CN104614210A - Method for pre-treatment of sample used in element organic form analysis and test - Google Patents

Abstract

The invention discloses a sample pretreatment method for the analysis and test of the organic form of elements. The specific steps are: weighing 2.00g of animal liver samples and 6.00g of quartz sand, fully stirring them evenly, putting them into an extraction pool, and then adding 100 μL of a standard mixture of the three arsenic species at a concentration of 1.00 mgmL ⁻¹ ; proceed to extraction. After the whole extraction process is completed, a total of 80-95mL of the extracted solution is obtained, and the volume is adjusted to 100mL with the extraction solvent; 30mL of the extracted sample solution is added to a 50mL polypropylene high-speed centrifuge tube. Centrifuge for 10 minutes to remove sediment; put it in the refrigerator and freeze at -20°C for 30 minutes to remove fat, use a sampling needle to draw 1mL sample solution in the middle of the centrifuge tube, and filter it through a 0.45μm organic filter membrane for testing. The invention can obtain a recovery rate of more than 80%.

Description

Sample pretreatment methods for analysis and testing of organic species of elements

technical field

The invention relates to an analysis and testing technology of organic forms of elements, in particular to a sample pretreatment method.

Background technique

In the analysis and test of the organic form of elements, due to the complex matrix and the low content of each form of compound, it is necessary to pre-treat the sample before the test, that is, to extract, purify and concentrate the sample. During the pre-treatment process, no should cause a change in the analytical composition of the sample. The speciation analysis of elements is to separate and detect the compounds of different forms in the sample. Obviously, the dry ashing, wet digestion and closed microwave digestion methods commonly used for the total amount of elements are not suitable for the analysis of the organic species of elements.

The sample matrix has a great influence on the extraction efficiency, and the more complex the matrix, the lower the extraction efficiency. In order to ensure the stability of the analyte in the pretreatment process, at present, the pretreatment technology widely used in elemental morphology analysis is the soft extraction method, that is, extraction by solvent, and the solvent system is generally methanol/water (or acetonitrile/water) , completed by stirring or ultrasonic steps, and continuous extraction can be used for complex samples. These traditional extraction methods have many disadvantages: (1) the amount of extraction solvent is relatively large; (2) the extraction time is long, and the analysis process takes an average of more than 1 hour for each sample; (3) the operation is relatively cumbersome, The degree of automation is low.

Contents of the invention

In order to overcome the above-mentioned prior art, the present invention proposes a sample pretreatment method for analysis and testing of organic forms of elements, using accelerated solvent extraction to extract organic arsenic in animal-derived food samples, and then proposes a method for several Optimization of extraction conditions for organic arsenic preparations.

The present invention proposes a sample pretreatment method for analysis and testing of organic forms of elements. Based on the selection of the 34mL stainless steel extraction pool equipped with the ASE300 accelerated solvent extraction system and the collection of extracts in 200mL glass bottles, the method includes the following steps:

Weigh 2.00g of animal liver sample and 6.00g of quartz sand, stir them evenly, put them into the extraction pool, and then add 100uL of three arsenic form mixtures with a concentration of 1.00mg mL-1 into the extraction pool;

Extraction was carried out under the following conditions: methanol/water solution with extraction solvent volume ratio of 3:7; static extraction time of 4 minutes; cycle times of 3 times; flushing rate of 60%; extraction temperature of 80°C; pressure of 1,500psi.

After the whole extraction process is completed, a total of 80-95mL of the extracted solution is obtained, and the volume of the extracted solution is adjusted to 100mL;

Take 30mL of the extracted sample solution and add it to a 50mL polypropylene high-speed centrifuge tube, and centrifuge at 12,000r/m for 10min at 4°C to remove the sediment;

Put it in the refrigerator and freeze it at -20°C for 30 minutes to remove fat, use a sampling needle to draw 1mL sample solution in the middle of the centrifuge tube, and filter it through a 0.45um organic filter membrane for testing.

