阅读说明：本技术 一种铁皮石斛中金属离子的检测方法 (Detection method for metal ions in dendrobium officinale ) 是由 曹君 宋晓玉 王淑玲 谢恬 张蕊 于 2019-08-27 设计创作，主要内容包括：本发明涉及中药材分析技术领域,公开了一种铁皮石斛中金属离子的检测方法,步骤为：制备铁皮石斛供试品母液；在铁皮石斛供试品母液中加入阴离子表面活性剂和电解质,调节pH得到铁皮石斛供试品溶液；配制背景缓冲液；配制络合剂溶液；设置毛细管电泳参数,依次用碱溶液、超纯水和背景缓冲液活化毛细管柱后,将络合剂溶液、铁皮石斛供试品溶液依次注入毛细管柱中,施加电压,进行在线络合和金属离子的富集；通过与标准品的电泳图对照,确定铁皮石斛供试品溶液中的金属离子浓度。本发明将在线络合和胶束到溶剂堆积巧妙结合,用于检测、分析中药铁皮石斛中的金属离子,可以大大提高检测的灵敏度,并减少络合剂的浪费和对环境的污染。(The invention relates to the technical field of analysis of traditional Chinese medicinal materials, and discloses a detection method for metal ions in dendrobium officinale, which comprises the following steps: preparing a dendrobium officinale sample mother solution; adding an anionic surfactant and electrolyte into the dendrobium officinale sample mother liquor, and adjusting the pH value to obtain a dendrobium officinale sample solution; preparing a background buffer solution; preparing a complexing agent solution; setting capillary electrophoresis parameters, activating a capillary column by using an alkaline solution, ultrapure water and a background buffer solution in sequence, then injecting a complexing agent solution and a dendrobium officinale sample solution into the capillary column in sequence, and applying voltage to perform online complexation and metal ion enrichment; and determining the concentration of metal ions in the dendrobium officinale test sample solution by comparing with an electrophoretogram of a standard product. The invention skillfully combines on-line complexation and micelle-to-solvent accumulation, is used for detecting and analyzing metal ions in the traditional Chinese medicine dendrobium officinale, can greatly improve the detection sensitivity, and reduces the waste of complexing agent and the pollution to the environment.)

1. A detection method of metal ions in dendrobium officinale is characterized by comprising the following steps:

(1) preparing a dendrobium officinale sample mother solution;

(2) adding an anionic surfactant and an electrolyte into the dendrobium officinale sample mother liquor to ensure that the concentration of the anionic surfactant is 4.2-16.2 mmol/L and the concentration of the electrolyte is 140-220 mmol/L, and adjusting the pH to 5-6 to obtain a dendrobium officinale sample solution;

(3) preparing a background buffer solution, wherein the background buffer solution is an electrolyte solution containing methanol, and the concentration of the electrolyte in the background buffer solution is the same as that of the electrolyte in the dendrobium officinale sample solution;

(4) preparing a complexing agent solution, wherein the concentration of the complexing agent in the complexing agent solution is 10-50 mmol/L;

(5) setting capillary electrophoresis parameters, activating a capillary column by using an alkaline solution, ultrapure water and a background buffer solution in sequence, then injecting a complexing agent solution and a dendrobium officinale sample solution into the capillary column in sequence, and applying voltage to perform online complexation and metal ion enrichment;

(6) and determining the concentration of metal ions in the dendrobium officinale test sample solution by comparing with an electrophoretogram of a standard product.

2. The method for detecting metal ions in dendrobium officinale according to claim 1, wherein the preparation method of the mother solution of the dendrobium officinale sample in the step (1) comprises the following steps: crushing and sieving the dendrobium officinale medicinal material, adding 68% concentrated nitric acid, uniformly mixing, and standing for 10-30 min to obtain a liquid mixture; digesting the liquid mixture at 100 ℃ until the liquid mixture is in a clear state to obtain a digestion solution; and cooling the digestion solution to room temperature, and performing centrifugal separation and then diluting the supernatant to obtain the dendrobium officinale sample mother solution.

