CN114778264A - Method for simultaneously accumulating, enriching and separating multiple arsenic forms in natural medicine on line based on micelle-solvent accumulation - Google Patents

Abstract

The invention discloses a method for simultaneously accumulating, enriching and separating various arsenic forms in natural medicines on line based on micelle-solvent accumulation. Two-step stacking capillary zone electrophoresis CZE of As (v), As (iii), MMA and DMA was performed efficiently and sensitively by electrokinetic injection and micelle-to-solvent stacking of MSS in co-electroosmotic flow co-EOF using zwitterionic surfactant PAPS. HDMB coats fused silica capillaries to meet the condition of the same EOF direction between anions and capillaries. The background solution is borax buffer solution. Specifically, micelles and target analytes are injected and stacked through a field enhanced sample under negative pressure; methanol was injected, with the analyte scanned being carried by the micelle to the MSS boundary. The invention utilizes the coating capillary electrophoresis from micelle to solvent accumulation method to detect arsenic in natural medicines, realizes the rapid detection of arsenic form and improves the sensitivity of on-line enrichment of capillary electrophoresis samples.

Description

A method for simultaneous online accumulation and separation of various arsenic species in natural medicines based on micelle-solvent accumulation

technical field

The invention belongs to the field of separation and enrichment of arsenic forms in natural medicines, and relates to a method for on-line enrichment of arsenic by capillary electrophoresis technology, in particular to a simultaneous online-on-line method for multiple arsenic forms in natural medicines based on micelle-solvent accumulation Stacking enrichment and separation methods.

Background technique

Arsenic (As) is a natural element in the earth's crust that contains more than 50 compounds of varying toxicity and is widely distributed in the atmosphere, water and land. As industrial pollution permeates every aspect of life, people are exposed to high concentrations of inorganic arsenic, the most toxic species. Chronic arsenic poisoning leading to cardiovascular disease, neurological disease, diabetes or skin, bladder and lung cancer can be caused by prolonged exposure to inorganic arsenic, mainly through drinking contaminated water, eating foods processed from this water, and irrigating with arsenic-rich water. food. Due to the serious effects of arsenic on the human body, it is listed by the World Health Organization (WHO) as one of the 10 chemicals that cause major public health problems. The latest WHO index on arsenic in drinking water is 0.01mg/L, and when the dose exceeds 6×10 ^-4 mg/L, there is a carcinogenic risk. However, due to measurement difficulties and practical difficulties in removing arsenic from drinking water, the development of this index value is only tentative. Although arsenic is extremely harmful to humans, it is used as an ingredient in certain pesticides and in poultry feed formulations to prevent pests and promote growth. Therefore, the accurate detection technology of arsenic content has been continuously studied, which is an extremely important and far-reaching research topic.

Arsenic exists in inorganic forms (arsenite and arsenate) and organic forms (arsenite, arsenocholine, monomethylarsinic acid and dimethylarsinic acid), and different species of arsenic have different toxicity, Among them, inorganic arsenic compounds are more toxic than organic arsenic compounds (such as arsenic compounds in seafood). To date, inductively coupled plasma mass spectrometry (ICP-MS) and hydride generation atomic fluorescence spectrometry (HG-AFS) are the most commonly used combined techniques for the separation and analysis of arsenic species. In addition, other important analytical methods have been developed to detect trace amounts of arsenic in real samples, such as high performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS), ICP optical emission spectrometry (ICP-OES) ), and quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS). Capillary electrophoresis (CE) has strong separation performance, but the short path length and small injection volume of capillary tubes reduce its sensitivity. To overcome these limitations, various in-line sample preconcentration techniques in CE are effective methods to enrich arsenic species, including enhanced in situ sample injection (FESI) and transient isotachophoresis (t-ITP). In addition, many techniques combined with CE, such as CE-Hydride Electrothermal Atomic Absorption Spectrometry (CE-HG-ETAAS), CE-ICP-MS, and CE-ESI-MS (CE-ESI-MS), are also Ideal way to get around these limitations. However, the direct detection of arsenic by capillary electrophoresis is highly attractive due to its high efficiency and simplicity. Therefore, the application of a new high-sensitivity pre-enrichment method for the detection of arsenic remains an interesting research topic.