Compared with the prior art, the recovery rate of more than 80% can be obtained for the sample of the analysis and test sample of the organic form of the element treated by the sample pretreatment method of the present invention.

Description of drawings

Fig. 1 is the overall flow schematic diagram of the present invention;

Fig. 2 is the relation figure of the viscosity of organic solvent and temperature;

Fig. 3 is the relationship figure of the surface tension of organic solvent and temperature;

Fig. 4 is the comparison chart of extraction efficiency of three kinds of extraction solvents of acetone-water, ethanol-water and methanol-water;

Figure 5 shows the effect of different extraction solvents on the extraction effect of three forms of arsenic in the spiked 1.0ug mL-1 sample. a) 100% methanol; b) 80:20v/v methanol/water; c) 50:50v/v methanol/water; d) 30:70v/v methanol/water;

Fig. 6 is the detection result diagram of different extraction solvent extraction samples; a:100% methanol; b:80:20 (v/v) methanol/water; c:50:50 (v/v) methanol/water; d:30 : 70(v/v) methanol/water; e: 20: 80(v/v) methanol/water; f: 100% water; see Table 4-2 for other testing conditions;

Figure 7 shows the effect of temperature on the extraction efficiency of different forms of arsenic. See Table 4-2 for other testing conditions.

Figure 8 shows the effect of standing extraction time on the extraction efficiency of different forms of arsenic. See Table 4-2 for other testing conditions.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but the implementation scope of the present invention is not limited thereto.

Taking the ASE system as an example, factors affecting the extraction efficiency of accelerated solvent extraction methods include:

1. The effect of temperature and pressure

First of all, temperature affects the properties of organic solvents and is one of the important factors affecting the ASE method.

As the temperature increases, the solubility of organic solvents increases (for example, when the temperature rises from 50°C to 150°C, the solubility of anthracene in an ideal organic solvent increases approximately 13 times). The solubility of water in non-polar organic solvents increases with the increase of temperature, for example, when the temperature rises from 25°C to 150°C, the solubility rate can increase by 2 to 10 times.

Second, increasing the temperature weakens and breaks the strong forces between the analyte and the sample matrix.

Forces such as van der Waals forces (dipole-dipole, dipole-induced dipole, diffusion) and hydrogen bonding depend on the chemical structure (e.g. functional groups) between the matrix and the analyte, and thermal energy can reduce the activation energy of the dissolution process.

Thirdly, increasing the temperature can reduce the viscosity of the organic solvent (as shown in Figure 1) and the surface tension (as shown in Figure 1), which can promote the penetration of the organic solvent into the sample micropores and matrix particles, and enhance the mass transfer. (For example, when the temperature rises from 25°C to 200°C, the viscosity of isopropanol decreases to 1/9 of its original value)

The high pressure used in the extraction cell is to keep the organic solvent in a liquid state at high temperature, well above its boiling point. Moreover, the high pressure can "push" the organic solvent into the tiny pores of the sample, fully contacting the analyte and improving the extraction efficiency.

2. The influence of the type of organic solvent

Many organic solvents can be used in the ASE system, except those that are spontaneously combustible in the range of 40°C to 200°C, such as carbon disulfide, ether, 1,4-dioxane, etc. In general, aggressive, strongly acidic solvents are not used]. Migrating the conditions of the traditional extraction method to the ASE method does not require changing the extraction solvent. ASE can use many extraction solvents that are considered inefficient in traditional methods. In the ASE method, the high-temperature and high-pressure environment increases the solvency of the solvent. The advantages of ASE over other extraction methods are evident, for example, for polymer samples. However, for environmental solid samples (such as soil, sediment, etc.), the matrix is relatively complex and contains many components with different concentrations, so it is still very difficult to extract. As the temperature and pressure increase, the extraction rate increases, but the selectivity becomes progressively poorer because more than the analyte is dissolved.

Although ASE can use many solvents that are considered to be inefficient in traditional methods as extractants, this may lead to the dissolution of matrix components that would not be dissolved in traditional extraction methods. This is why the selectivity of the ASE method cannot be easily improved for environmental samples.