3. The method for detecting metal ions in dendrobium officinale according to claim 2, wherein the ratio of the dendrobium officinale medicinal material to the concentrated nitric acid in the liquid mixture is 1 g: (40-50) mL.

4. The method as claimed in claim 1, wherein the anionic surfactant in step (2) is sodium dodecyl sulfate, and the electrolyte is sodium acetate.

5. The method for detecting metal ions in dendrobium officinale according to claim 1, wherein the mass content of methanol in the background buffer solution prepared in the step (3) is 30-70%.

6. The method for detecting metal ions in dendrobium officinale according to claim 1, wherein the complexing agent in the complexing agent solution prepared in step (4) is one of 1, 10-phenanthroline, 18-crown-6, L-cysteine and imidazole.

7. The method for detecting metal ions in dendrobium officinale according to claim 6, wherein when the complexing agent in the complexing agent solution prepared in the step (4) is 1, 10-phenanthroline, the solvent is methanol; when the complexing agent is 18-crown-6, L-cysteine and imidazole, the solvent is water.

8. The method for detecting metal ions in dendrobium officinale according to claim 1, wherein before the complexing reagent is injected in the step (5), the capillary column is activated by sequentially using 1.0mol/L sodium hydroxide solution, 0.1mol/L sodium hydroxide solution, ultrapure water and background buffer solution, wherein the activation time is 10min, 5min and 5 min.

9. The method for detecting metal ions in Dendrobium officinale according to claim 1 or 8, wherein the sample injection time of the complexing agent solution in step (5) is 2-10 s, and the sample injection time of the test sample solution of Dendrobium officinale is 40-100 s.

10. The method according to claim 1 or 8, wherein the complexing agent solution and the test solution of Dendrobium officinale Kimura et Migo in step (5) have an injection pressure of 50mbar and an applied voltage of 16 kV.

Technical Field

The invention relates to the technical field of analysis of traditional Chinese medicinal materials, in particular to a detection method of metal ions in dendrobium officinale.

Background

Traditional Chinese medicine Dendrobium officinale (Dendrobium officinale Kimura et Migo), a traditional genuine medicinal material in Zhejiang province, belongs to fresh or dry stems of Dendrobium officinale in Orchidaceae, is also called as 'Equisetum nigrum' or 'Feishilan', has the effects of nourishing stomach and strengthening spleen, nourishing yin and tonifying kidney, moistening lung and promoting fluid production, and is classified as new Zhejiang eight flavors. The problem of heavy metal pollution also exists widely in traditional Chinese medicinal materials due to factors such as mining, waste gas emission, sewage irrigation and the like. High doses of metal ions are potentially harmful to ecosystems and human health due to their bioaccumulation and non-biodegradability. For example, nickel is a trace element essential to the human body that is potentially toxic. The most common symptoms caused by nickel are respiratory diseases and dermatitis. Certain compounds of nickel are even carcinogenic. The main effects of cobalt on the skin are allergic or irritant dermatitis, which also has some effect on the respiratory system. Excess copper poses potential risks to the kidneys, gastrointestinal tract, motor and sensory nerves. The highly toxic properties of mercury are now well known. Exposure to high mercury may lead to brain damage, inflammation, autoimmunity, and affect development of the nervous system. Exposure to part of the cadmium produced by industrial discharge or smoking can lead to kidney and liver toxicity and affect the normal growth of embryos. Therefore, it is necessary to develop a sensitive and efficient method for measuring metal ions in Chinese medicinal materials.

Currently, various analytical techniques such as colorimetric determination, fluorescence sensor array voltammetry, flame atomic absorption spectrometry, resonance raman, and the like are developed for the determination of metal ions. However, the wider application of these techniques is limited due to the requirement of complex instrumentation and cumbersome pre-processing procedures. In recent years, capillary electrophoresis methods equipped with diode array detectors have become increasingly popular in the detection of metal ions, and have the characteristics of high efficiency, easy control and low solvent consumption. For example, the chinese patent document discloses "a method for capillary electrophoresis detection of metal cations in a liquid", which is published under the publication number CN107389774A, and comprises the following steps: respectively preparing single-standard solutions of each ion, and obtaining the peak output time of each ion through a capillary electrophoresis apparatus; preparing mixed standard solutions of ions with different concentrations, and obtaining a linear characteristic relation between the concentration of each ion and an emergent area through a capillary electrophoresis apparatus; and detecting the sample to be detected to obtain a peak area, and calculating to obtain the concentration of each ion in the sample to be detected. The method has the advantages of large peak interval, wide linear range, high accuracy and high detection speed.