Typical methods for in-line preconcentration of CE are FESI, t-ITP and dynamic pH ligation. In addition to the above enrichment techniques, micelle-solvent stacking (MSS) is one of the emerging online sample enrichment techniques in recent years. Furthermore, transient capture, focusing of analytes by micelle collapse (AFMC), and micelle-cyclodextrin stacking (MCDS) are advantageous for similarly enhancing detection sensitivity. MSS, first proposed by Joselito P. Quirino in 2009, is achieved by reversing the effective electrophoretic migration direction of organic cations using micelles in the sample solution and organic solvents in the background solution (BGS). The focusing effect is achieved by the inversion of the effective electrophoretic mobility of the target analyte in the boundary region between the micelles and the organic solvent-modified BGS. The superior detection sensitivity of MSS has aroused widespread attention and continues to innovate on this basis. Innovations have focused on the type of micelles and the location of organic solvents and micelles. In general, most previous studies have focused on the detection and separation of anionic analytes in MSS using cationic surfactants. There are generally three models in MSS: one-step stacking of organic solvent in BGS and mixed sample in micelles, two-step stacking of organic solvent and sample and micelle injection in BGS, and three-step stacking of organic solvent, analyte and micelle injection , respectively, for the detection of various compounds. Choosing an injection model between electric and hydrodynamic injection can also significantly improve performance, especially in achieving the lowest detection limits. According to the above, the development of a more sensitive and efficient MSS technique is worth exploring.

The present invention establishes a new two-step superposition model for detecting four kinds of arsenic in coated capillary zone electrophoresis (CZE). Coating of capillary walls with hexamethylene bromide (HDMB) is an important step in reversing electroosmotic flow. The zwitterionic surfactant 3-(N,N-dimethylpalmitamido)propanesulfonate (PAPS) was mixed as micelles with the target analyte and introduced as a sample solution by electrokinetic injection at a negative voltage, and then injected into the organic solution separately , which facilitates the formation of MSS boundaries to achieve a critical step in sample stacking. Various optimized conditions were investigated, including micelle type and concentration, methanol percentage, borax buffer concentration, and injection time to improve stacking efficiency and resolution. This method was used for the determination of arsenic in kelp.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for simultaneous online accumulation and separation of various arsenic forms in natural medicine based on micelle-solvent accumulation, which is more rapid, efficient and sensitive than previous enrichment methods, and is suitable for those with Detection of arsenic in natural medicines in complex matrices.

The inventive method adopts the following technical solutions to realize:

Step (1), target analyte sample preparation:

Wash, dry, pulverize, sieve and mix with HNO ₃ , ultrasonicate for 0.5-1h at 40-45°C, shake at room temperature for 1.5-2h, and finally centrifuge at high speed for 10-15min, and remove the supernatant. The supernatant was neutralized with NaOH to obtain the target analyte sample;

Preferably, the natural medicine refers to kelp;

Preferably, the mass-volume ratio of natural medicine and HNO ₃ is 200mg: 5mL;

Preferably, the concentration of HNO ₃ is 2% (w/v, mg/mL);

Preferably, the centrifugal speed is 4000-5000r/min;

Step (2), target analyte sample enrichment and separation:

2-1. Activated capillary column

A 1% (w/v, g/mL) hexamethylene bromide (HDMB)-coated fused silica capillary was used for 20-25 min to satisfy the condition of the same EOF orientation between the anion and the capillary.

2-2. Rinse the activated capillary column with borax buffer for 5-10 minutes before injection.

Preferably, the concentration of the borax buffer is 25-100 mM, preferably 100 mM.

Preferably, the pH of the borax buffer is 8.8-9.4, more preferably 9.2.

