant, adding 0.8mL of each of 100g/L potassium ferrocyanide solution and 220g/L zinc acetate solution, shaking up, and centrifuging at 8000rpm at 6 ℃ for 8min. The whole supernatant was transferred, the pH was adjusted to 11 with 100g/L potassium hydroxide, and the mixture was centrifuged again (9000 rpm at 6 ℃ C. For 7 min).

Example 3

The chlorate content in yam was determined according to the method of example 1, except that in the first step, 10g of yam sample was precisely weighed into a 250mL conical flask, 100mL of ultrapure water was added to the flask and dissolved and extracted by ultrasound (frequency 35Hz, time 22 min) in an ice water bath, and centrifuged at 10000rpm for 5min at 8 ℃. Taking 50mL of supernatant, adding 2mL of each of 100g/L potassium ferrocyanide solution and 220g/L zinc acetate solution, shaking up, and centrifuging at 10000rpm for 10min at 8 ℃. The whole supernatant was transferred, the pH was adjusted to 12 with 100g/L potassium hydroxide, and the mixture was centrifuged again (8 ℃ centrifugation at 8000rpm for 10 min).

Example 4

The measurement was carried out in accordance with the procedure of example 1, except that instead of adding potassium ferrocyanide and zinc acetate, 5mL of acetonitrile and 2g of sodium chloride were added for protein precipitation.

Example 5

The assay was carried out as in example 1, except that instead of adding potassium ferrocyanide and zinc acetate, 5mL of a 10wt% trichloroacetic acid solution was added for protein precipitation.

Comparative example 1

The measurement was carried out in the same manner as in example 1, except that the sonication was not carried out in an ice-water bath, but carried out at ordinary temperature.

TABLE 2

The results in table 2 show that the method provided by the present invention has good effect, high recovery rate of chlorate under low, medium and high loading concentration conditions, and low relative deviation (the recovery rate of extraction is between about 50-120%, the result of the method can be considered as credible), which indicates that the method of the present invention has good recovery rate result and high accuracy. In addition, the present inventors have found that the effect is slightly inferior when the potassium ferrocyanide solution and zinc acetate are replaced with other protein precipitants.

Application example

The ion chromatography detection method for chlorate content in yam samples provided in example 1 is adopted, except that the yam samples added with the standard are replaced with commercially available yam samples, that is, the chlorate content in the commercially available yam samples is detected, and the detection results are shown in fig. 3, fig. 4, fig. 5 and table 3.

TABLE 3

As can be seen from Table 3, the detection of the yam sample by the method of the invention solves the technical problems that the yam sample has high starch and mucin content and the chlorate content in the yam sample can not be directly analyzed and determined by sample injection in the prior art. The method has good repeatability and stability, and the selected high-throughput AS19 type anion exchange chromatographic column with good hydrophilic performance and high selectivity on the leacheate OH < - > is strong in specificity and high in accuracy.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including various technical features being combined in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (10)

1. A method for measuring chlorate in Chinese yam is characterized by comprising the following steps:

(1) Mixing Chinese yam with water, performing ultrasonic treatment in ice water, performing first solid-liquid separation, mixing the obtained first clear liquid with a protein precipitator to precipitate protein in the first clear liquid, performing second solid-liquid separation, and purifying the obtained second clear liquid to obtain a purified liquid phase;

(2) Preparing chlorate standard solutions with different concentrations, analyzing the content of chlorate ions by adopting ion chromatography, and drawing a standard curve to obtain a regression equation;

(3) And carrying out ion chromatography analysis on the purified liquid phase, and substituting the obtained result into a regression equation to obtain the content of the chlorate.

2. The method as claimed in claim 1, wherein in step (1), the solid-to-liquid ratio of yam to water is (5-10) g: (50-100) mL;

and/or the frequency of the ultrasonic wave is 33-40Hz, and the time is 20-30min.

3. The method according to claim 1 or 2, wherein in step (1), the first solid-liquid separation mode is centrifugation, and the centrifugation conditions comprise: the temperature is 4-8 deg.C, the rotation speed is 8000-10000rpm, and the time is 3-5min;

and/or the amount of the protein precipitator is 2-15g per liter of the first clear liquid;

and/or, the protein precipitating agent comprises potassium ferrocyanide and zinc acetate.

4. The method according to claim 3, wherein in the step (1), the amount of potassium ferrocyanide is 2-4g and the amount of zinc acetate is 4.4-8.8g per liter of the first clear solution.

5. The method according to any one of claims 1-4, wherein in step (1), the second solid-liquid separation mode is centrifugation, and the conditions of the centrifugation comprise: the temperature is 4-8 deg.C, the rotation speed is 8000-10000rpm, and the time is 5-10min.

6. The method according to any one of claims 1 to 5, wherein in step (1), the purification treatment comprises: mixing the second clear solution with a pH adjusting agent.

7. The method according to claim 6, wherein the pH value of the mixed solution obtained by mixing the second clear solution and the pH regulator is 11-12;

preferably, the pH adjusting agent is selected from sodium hydroxide and/or potassium hydroxide.

8. The method of claim 6, wherein the method further comprises: and carrying out third solid-liquid separation on the mixed solution obtained by mixing the second clear solution and the pH regulator, and filtering the third clear solution through a filter membrane after sequentially passing through an IC-Ag column and an IC-Na column.

9. The method according to claim 8, wherein the IC-Ag column and the IC-Na column are activated in advance before passing the third clear solution through the IC-Ag column and the IC-Na column in this order.

10. The process as claimed in any one of claims 1-9, wherein in step (2), the concentration of the chlorate standard solution is in the range of 0.05-5 μ g/mL;

and/or, the ion chromatography conditions comprise:

a chromatographic column: a Dionex IonPacAS19 analytical column and an IonPacAS19 protective column;

mobile phase: DIONEXEG50 automatic drip washing liquid generator, OH-type;

column temperature: 30 plus or minus 0.5 ℃;

flow rate: 1 plus or minus 0.1mL/min;

sample injection amount: 50 +/-0.5 mu L;

a suppressor: dionexassrs 4mm anion suppressor;

a detector: a conductivity detector.

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