However, when the capillary electrophoresis method is used for detecting metal ions, because some metals have no chromophore under ultraviolet and visible light, complexing agents are required to be used in front of or on a capillary column, and the reported complexing agents include imidazole, 1, 10-phenanthroline, porphyrin and the like. In the prior art, the complexing agents are all dissolved in the background buffer solution, as is well known, the background buffer solution has a relatively large usage amount, but only a small amount of the complexing agents are actually needed to react with the analyte, the complexing agents are dissolved in the background buffer solution, the complexing agents are wasted, the complexing agents are all organic matters, the usage amount is large, the environment is polluted, and the detection result and the sensitivity are influenced. In addition, when the capillary electrophoresis method is used for detecting metal ions, the measurement sensitivity is low due to the limited injection amount and the short optical path.

Disclosure of Invention

The invention aims to overcome the defects that when a capillary electrophoresis method is used for detecting metal ions in the prior art, complexing agents are dissolved in a background buffer solution, the consumption of the background buffer solution is relatively large, the complexing agents are wasted, the environment is easily polluted, and the detection result and the sensitivity are influenced; in addition, when the capillary electrophoresis method is used for detecting metal ions, due to the limited injection amount and short light path, the problem of low detection sensitivity is solved, the method for detecting the metal ions in the dendrobium officinale is provided, and an enrichment method of on-line complexation and micelle-to-solvent accumulation is applied to the capillary electrophoresis method, so that the waste of complexation agents and the pollution to the environment in the detection process are greatly reduced, and the detection sensitivity is improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

a detection method of metal ions in dendrobium officinale comprises the following steps:

(1) preparing a dendrobium officinale sample mother solution;

(2) adding an anionic surfactant and an electrolyte into the dendrobium officinale sample mother liquor to ensure that the concentration of the anionic surfactant is 4.2-16.2 mmol/L and the concentration of the electrolyte is 140-220 mmol/L, and adjusting the pH to 5-6 to obtain a dendrobium officinale sample solution;

(3) preparing a background buffer solution, wherein the background buffer solution is an electrolyte solution containing methanol, and the concentration of the electrolyte in the background buffer solution is the same as that of the electrolyte in the dendrobium officinale sample solution;

(4) preparing a complexing agent solution, wherein the concentration of the complexing agent in the complexing agent solution is 10-50 mmol/L;

(5) setting capillary electrophoresis parameters, activating a capillary column by using an alkaline solution, ultrapure water and a background buffer solution in sequence, then injecting a complexing agent solution and a dendrobium officinale sample solution into the capillary column in sequence, and applying voltage to perform online complexation and metal ion enrichment;

(6) and determining the concentration of metal ions in the dendrobium officinale test sample solution by comparing with an electrophoretogram of a standard product.

According to the invention, the complexing agent is not dissolved in the background buffer solution, but the complexing agent and the dendrobium officinale sample solution are directly injected, so that the complexing agent and metal ions in the dendrobium officinale sample solution are subjected to online complexing, the using amount of the complexing agent is greatly reduced, the waste and pollution are reduced, and the detection sensitivity is improved.

Meanwhile, the invention also utilizes a micelle-to-solvent accumulation method to enrich the metal ions. The emphasis of micelle-to-solvent stacking is based on the reversal of the effective electrophoretic mobility of the charged analyte at the micelle and solvent boundaries, the essential conditions for micelle-to-solvent stacking are a micelle-containing sample solution and a background buffer containing an organic solvent, and the micelles in the sample solution are required to have an opposite charge to the charged analyte to bind the analyte and transport the analyte to the micelle and solvent boundaries, and the amount of organic solvent in the background buffer must be sufficient to reduce the interaction of the micelles with the analyte upon passing the boundaries, thereby releasing the analyte.