2-3. In the capillary column, the micelles and the target analyte are mixed and injected into the stack as a sample solution under negative pressure for 30-240s, and then an aqueous methanol solution as an organic solvent is injected under negative pressure. The scanned analyte is bound by the micelles. to the MSS boundary to achieve sample stacking and separation; wherein the micelles use 3-(N,N-dimethylpalmitamido)propane sulfonate PAPS.

Preferably, the capillary electrophoresis conditions: the detection wavelength is 192nm, the separation negative pressure is -20kV, and the temperature is 20°C.

Preferably, the micelle concentration is 10-40 mM, more preferably 20 mM.

Preferably, the injection time of the micelle is 180s.

Preferably, the injection negative pressure of the micelle is -10kV.

Preferably, the injection parameters of the methanol aqueous solution are injection power-10kV, injection pressure 50mbar, and injection time 5s.

Preferably, the volume content of the methanol aqueous solution is 40-100% (v/v), preferably 60% (v/v).

The enrichment factors of the above-mentioned various forms of arsenic are [As(V), As(III), MMA and DMA] 1230, 840, 3820 and 1450, respectively. The detection limit (S/N=3) ranged from 0.382 to 0.911 ng/mL. Intracapillary repeatability (% RSD, N=3) migration time was 0.5-1.0% and peak area was 0.3-0.9%.

The advantages of the present invention are:

(1) The present invention proposes an electrodynamic sampling assisted MSS in the coated CE.

(2) The present invention proposes the zwitterionic surfactant 3-(N,N-dimethylpalmitamido)propane sulfonate PAPS as a micellar solution in MSS, and then reverses EOF with trimetium bromide HDMB.

(3) Compared with the typical injection in CZE, the present invention proposes that the enrichment factors of As(V), As(III), MMA and DMA are 1230, 840, 3820 and 1450, respectively.

(4) The present invention utilizes coating capillary electrophoresis to detect arsenic in natural medicines from micelles to solvent accumulation. This method not only realizes the rapid detection of arsenic forms, but also greatly improves the sensitivity of capillary electrophoresis sample online enrichment. Compared with traditional arsenic detection methods, this method achieves efficient detection and separation of typical arsenic species.

Description of drawings

Figure 1 is a diagram of the enrichment mechanism of the present invention.

Figure 2 is the effect of micelle species (PAPS, CTAB, CTAC); CE conditions: micelle concentration in the sample solution is 10mM, analyte concentration is 10mg/L, injection at -10kV for 60s; MeOH (100%) at 50mbar down injection for 5 s; background solution: 100 mM borax buffer.

Figure 3 is the effect of PAPS concentration (0, 5, 10, 20, 30, 40 mM); other CE conditions are the same as in Figure 2.

Figure 4 is the effect of methanol content (40%, 60%, 80%, 100%); CE conditions: same as Figure 2.

Figure 5 is the effect of borax concentration (25, 50, 75, 100 mM); A: electropherogram, 1-As(V), 2-MMA, 3-DMA, 4-As(III); (B) apparent mobility , CE conditions: PAPS concentration in sample solution is 20mM, analyte concentration is 10mg/L, injection at -10kV for 60s; MeOH (60%) injection at 50mbar for 5s; background solution: 100mM borax buffer.

Figure 6 is the effect of buffer pH (8.8, 9.0, 9.2, 9.4); other conditions are the same as in Figure 5.

Figure 7 is the effect of sample injection time (30, 60, 120, 180, 240s); the analyte concentration is 1 mg/L, and other conditions are the same as in Figure 6.

Figure 8A is a spiked electropherogram 60s (a) and 5s (b) of the analysis of kelp under optimal conditions.

Figure 8B is an electropherogram overlaid by a two-step method and a typical injection of a standard solution; two-step stack: a sample solution containing 1 mg/L analyte mix and 20 mM PAPS was injected at -10 kV for 180 s. Methanol (60%) was injected at 50 mbar for 5 s, typical injection: target analyte without micelles was injected at 50 mbar for 3 s, and the mixed arsenic concentration was 10.0 mg/L.