3. The influence of matrix composition

The influence of the sample matrix depends on the composition of the sample. Environmental solid samples, such as sediments, soils, etc., vary greatly in their physical and chemical properties, composition types, and particle sizes. These parameters affect the adsorption and retention characteristics of the analyte. As the number of organic components in the sample increases, the complexity of the analysis increases. In order to dissolve the analyte in the sample during the extraction process, we need to find appropriate conditions (such as solvent, temperature, pressure, etc.) to overcome the force between the organic debris (such as wax, humus, etc.) and the analyte. This often results in co-extraction of components in the sample matrix that need to be removed prior to analysis.

Specific embodiments of the present invention are described as follows:

1. Selected experimental instruments and reagents

Accelerated solvent extraction system (ASE300, Dionex, USA), Mettler-Toledo 320-S pH meter (Greifensee, Switzerland), Avanti J-26XPI high-speed centrifuge (Beckman, USA), high-speed homogenizer (IKA, Germany) , Vortex Mixer (Fisher Company, USA), Ultrapure Water Meter (Milli-Q Company, USA); Ultrasonic Water Bath (Type 300, NEY Company, USA).

Methanol, ethanol and acetone (U.S. Dikma Company, chromatographic grade), and quartz sand (Shanghai Tianlian Chemical Reagent Co., Ltd.) were used as extraction solvents in three different combinations and proportions of methanol/water, ethanol/water, and acetone/water.

2. Experimental method

Pork liver and chicken liver purchased in the market within 24 hours were selected as the tested samples and smashed before the experiment.

3. Traditional extraction methods

Three arsenic compounds in food of animal origin were extracted by traditional extraction methods. First, pork liver and chicken liver samples were cut into small portions, freeze-dried and homogenized. Weigh 2.00g of pig liver and 2.00g of chicken liver and place them in polypropylene high-speed centrifuge tubes respectively. Before extraction, add 50uL of ASA, NIT and ROX arsenic form mixture with a concentration of 1.0mg mL ^-1 to the sample. The sample was extracted by 30mL methanol/water mixed solution (1:1, volume ratio v/v) for 20min with the assistance of ultrasonic water bath at 40°C. The obtained sample solution was centrifuged at a high speed of 12,000 r/m for 10 min at 4° C., and the supernatant was taken. Add 10 mL of extraction solvent to the remaining solution, centrifuge again at high speed for 10 min under the above conditions, take the supernatant, mix it with the supernatant obtained above, and dilute to 25 mL. In order to remove the fat in the obtained solution, put it in the refrigerator and freeze it at -20°C for 30 minutes, use a sampling needle to draw 1mL of the sample solution in the middle of the centrifuge tube, and filter it through a 0.45um organic filter membrane for testing.

Four, adopt the accelerated solvent extraction method of the present invention to carry out sample pretreatment

The 34mL stainless steel extraction pool and 200mL glass bottle equipped with the ASE300 accelerated solvent extraction system were selected to collect the extract. Weigh 2.00g of animal liver samples and 6.00g of quartz sand, stir well, put them into the extraction pool, and then add 100uL of three arsenic form mixtures with a concentration of 1.00mg mL-1 into the extraction pool.

Extraction conditions: methanol/water solution with an extraction solvent volume ratio of 3:7; static extraction time 4min; cycle times 3 times; flushing rate 60% (the sample solution is extracted three times, and the volume of each discharged solution is 60% of the volume of the extraction pool, Then add new extraction solvent); extraction temperature 80°C; pressure 1,500psi. After the whole process is completed, a total of 80-95 mL of the extracted solution is obtained, and the volume of the extracted solution is adjusted to 100 mL. Take 30mL of the extracted sample solution and add it to a 50mL polypropylene high-speed centrifuge tube, centrifuge at 4°C and 12,000r/m for 10min at high speed to remove sediment, put it in the refrigerator at -20°C for 30min to remove fat, and use The sampling needle draws 1mL of the sample solution in the middle of the centrifuge tube, and filters it through a 0.45um organic filter membrane for testing.