The metal ions to be detected in the invention are cations, so that an anion surfactant and electrolyte are added into the dendrobium officinale sample mother liquor to prepare a dendrobium officinale sample solution, the charges of the metal ions and the anion surfactant are opposite, under the condition of applying forward voltage, due to the interaction of electrostatic force, the anion micelle carries the cations to be detected to move to a positive electrode (inlet end), when the metal ions and the anion surfactant are contacted with a background buffer solution, the organic solvent methanol can reduce the interaction of the micelle and the ions to be detected, so that the cations to be detected are released from the micelle, the electrophoretic migration direction of the cations to be detected is changed, the cations are changed from the positive electrode to the negative electrode and move to the outlet end, the micelles in the background buffer solution carry the ions to be detected to continue to move to the positive electrode, the. The micelle carries metal cations to continuously move towards the negative electrode and continuously accumulate, and when all the cations are completely released from the micelle, the accumulation is finished. Finally, ions to be detected can be separated and detected in a proper capillary electrophoresis mode. In order to ensure that the aggregation of metal ions is caused by the effect of micelle to solvent accumulation rather than other field amplification effects, the electrolyte concentration in the dendrobium officinale sample solution is consistent with that in the background buffer solution.

Therefore, the invention skillfully combines the 'on-line complexation' as a sensitization means and the 'micelle-to-solvent accumulation' as an enrichment means for detecting and analyzing metal ions in the traditional Chinese medicine dendrobium officinale, can greatly improve the detection sensitivity, and reduces the waste of a complexing agent and the pollution to the environment.

Preferably, the preparation method of the dendrobium officinale sample mother liquor in the step (1) comprises the following steps: crushing and sieving the dendrobium officinale medicinal material, adding 68% concentrated nitric acid, uniformly mixing, and standing for 10-30 min to obtain a liquid mixture; digesting the liquid mixture at 100 ℃ until the liquid mixture is in a clear state to obtain a digestion solution; and cooling the digestion solution to room temperature, and performing centrifugal separation and then diluting the supernatant to obtain the dendrobium officinale sample mother solution. The method for preparing the dendrobium officinale sample mother liquor can effectively destroy organic matters and dissolve particles, and oxidize elements to be detected with various valence states into a single high valence state, thereby being beneficial to subsequent detection.

Preferably, the ratio of the dendrobium officinale medicinal material to the concentrated nitric acid in the liquid mixture is 1 g: (40-50) mL. By adopting the proportion range, the dendrobium officinale medicinal material can be fully digested.

Preferably, the anionic surfactant in step (2) is Sodium Dodecyl Sulfate (SDS), and the electrolyte is sodium acetate. SDS is an anionic surfactant, the electric charge of the SDS is opposite to that of metal ions, and the SDS is added into a dendrobium officinale sample solution containing sodium acetate, so that the metal ions can effectively realize the accumulation of micelles to a solvent.

Preferably, the mass content of methanol in the background buffer solution prepared in the step (3) is 30-70%. Within this content range, the interaction of the micelle with the metal ion can be reduced, thereby allowing the metal ion to be efficiently released.

Preferably, the complexing agent in the complexing agent solution prepared in the step (4) is one of 1, 10-phenanthroline, 18-crown-6, L-cysteine and imidazole. These complexing agents have strong binding force with metal ions and can enhance the ultraviolet absorption of various metal ions.

Preferably, when the complexing agent in the complexing agent solution prepared in the step (4) is 1, 10-phenanthroline, the solvent is methanol; when the complexing agent is 18-crown-6, L-cysteine and imidazole, the solvent is water. The proper solvent can ensure that the complexing agent is fully dissolved and effectively plays a role of the complexing agent.

Preferably, before the complexing reagent is injected in the step (5), the capillary column is activated by sequentially using 1.0mol/L sodium hydroxide solution, 0.1mol/L sodium hydroxide solution, ultrapure water and background buffer solution, wherein the activation time is 10min, 5min and 5min respectively. The inner surface of the capillary column can be smoothened by activating the capillary column with a sodium hydroxide solution, the silicon hydroxyl group is activated, then the sodium hydroxide solution is washed away by water, and the capillary column is balanced by a background buffer solution, so that the detection precision is ensured.