Detailed ways

As mentioned above, in view of the deficiencies of the prior art, the inventor of the present invention, through long-term research and a large number of practices, has proposed the technical solution of the present invention, which is mainly based on at least including: 1) the present invention utilizes a zwitterionic surfactant 3-( N,N-Dimethylpalmitamino)propanesulfonate (PAPS) in co-electroosmotic flow (co-EOF) via electrokinetic injection and micelle-to-solvent stacking (MSS) on four arsenic species [As(V), As(III), sodium monomethylarsinate (MMA) and sodium dimethylarsinate (DMA)] for efficient and sensitive two-step stacking capillary zone electrophoresis (CZE). (2) Coating the capillary wall with hexamethylene bromide (HDMB) reverses the EOF to satisfy the condition of the same EOF direction between the anion and the capillary. (3) In the present invention, the zwitterionic surfactant 3-(N,N-dimethylpalmitoamino)propane sulfonate (PAPS) is mixed with the target analyte as a micelle and introduced as a sample solution by electrokinetic injection at a negative voltage, The organic solution is then injected separately to form MSS boundaries for sample stacking. When a negative separation voltage is applied, all compounds migrate towards the anode under strong reverse EOF. The presence of organic solvents reduces the retention factor (κ). When κ is lowered to the point where the effective electrophoretic mobility of the analyte [μ _ep (a′)] is reversed and directly transferred to the anode, the effective electrophoretic mobility of the anionic analyte [μ _ep ^* (a′)] can be obtained from equation (1) get:

In the formula, μ _ep (a′) is the electrophoretic mobility of the analyte, and μ _ep (mc) is the electrophoretic mobility of the micelle. When the κ of _μep ^* (a′) is 0 due to the collapse of the micelles, _μep (a′) points to the cathode and the analyte accumulates at the MSSB. Thus, formula (1) can be simplified to:

Finally, under the continuous electric field, all surfactants pass through the MSSB and the stacked analytes are separated by CZE. Since the four As species have different migration rates in electrophoresis, they are sequentially detected and analyzed by a UV detector.

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

Fig. 1 is the enrichment mechanism diagram of the present invention, a kind of on-line accumulation enrichment and separation method for multiple arsenic forms in natural medicines based on micelle-solvent accumulation, comprising the following steps:

Step (1), target analyte sample preparation:

Wash, dry, pulverize, sieve and mix with HNO ₃ , ultrasonicate for 0.5-1h at 40-45°C, shake at room temperature for 1.5-2h, and finally centrifuge at high speed for 10-15min, and remove the supernatant. The supernatant was neutralized with NaOH to obtain the target analyte sample;

Preferably, the natural medicine refers to kelp;

Preferably, the mass-volume ratio of natural medicine and HNO ₃ is 200mg: 5mL;

Preferably, the concentration of HNO ₃ is 2% (w/v);

Preferably, the centrifugal speed is 4000-5000r/min;

Step (2), target analyte sample enrichment:

2-1. Activated capillary column

A 1% (w/v, g/mL) hexamethylene bromide (HDMB)-coated fused silica capillary was used for 20-25 min to satisfy the condition of the same EOF orientation between the anion and the capillary.

2-2. Rinse the activated capillary column with borax buffer for 5-10 minutes before injection.

Preferably, the concentration of the borax buffer is 25-100 mM, preferably 100 mM.

Preferably, the pH of the borax buffer is 8.8-9.4, more preferably 9.2.

2-3. In the capillary column, the micelles and the target analyte are mixed and injected into the stack as a sample solution under negative pressure for 30-240s, and then an aqueous methanol solution as an organic solvent is injected under negative pressure. The scanned analyte is bound by the micelles. to the MSS boundary to achieve sample stacking and separation; wherein the micelles use 3-(N,N-dimethylpalmitamido)propane sulfonate PAPS.