5. Optimization of accelerated solvent extraction conditions

Extraction solvent, extraction temperature, standing time, washing volume and extraction times are the most important conditions in the ASE method, and their selection has a great influence on the extraction efficiency. During optimization, one of the conditions was changed each time, and the optimal conditions for simultaneously extracting ASA, NIT and ROX were studied. In the optimization process of the following parameters, the above three arsenic compounds in real samples were selected as target extracts, and the extraction effect of ASE method on real samples was studied. For sample pretreatment, adopt the method described above, that is: weigh 2.00g of the treated animal liver sample and 6.00g of quartz sand, stir well, put it into a 34mL extraction pool, add 100uL concentration of 1.00mg mL-1 Mixture of three arsenic species.

1. Selection of extraction solvent

The extraction solvent is crucial to the extraction effect of the analyte, so it is the primary research factor. At present, commonly used extraction solvents mainly include methanol-water, ethanol-water, and acetone-water. The present invention selects three systems of methanol-water, ethanol-water, and acetone-water as the extraction solvent (volume ratio is 1:1), Pig liver samples spiked with 1ug mL-1 of arsanic acid, shofenic acid, and roxarsone were used as test samples to investigate the effects of these three different organic solvent-water systems on different forms of arsenic compounds in animal-derived matrix samples. extraction effect. Other parameters of the accelerated solvent extraction: the extraction temperature is set to 80°C, the delicate extraction time is set to 4min, the washing volume is set to 60%, and the extraction cycle is 3 times. After extraction, the sample is tested by the HG-AFS method, and the instrument conditions are set according to Table 4-2. The extraction efficiency is shown in Figure 3.

Table 4-2 HPLC-HG-AFS system parameter conditions

As can be seen from Figure 3, methanol-water solution has the best extraction efficiency as the extractant, followed by acetone-water, and ethanol-water solution has the lowest extraction efficiency. Acetone is more polar, and acetone-water solution has a stronger penetrating power as an extractant. Usually, there are more fats in animal-derived matrix samples, and acetone-water solution extracts part of the fat while extracting the analyte. It is beneficial to the purification and concentration of the extract, and it will cause the pollution of the chromatographic column and the broadening of the test spectrum, which will affect the experimental effect. The ethanol-water solution extracts a lot of organic matter in the sample solution, and the extract becomes turbid and viscous, which also affects the extraction efficiency of the analyte, and the extraction efficiency is not high. Methanol-water solution was used as the extractant, and the extract solution was relatively clear. Under the same instrument conditions, the peak broadening was small, and the experimental effect was better. Therefore, methanol-water solution was selected as the extractant.

For the volume ratio of methanol-water solution, A) 100% methanol; B) 80:20 (v/v) methanol/water; C) 50:50 (v/v) methanol/water; D) 30:70 (v/v) Methanol/water was used as the extraction solvent, and pig liver samples spiked with 1ug mL-1 of arsanic acid, phenylarsenic acid, and roxarsone were used as test samples to investigate the extraction effects of different ratios of extraction solvents . The extraction temperature was selected to be 80°C, the delicate extraction time was set to 4min, the washing volume was set to 60%, and the extraction was repeated 3 times. After extraction, the sample is tested by the HG-AFS method, and the instrument conditions are set according to Table 4-2.

It can be seen from Figure 4 and Figure 5 that in the case of methanol/water=3:7 (v/v) as the extraction solvent, the extraction efficiency of ASA, NIT and ROX is the highest, while the extraction efficiency of pure methanol is the lowest . Because for pure methanol, the extraction efficiency is higher under the traditional extraction method, but for the ASE method, due to the high temperature and high pressure, pure methanol will extract the analyte together with the sample matrix, and the sample matrix will be tested. Surrounded by substances, on the one hand, the resolution of high performance liquid chromatography is affected, and on the other hand, the reaction efficiency of the analyte in contact with the hydrogenation reagent is low, resulting in a low final detection value. Based on this, 3:7 (v/v) methanol/water solution was selected as the extraction solvent, and the optimization of the following parameters was based on this.