Preferably, the sample introduction time of the complexing agent solution in the step (5) is 2-10 s, and the sample introduction time of the dendrobium officinale sample solution is 40-100 s. Within the sample introduction time range, the peak area and the peak height of the metal ions are optimal.

Preferably, the injection pressure of the complexing agent solution and the dendrobium officinale sample solution in the step (5) is 50mbar, and the applied voltage is 16 kV. Under the injection pressure and voltage, effective complexation and enrichment can be achieved.

Therefore, the invention has the following beneficial effects:

(1) the method combines the online complexation with the method of accumulating and enriching from micelle to solvent, and is applied to the detection of metal ions in the traditional Chinese medicinal materials, so that the detection sensitivity of an instrument to the metal ions is greatly improved, the waste of complexing agent and the pollution to the environment are reduced, and the method belongs to an environment-friendly green detection method;

(2) in the early stage, only pretreatment is needed to be carried out on the dendrobium officinale samples, only parameters of capillary electrophoresis are needed to be set for subsequent complexation and enrichment, detection of metal ions can be flexibly completed by utilizing an automatic sample introduction method of an instrument, the operation environment is safe, and the operation steps are simple and easy to control;

(3) the method has wide application range, and can be used for detecting metal ions in dendrobium officinale or other Chinese medicinal materials.

Drawings

FIG. 1 is a schematic representation of the combination of in-line complexation and micelle-to-solvent stacking of the present invention;

FIG. 2 is a graph showing experimental validation of on-line complexation and micelle-to-solvent stacking effects in capillary electrophoresis;

FIG. 3 is an electropherogram examining different complexing agent species;

FIG. 4 is a line graph for examining the enrichment effect of different complexing agent concentrations;

FIG. 5 is a line graph for examining the enrichment effect of complexing agents at different injection times;

FIG. 6 is a line graph illustrating the enrichment effect of different SDS concentrations in sample solutions;

FIG. 7 is a line graph illustrating the enrichment effect of different sodium acetate contents in a sample solution;

FIG. 8 is an electrophoretogram examining different sodium acetate contents in sample solutions;

FIG. 9 is a line graph illustrating the enrichment effect of different methanol contents in the background buffer;

FIG. 10 is an electrophoretogram examining different methanol contents in background buffer;

FIG. 11 is a line diagram for examining the enrichment effect of the sample solution to be tested at different sampling times;

FIG. 12 is an electrophoretogram for examining different sample injection times of a sample solution to be tested;

FIG. 13 is an electrophoresis chart of the Dendrobium officinale sample solution in the examples.

Detailed Description

The invention is further described with reference to the following detailed description and accompanying drawings.

The preparation method of the metal ion reference substance solution related in the embodiment of the invention comprises the following steps: accurately weighing proper amount of nickel nitrate (Ni) hexahydrate²⁺) Cobalt nitrate hexahydrate (Co)²⁺) Copper nitrate trihydrate (Cu)²⁺) Mercuric nitrate monohydrate (Hg)²⁺) Cadmium nitrate tetrahydrate (Cd)²⁺) Dissolving the standard substance in 1.5mL centrifuge tube with appropriate amount of water by ultrasonic wave to obtain 1000mg/mL sample mother solution (100 mg/mL due to poor water solubility of mercury nitrate monohydrate), and diluting before use to obtain control solution with desired concentration.

The preparation method of the complexing agent solution related in the embodiment of the invention comprises the following steps: precisely weighing a proper amount of complexing reagent comprising 1, 10-phenanthroline, 18-crown ether-6, L-cysteine and imidazole, respectively ultrasonically dissolving the complexing reagent with a proper solvent (methanol for 1, 10-phenanthroline and water for the rest) to prepare a mother solution of 100mmol/L, and diluting the mother solution with a proper solvent (methanol for 1, 10-phenanthroline and water for the rest) before use to obtain a complexing agent solution with a required concentration.