Preferably, the capillary electrophoresis conditions: the detection wavelength is 192nm, the separation negative pressure is -20kV, and the temperature is 20°C.

Preferably, the micelle concentration is 10-40 mM, more preferably 20 mM.

Preferably, the injection time of the micelle is 180s.

Preferably, the injection negative pressure of the micelle is -10kV.

Preferably, the injection parameters of the methanol aqueous solution are injection power-10kV, injection pressure 50mbar, and injection time 5s.

Preferably, the volume content of the methanol aqueous solution is 40-100% (v/v), preferably 60% (v/v).

The technical solution of the present invention will be further explained below with reference to some preferred embodiments, but the experimental conditions and setting parameters therein should not be regarded as limitations to the basic technical solution of the present invention. And the protection scope of the present invention is not limited to the following embodiments.

CE conditions are:

Detection wavelength: 192nm. Column temperature: 20°C.

Capillary column: inner diameter 50 μm, outer diameter 375 μm, length 60 cm, effective length 51.5 cm. Before the first use, the new capillary column should be rinsed with 1.0M NaOH solution for 20 minutes, 0.1M NaOH solution for 15 minutes, pure water for 10 minutes, 1% HDMB coating for 30 minutes, and running buffer for 10 minutes. To achieve good reproducibility, the needles were rinsed with 0.1M NaOH solution for 2 minutes, pure water for 2 minutes, and running buffer for 5 minutes.

Separation voltage: -20kV.

Data logging: HP ChemStation (Agilent).

Embodiment 1. Investigate the influence of micelle species on detection effect

The kelp was washed and dried, crushed and passed through a 100-mesh sieve, then 200 mg of each sample was accurately weighed and placed in a 15 mL centrifuge tube, 5 mL of 2% HNO ₃ was added, sonicated for 1 h (40°C), and then shaken at room temperature for 2 h, Finally, centrifuge at 4000 r/min for 15 min. Extracts were neutralized with NaOH prior to analysis.

Fused silica capillaries were coated with 1% hemexonium bromide (HDMB) for 20 min to satisfy the condition of the same EOF orientation between anions and capillaries. The activated capillary column was rinsed with 100 mM borax buffer pH 9.2 for 5 minutes before injection. In the capillary column, 10mM micelles and 10mg/L target analyte were mixed and injected into the stack as a sample solution at -10kV for 60s, and then 100% MeOH5s was injected at -10kV at 50mbar, the scanned analytes were brought from the micelles to the MSS boundary, In order to achieve sample stacking and separation; wherein the micelle adopts 3-(N,N-dimethylpalmitamido)propane sulfonate PAPS. Capillary electrophoresis conditions: the detection wavelength is 192 nm, the separation negative pressure is -20 kV, and the temperature is 20 °C.

Cationic surfactant is a key factor in the formation of MSS, which significantly affects the enrichment and separation efficiency. In this example, three surfactant solutions, PAPS, CTAB or CTAC, were used as micelles to evaluate the effect of micelles on the stacking efficiency of the four target analytes.

As shown in Fig. 2, longer migration time and higher sensitivity can be obtained with CTAB (b) than with CTAC (c), which has a more heterogeneous baseline than CTAB. Meanwhile, when CTAB and CTAC act as micelles, the As(V) peak is not obvious. Clearly, when PAPS(a) is applied, the four analytes have the highest detection sensitivity and the highest satisfactory resolution. This may be due to the fact that PAPS is a quaternary ammonium amphoteric surfactant containing quaternary ammonium salt cations and sulfonic acid anions. With the lowest CMC (0.79×10-4M), zwitterionic surfactants can reverse the EOF and reduce the adsorption of analytes and the coated walls. It has good surface properties and a clear synergistic effect with the analyte. Therefore, PAPS was selected as the micelle for the follow-up work.

Embodiment 2. investigate the influence of PAPS concentration on detection effect

On the basis of Example 1, PAPS was selected as the micelle, and the PAPS concentration was changed to 0, 5, 10, 20, 30, and 40 mM, and other conditions remained unchanged.