2. Optimization of extraction temperature

Temperature is a very important factor for the extraction efficiency, select several typical values of temperature, and study the optimal extraction temperature for ASA, NIT and ROX through a series of experiments. In this process, the 3:7 (v/v) methanol/water solution proposed in the previous step was selected as the extraction solvent, the extraction time was left to stand for 4 minutes, the volume of washing was 60%, and the number of cycles was 3 times. The test results of the extracts at different temperatures are shown in Figure 5-8. It can be seen from the figure that the signal intensity of ASA, NIT and ROX increased at the beginning, and the change was not particularly significant until 120°C. After 120°C, the three arsenides The signal strength of all decreased rapidly, especially for ASA, the decrease was the most obvious. The reason may be that the three arsenic compounds degrade when the temperature is higher than 120 °C, and the fluctuation of the curve may be due to the deviation introduced during the sample weighing, preparation or extraction process. These three arsenides do not change greatly when the temperature is lower than 120°C, indicating that they are relatively stable in this temperature range. However, when the temperature is higher than 120°C or even higher, the detection intensities of these three arsenides drop significantly. Among them, the strength of ASA decreased most dramatically, indicating that ASA was the most unstable at high temperature. In summary, the present invention chooses 80°C as the extraction temperature, because at this temperature, the detected intensity of the three arsenic compounds is relatively high, that is to say, at this temperature, the extraction efficiency is the best.

3. Optimization of static extraction time

We choose different standing extraction time (2, 4, 6, 8min) to study the effect of standing time on the extraction efficiency, using 3:7 (v/v) methanol/water solution as the extraction solvent, the extraction temperature is 80 ° C, rinse The volume is 60%, and the number of cycles is 3 times. The detection results of samples extracted under different static extraction times are shown in Figure 6. The effect of increasing the extraction time from 2min to 8min on the extraction efficiency of the three arsenic compounds was not very obvious. When the standing time increases to 4min, the extraction efficiency of ASA, NIT and ROX has reached the highest, so when the number of cycles is 3, the selected standing time is 4min.

We found that changing the flushing volume to 20%, 40%, 60%, and 80% had little effect on the extraction efficiency of ASA, NIT, and ROX. Therefore, the flushing volume was appropriately selected as 60%.

For the optimization of the number of cycles, we used the above-mentioned optimized conditions, under the same conditions, a sample was extracted three times continuously, and the influence of the number of cycles on the extraction efficiency was investigated. During the experiment, we found that when the number of cycles was 3, the three arsenic compounds had been fully extracted in the sample, and increasing the number of cycles could no longer improve the extraction efficiency, on the contrary, the intensity decreased. This is due to the increase in the number of cycles and the excessive dilution of the extracted sample, which reduces the detection sensitivity.

In order to evaluate the stability of the ASE method, we divided the same sample into three parts, tested them at intervals of 3 hours and 24 hours respectively under optimized conditions, compared the test results, and finally concluded that the concentration of three arsenic compounds, ASA, NIT and ROX The standard deviations (RSD) of the detection values were all lower than 5%, which indicated that the ASE method was reliable for the detection of these three arsenic compounds under this condition.

The present invention compares with traditional extraction method:

The samples spiked at 1 ug/mL were extracted by the ultrasonic centrifugal extraction method and the rapid solvent extraction method under optimized conditions respectively, both extraction methods were carried out under optimized conditions, and the HPLC-HG-AFS system was used for determination. Refer to Table 4-2 for the setting of each parameter. For the same sample, the two extraction methods were used to extract 4 times to test the stability of the extraction efficiency.

The recovery results of various forms of arsenic compounds are shown in Table 5-1 and Table 5-2 respectively.

Table 5-1 Sample recovery rate of ultrasonic centrifugal extraction method

Table 5-2 Sample recovery rate of fast solvent extraction method

It can be seen from Table 5-2 that the accelerated solvent extraction method is used to extract the three different forms of arsenic compounds in the spiked samples with high efficiency, and the average recovery rates of arsanic acid, shofenic acid and roxarsone are respectively 91.5%, 90.5% and 89.25% were achieved. Moreover, the stability is also good, and the standard deviation is controlled within 2% for the 4 tests of the same sample, which shows that this method is relatively stable and reliable.