The preparation methods of the anionic surfactant and the electrolyte solution related in the embodiment of the invention are respectively as follows: accurately weighing appropriate amount of SDS, and ultrasonically dissolving with water to obtain 50mmol/L SDS mother solution; precisely weighing appropriate amount of sodium acetate, and dissolving with water by ultrasonic wave to obtain 500mmol/L sodium acetate mother liquor. The mother liquor is used to prepare the required concentration.

The conditions of capillary electrophoresis in the present invention are: uncoated fused silica capillary was 41.5cm long by 50 μm.d. inner diameter, and the effective column was 33cm long. The column temperature is 25 ℃, the separation voltage is 16kV, the DAD detection wavelength is 214nm, the broadband is 4nm, and no reference wavelength exists.

As shown in fig. 1, a model diagram of the on-line complexation coupled with micelle-to-solvent stacking, in which: A) in the initial situation: sequentially injecting a complexing agent and a sample solution into a capillary; (B) voltage application: the direction of electroosmotic flow is towards the negative electrode, the direction of the analyte in the sample solution is towards the positive electrode, and meanwhile, the analyte and the complexing agent are combined with each other; (C-D) continuing to apply the voltage: all of the micelle bound analyte is transported to the micelle-solvent boundary. At the micelle-solvent boundary, the inclusion of organic solvent in the background buffer reduces the affinity of the anionic micelle for the cationic analyte and results in a reversal of the effective electrophoretic mobility of the analyte at the boundary towards the cathode. The cationic analyte is then released and separated.

As shown in fig. 2, the experimental verification diagram of the on-line complexation and micelle-to-solvent stacking effect in capillary electrophoresis is shown, wherein: (a) sample introduction is typically performed, and the sample solution is injected at 50mbar for 5 s; (b) injecting sample solution in large volume, and injecting the sample solution at 50mbar for 60 s; (c) micelle to solvent accumulation and sample solution injection at 50mbar for 60 s; (d) simple on-line complexing, namely injecting a complexing agent for 3s at 50mbar and then performing typical sample injection; (e) online complexing and large-volume sample injection; (f) in-line complexation and micelle-to-solvent stacking. Typical sample solutions for injection and bulk injection contain 180mM sodium acetate. The sample solution from the micelle-to-solvent stack contained 7.2mM SDS and 180mM sodium acetate. All background buffer used was 180mM sodium acetate solution containing 50% methanol. The separation voltage is set at 16kV, and the detection wavelength is 214 nm.

Firstly, investigating the type of a complexing agent:

1. taking 9 clean capillary electrophoresis sample bottles, adding 1mol/L sodium hydroxide, 0.1mol/L sodium hydroxide, ultrapure water, background buffer solutions (4, 5 and 6), complexing agent solutions and metal ion reference substance solutions (which are filtered by 0.22 mu m microporous filter membranes before sample loading) into No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-8 bottles respectively, and taking the No. 9 bottle as a waste liquid bottle.

1mol/L of sodium hydroxide added into the bottle No. 1 is from an Agilent standard solution; the 0.1mol/L sodium hydroxide added into the No. 2 bottle is diluted by 1mol/L sodium hydroxide and ultrapure water; ultrapure water added into the bottle No. 3 and water used for preparing the solution in the experimental process are both in HPLC level; 4. background buffer added into bottle No. 5 and 6The solution is 180mmol/L sodium acetate solution containing 50% methanol, and the liquid level of bottles 5 and 6 are kept consistent; the types of complexing agents in the complexing agent solution added into the bottle No. 7 are respectively 30mmol/L of 1, 10-phenanthroline, 18-crown ether-6, L-cysteine and imidazole; metal ion reference solution added into No. 8 bottle contains Ni²⁺，Co²⁺，Cu²⁺，Hg²⁺And Cd²⁺4. mu.g/mL, 8. mu.g/mL and 8. mu.g/mL, respectively, the concentration of SDS was 7.2mmol/L and the concentration of sodium acetate was 180 mmol/L.