The concentration of micelles is a key parameter affecting the formation of micelles to the solvent packing boundary (MSSB). Therefore, different PAPS concentrations in the sample solution were analyzed, and the results are shown in Figure 3. When the surfactant concentration was varied from 0 to 20 mM, the peak areas of As(V), MMA and DMA gradually increased, while the peak area of As(III) decreased sharply when the micelle concentration was varied from 5 to 10 mM. While the PAPS concentration was further increased from 20 mM to 40 mM, the peak heights for all four analytes decreased slightly. It was demonstrated that an increase in PAPS concentration negatively affects the formation of micelles during MSS, as low concentrations of organic solvents are not eligible to reverse μ*ep(a') for high-κ analytes. If its concentration is too high, the affinity between the zwitterionic surfactant and the analyte is too strong to reverse the μep(a') at MSSB, resulting in failure of MSS. As a result, 20 mM PAPS was selected in the sample matrix.

Embodiment 3. investigate the influence of methanol content on detection effect

On the basis of Example 1, PAPS was selected as the micelle, and the concentration of MeOH was changed to 40, 60, 80, 100% (v/v), and other conditions remained unchanged.

Methanol is usually an organic solvent in MSS, which affects the interaction between analyte and micelles, reversing the effective electrophoretic mobility for enrichment. This example superimposes two steps of MeOH injection alone, and optimizes the percentage of MeOH in the range of 40-100% (v/v) to obtain the best resolution and enrichment of analytes under other conditions.

The results obtained are shown in Figure 4. Except As(III), when the methanol concentration was 40% (v/v), the peak areas of the other three compounds were all smaller than those when 60% was used. However, when the MeOH content was 60%-100% (v/v), the peak intensities of the three compounds [As(V), MMA, DMA] decreased, and the migration time of the analytes was prolonged with the increase of MeOH percentage. The reason for this can be proven that the analyte has a weaker affinity for micelles, resulting in a lower κ value, which occurs in the presence of organic solvents. As the percentage of methanol increases, the κ value decreases due to the reduced affinity of the analyte for the micelles, thereby expanding the length of the scan region. However, the detection sensitivity of As(III) was highest when 80% (v/v) MeOH was applied. Therefore, 60% (v/v) MeOH was selected for further study.

Embodiment 4. investigate the influence of borax concentration on detection effect

On the basis of Example 1, PAPS was selected as the micelle, and the concentration of borax buffer was changed to 25, 50, 75, and 100 mM, and other conditions remained unchanged.

Borax buffer was used as electrolyte in BGS, and this example explores that the concentration of electrolyte in BGS has a significant effect on migration time and peak intensity.

The electropherogram of Figure 5A shows that the apparent mobility ([mu]app) decreases from 25 to 100 mM, inversely proportional to the migration time, and the results are shown in Figure 5B. Notably, the increase in solution current is due to the increase in concentration. It was demonstrated that EOF was affected by borax buffer concentration, which decreased with increasing borax buffer concentration in BGS. Additionally, the increased current generates more Joule heat, which will result in an increase in baseline noise (16-65 μA). Comprehensive consideration, the present invention prefers 100mM borax buffer.

Embodiment 5. Investigate the influence of buffer pH on detection effect

On the basis of Example 1, PAPS was selected as the micelle, and the pH of the borax buffer was changed to 8.8, 9.0, 9.2, and 9.4, and other conditions remained unchanged.

The migration of arsenic compounds in CE is also affected by their pKa values, so it is necessary to study the effect of BGS pH on the analysis of arsenic species. Arsenic species in the present invention are polygenic acids whose apparent charge is related to their own pKa and pH of BGS. The BGS pH was 8.8-9.4 because pre-experimental studies showed that a two-step overlay was more efficient for BGS analysis at alkaline pH. Furthermore, alkaline conditions ensure that all As migrates to the anode in the form of anions.