In comparison, for the traditional ultrasonic centrifugal extraction method, it can be seen from Table 5-1 that, using the same detection method and conditions, the average spiked recoveries of arsanic acid, shofenic acid and roxarsone are respectively 83%, 73.75% and 77.5%, indicating that the losses of the three forms of arsenic in the pretreatment stage are relatively large; moreover, the standard deviations of the four tests are relatively large, all higher than 5%.

The extraction efficiency of the ultrasonic centrifugal extraction method is not high, mainly because more processing steps have been passed in the pretreatment stage, and each step may cause sample loss, such as homogenization and vortexing. These two operations require the sample solution to be mixed with the The contact of instruments and stirring utensils will inevitably cause the loss of samples. Moreover, because it is purely manual operation, the amount of loss in each treatment is random, so the standard deviation of the final test will be relatively large.

The accelerated solvent extraction method has a high degree of automation, the whole process is automatically completed, and the consistency is good, and since the entire extraction process is carried out in the extraction pool, the extraction solvent is added first, the air in the extraction pool is discharged, and then the extraction process is carried out. The warm and pressurized extraction avoids the oxidation of the sample in contact with air during this process, coupled with multiple purging and flushing under optimized conditions, so the loss of the sample can be minimized.

In summary, for the extraction of different forms of arsenic, the accelerated solvent extraction method has obvious advantages over the traditional extraction method.

Although the present invention has been described above in conjunction with the drawings, the present invention is not limited to the above-mentioned specific embodiments, and the above-mentioned specific embodiments are only illustrative, rather than restrictive. Under the inspiration, many modifications can be made without departing from the gist of the present invention, and these all belong to the protection of the present invention.

Claims (6)

1. A sample pretreatment method for the analytical test of an elemental organic form, based on selecting the equipped 34mL stainless steel extraction pool of the ASE300 accelerated solvent extraction system and collecting the extract in a 200mL glass bottle, it is characterized in that the method comprises the following steps: Weigh 2.00g of animal liver sample and 6.00g of quartz sand, stir them evenly, put them into the extraction pool, and then add 100uL of three arsenic form mixtures with a concentration of 1.00mg mL-1 into the extraction pool; Extraction was carried out under the following conditions: methanol/water solution with extraction solvent volume ratio of 3:7; static extraction time of 4 minutes; cycle times of 3 times; flushing rate of 60%; extraction temperature of 80°C; pressure of 1,500psi. After the whole extraction process is completed, a total of 80-95 mL of the extracted solution is obtained, and the volume is adjusted to 100 mL with the extraction solvent; Take 30mL of the extracted sample solution and add it to a 50mL polypropylene high-speed centrifuge tube, and centrifuge at 12,000r/m for 10min at 4°C to remove the sediment; Put it in the refrigerator and freeze it at -20°C for 30 minutes to remove fat, use a sampling needle to draw 1mL sample solution in the middle of the centrifuge tube, and filter it through a 0.45um organic filter membrane for testing. 2. The sample pretreatment method of the analytical test of elemental organic form as claimed in claim 1, is characterized in that, the optimization selection methanol-water solution of described extraction solvent. 3. The sample pretreatment method for the analysis and test of the organic form of elements as claimed in claim 1, characterized in that the optimal selection of the extraction temperature is 80°C. 4. the sample pretreatment method of the analytical test of elemental organic form as claimed in claim 1, is characterized in that, the optimized selection of described standing time is selected standing time as 4min 5. The sample pretreatment method for the analysis and testing of elemental organic forms as claimed in claim 1, characterized in that the optimal selection of the flushing volume is 60%. 6. The sample pretreatment method for the analysis and test of the organic form of elements as claimed in claim 1, characterized in that, the number of extraction cycles is 3 times.