2. Before the start of the run, the capillary column was activated with 1.0mol/L sodium hydroxide (10 min), 0.1mol/L sodium hydroxide (10 min), ultra pure water (5 min), and background buffer solution (5 min) in this order.

3. And selecting a pressure injection mode, injecting a complexing agent solution for 2s and a metal ion reference substance solution for 60s under the condition of 50mbar, and applying positive pressure of 16kV at an inlet end. The operation starts.

The results are shown in FIG. 3, and it can be seen from FIG. 3 that five metal ions, Ni²⁺，Co²⁺，Cu²⁺，Hg²⁺And Cd²⁺Can be determined by complexation with 1, 10-phenanthroline and 18-crown-6. However, when L-cysteine and imidazole were injected separately as complexing agents, only Ni appeared²⁺，Co²⁺，Cu²⁺，Hg²⁺And Ni²⁺，Co²⁺，Cu²⁺，Cd²⁺Peak of (2). In addition, the five metal ions complexed by 1, 10-phenanthroline responded the highest compared to the other three complexing agents. This phenomenon should be due to differences in the binding force between the complexing agent and the metal ion, as well as differences in the ultraviolet absorption of the complexing agent itself. Therefore, the detection sensitivity of the five metal ions is improved most obviously by adopting the 1, 10-phenanthroline as the complexing agent.

Secondly, investigating the concentration of the complexing agent:

1. taking 9 clean capillary electrophoresis sample bottles, adding 1mol/L sodium hydroxide, 0.1mol/L sodium hydroxide, ultrapure water, background buffer solutions (4, 5 and 6), complexing agent solutions and metal ion reference substance solutions (which are filtered by 0.22 mu m microporous filter membranes before sample loading) into No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-8 bottles respectively, and taking the No. 9 bottle as a waste liquid bottle.

1mol/L of sodium hydroxide added into the bottle No. 1 is from an Agilent standard solution; the 0.1mol/L sodium hydroxide added into the No. 2 bottle is diluted by 1mol/L sodium hydroxide and ultrapure water; ultrapure water added into the bottle No. 3 and water used for preparing the solution in the experimental process are both in HPLC level; 4. the background buffer solution added into the bottles 5 and 6 is 180mmol/L sodium acetate solution containing 50% methanol, and the liquid level heights of the bottles 5 and 6 are kept consistent; the complexing agent in the complexing agent solution added into the bottle No. 7 is 1, 10-phenanthroline, and the concentrations are 10mmol/L, 20mmol/L, 30mmol/L, 40mmol/L and 50mmol/L respectively; metal ion reference solution added into No. 8 bottle contains Ni²⁺，Co²⁺，Cu²⁺，Hg²⁺And Cd²⁺4. mu.g/mL, 8. mu.g/mL and 8. mu.g/mL, respectively, the concentration of SDS was 7.2mmol/L and the concentration of sodium acetate was 180 mmol/L.

2. Before the start of the run, the capillary column was activated with 1.0mol/L sodium hydroxide (10 min), 0.1mol/L sodium hydroxide (10 min), ultra pure water (5 min), and background buffer solution (5 min) in this order.

3. And selecting a pressure injection mode, injecting a complexing agent solution for 2s and a metal ion reference substance solution for 60s under the condition of 50mbar, and applying positive pressure of 16kV at an inlet end. The operation starts.

As shown in FIG. 4, it can be seen from FIG. 4 that Ni increases with the concentration from 10mmol/L to 30mmol/L²⁺，Co²⁺And Cu²⁺Then Ni when the concentration of the complexing agent is increased from 30mmol/L to 50mmol/L²⁺，Co²⁺And Cu²⁺The peak area of (a) gradually decreases. However, for Hg²⁺And Cd²⁺The peak area increased with the amount of 1, 10-phenanthroline reaching 40mmol/L, and remained substantially constant above 40 mmol/L. This phenomenon is attributed to Ni²⁺，Co²⁺，Cu²⁺And Hg²⁺，Cd²⁺Different ultraviolet absorption intensity of (c). In order to realize high-response detection on five metal ions at the same time, 30mmol/L is selected as the optimal complexing agent concentration.