The results showed that the apparent mobility reached a maximum at pH 9.0 (Figure 6). When the pH increased from 9.0 to 9.4, the apparent mobility started to decrease and the migration time prolonged. In the co-electroosmotic flow mode, the As species migrates in the same direction as the EOF because the surface of the fused silica is decorated with HDMB. Therefore, the separation of the analytes is faster. Meanwhile, when the pH of BGS was 9.2, the resolution between the four analytes was good, and the maximum peak was obtained at pH 9.2. For higher sensitivity, when the apparent mobility is lower than 9.0, it is better to choose pH 9.2 as BGS.

Embodiment 6. Investigate the influence of sample injection time on detection effect

On the basis of Example 1, PAPS was selected as the micelle, and the injection time of the sample solution was changed to 30, 60, 120, 180, and 240 s, and other conditions remained unchanged.

The injection time affects the detection sensitivity, satisfactory separation efficiency, and high stacking performance of analytes in MSS by directly changing the injection volume. The injection time of 60% methanol was kept at 5 s (50 mbar), and the injection time of the sample solution was varied from 30 to 240 s.

As can be seen from Figure 7, it can be seen from the histogram that when the injection time is 30-180 s, by enlarging the injection volume, the peak area of the target analyte is increased, and a satisfactory stacking efficiency is obtained. However, as the injection time gradually increased, the peak shape broadened, the baseline was turbulent, and the analysis time was relatively long. At the 240s injection, with no further expansion of the peak area compared to the 180s, a minimum resolution of four analyte peaks (1.17) occurred, leading to catastrophic separation of the analytes. Based on the above results, 180 s is preferred as the optimal implantation time to obtain satisfactory stacking efficiency and good resolution.

Repeated study

Under the optimal experimental conditions described above, linearity, limits of detection (LODs) and quantifications (LOQs) were calculated for the intracapillary and intercapillary reproducibility of the four arsenic species (n=3). A summary of the analytical figures on performance is presented in Table 1. Standard sample solutions containing 0.05, 0.10, 0.25, 0.50, 1.00 mg/L and 20 mM PAPS were prepared to obtain a calibration curve. The coefficients of determination (R2 ⁾ calculated from the peak areas of As(V), MMA, DMA and As(III) were 0.9992, 0.9990, 0.9990 and 0.9928, respectively. The reproducible RSD % of migration time and peak area were both below 2.7%. The concentration of the standard solution was 1 mg/L, and it was used to calculate the reproducible RSD% in capillaries. The intracapillary migration time RSD% of As(V), MMA, DMA and As(III) were 1.0, 0.8, 0.7 and 0.5, respectively. The % intracapillary RSD for the peak area of target analytes ranged from 0.3 to 0.7. A standard solution (10 mg/L) was used to obtain % RSD reproducibility between capillaries. The intercapillary RSD percentages for the migration times of As(V), MMA, DMA and As(III) were 1.1, 1.6, 2.3 and 2.7, respectively. The inter-capillary RSD% of the peak areas of the four arsenic species ranged from 0.5 to 0.9%. The reproducible RSD % of both migration time and peak area were satisfactory. The peak area LODs (S/N=3) of As(V), MMA, DMA and As(III) were 0.382, 0.633, 0.911 and 0.659 ng/mL, respectively. The LOQ ranged from 1.27 to 3.04 ng/mL, calculated as a signal-to-noise ratio of 10. The enrichment factors of As(V), As(III), MMA and DMA were 1230, 840, 3820 and 1450, respectively.

Table 1. Linear regression data, LOD and LOQ

Recovery experiment

The practicability of the developed technique was evaluated by analyzing the addition of kelp at 1 mg/L as a standard solution. According to the sample detection results, As(III) and As(V) were not detected in the kelp samples but contained trace amounts of MMA and DMA. The analytical results of the spiked samples are shown in Table 2. The recovery rate of kelp was 89.3-120.6%. The purpose of three parallel experiments is to obtain the reproducibility of this method. The RSD% of migration time and peak area varied from 5.3 to 6.2 and 0.9–4.1, respectively.

Table 2. Analysis results of spiked samples

Claims (10)

1. based on micelle-solvent stacking to multiple arsenic forms on-line stacking enrichment, separation method simultaneously in natural medicine, it is characterized in that comprising the following steps: Step (1), target analyte sample preparation: Wash and dry the natural medicine, crush it, sieve it and mix it with HNO ₃ , ultrasonicate for 0.5-1h at 40-45°C, shake it for 1.5-2h at room temperature, and finally centrifuge at high speed for 10-15min, and take out the supernatant; The supernatant is neutralized with NaOH to obtain the target analyte sample; Step (2), target analyte sample enrichment: 2-1. Activated capillary column The fused silica capillaries were coated with hexamethylene bromide HDMB for 20-25 minutes to meet the condition of the same EOF direction between the anion and the capillary; 2-2. Rinse the activated capillary column with borax buffer for 5-10 minutes before injection; 2-3. In the capillary column, the micelles and the target analyte are mixed and injected into the stack as a sample solution under negative pressure for 30-240s, and then an aqueous methanol solution as an organic solvent is injected under negative pressure. The scanned analyte is bound by the micelles. to the MSS boundary to achieve sample stacking and separation; wherein the micelles use 3-(N,N-dimethylpalmitamido)propane sulfonate PAPS. 2. a kind of based on micelle-solvent stacking as claimed in claim 1 to simultaneously online stacking enrichment, separation method for multiple arsenic forms in natural medicine, it is characterized in that the natural medicine described in step (1) is kelp. 3. a kind of based on micelle-solvent stacking as claimed in claim 1 to simultaneously online stacking enrichment, separation method for multiple arsenic forms in natural medicine, it is characterized in that the mass volume of natural medicine and HNO described in step ( ₁ ) The ratio is 200 mg: 5 mL; wherein the mass volume concentration of HNO ₃ is 2%. 4. a kind of based on micelle-solvent stacking as claimed in claim 1 to simultaneously online stacking enrichment, separation method for multiple arsenic forms in natural medicine, it is characterized in that the described borax buffer concentration of step (2-2) is 25 -100mM, pH 8.8-9.4. 5. a kind of based on micelle-solvent stacking as claimed in claim 4 to simultaneously online stacking enrichment, separation method for multiple arsenic forms in natural medicine, it is characterized in that the described borax buffer concentration of step (2-2) is 100mM , the pH is 9.2. 6. a kind of based on micelle-solvent stacking as claimed in claim 1 to simultaneously online stacking enrichment, separation method for multiple arsenic forms in natural medicine, it is characterized in that the described capillary electrophoresis condition of step (2-3): detection wavelength is 192nm, the separation negative pressure is -20kV, and the temperature is 20℃. 7. a kind of based on micelle-solvent stacking as claimed in claim 1 to simultaneously online stacking enrichment, separation method for multiple arsenic forms in natural medicine, it is characterized in that the described micelle concentration of step (2-3) is 10- 40mM. 8. a kind of based on micelle-solvent stacking as claimed in claim 7 to simultaneously online stacking enrichment, separation method for multiple arsenic forms in natural medicine, it is characterized in that the described micelle concentration of step (2-3) is 20mM, The injection time was 180s, and the injection negative pressure was -10kV. 9. a kind of based on micelle-solvent stacking as claimed in claim 1 to simultaneously online stacking enrichment, separation method for multiple arsenic forms in natural medicine, it is characterized in that the described methanol aqueous solution volume content of step (2-3) is 40. -100%, injection parameters are injection power -10kV, injection pressure 50mbar, injection time 5s. 10. A method for simultaneous online accumulation and separation of multiple arsenic forms in natural medicines based on micelle-solvent accumulation as claimed in claim 1, characterized in that As(V), As(III), monomethylarsine The enrichment factors of various arsenic forms of sodium MMA and sodium dimethylarsine DMA were 1230, 840, 3820 and 1450, respectively.

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