Thirdly, inspecting the sample injection time of the complexing agent:

1. taking 9 clean capillary electrophoresis sample bottles, adding 1mol/L sodium hydroxide, 0.1mol/L sodium hydroxide, ultrapure water, background buffer solutions (4, 5 and 6), complexing agent solutions and metal ion reference substance solutions (which are filtered by 0.22 mu m microporous filter membranes before sample loading) into No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-8 bottles respectively, and taking the No. 9 bottle as a waste liquid bottle.

1mol/L of sodium hydroxide added into the bottle No. 1 is from an Agilent standard solution; the 0.1mol/L sodium hydroxide added into the No. 2 bottle is diluted by 1mol/L sodium hydroxide and ultrapure water; ultrapure water added into the bottle No. 3 and water used for preparing the solution in the experimental process are both in HPLC level; 4. the background buffer solution added into the bottles 5 and 6 is 180mmol/L sodium acetate solution containing 50% methanol, and the liquid level heights of the bottles 5 and 6 are kept consistent; the complexing agent in the complexing agent solution added into the bottle No. 7 is 30 mmol/L1, 10-phenanthroline; metal ion reference solution added into No. 8 bottle contains Ni²⁺，Co²⁺，Cu²⁺，Hg²⁺And Cd²⁺4. mu.g/mL, 8. mu.g/mL and 8. mu.g/mL, respectively, the concentration of SDS was 7.2mmol/L and the concentration of sodium acetate was 180 mmol/L.

2. Before the start of the run, the capillary column was activated with 1.0mol/L sodium hydroxide (10 min), 0.1mol/L sodium hydroxide (10 min), ultra pure water (5 min), and background buffer solution (5 min) in this order.

3. And selecting a pressure injection mode, injecting a complexing agent solution and a metal ion reference substance solution in sequence under the condition of 50mbar, wherein the sample injection time of the complexing agent solution is respectively 2, 4, 6, 8 and 10s, the sample injection time of the metal ion reference substance solution is 60s, and the positive pressure of 16kV is applied to the complexing reagent at the inlet end. The operation starts.

As a result, as shown in fig. 5, it can be seen from fig. 5 that the peak areas of the five metal ions increase as the injection time of the complexing agent increases, but the peak area increases more slowly when the injection time reaches 6 s. This may be due to the effect of the organic solvent contained in the complexing agent on the micelle-to-solvent stacking effect. Therefore, 8s of complexing reagent is the optimal condition.

Fourthly, investigating the concentration of SDS:

1. taking 9 clean capillary electrophoresis sample bottles, adding 1mol/L sodium hydroxide, 0.1mol/L sodium hydroxide, ultrapure water, background buffer solutions (4, 5 and 6), complexing agent solutions and metal ion reference substance solutions (which are filtered by 0.22 mu m microporous filter membranes before sample loading) into No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-8 bottles respectively, and taking the No. 9 bottle as a waste liquid bottle.

1mol/L of sodium hydroxide added into the bottle No. 1 is from an Agilent standard solution; the 0.1mol/L sodium hydroxide added into the No. 2 bottle is diluted by 1mol/L sodium hydroxide and ultrapure water; ultrapure water added into the bottle No. 3 and water used for preparing the solution in the experimental process are both in HPLC level; 4. the background buffer solution added into the bottles 5 and 6 is 180mmol/L sodium acetate solution containing 50% methanol, and the liquid level heights of the bottles 5 and 6 are kept consistent; the complexing agent in the complexing agent solution added into the bottle No. 7 is 30 mmol/L1, 10-phenanthroline; metal ion reference solution added into No. 8 bottle contains Ni²⁺，Co²⁺，Cu²⁺，Hg²⁺And Cd²⁺4. mu.g/mL, 8. mu.g/mL and 8. mu.g/mL, respectively, the concentration of SDS was 4.2mmol/L, 7.2mmol/L, 10.2mmol/L, 13.2mmol/L and 16.2mmol/L, respectively, and the concentration of sodium acetate was 180 mmol/L.

2. Before the start of the run, the capillary column was activated with 1.0mol/L sodium hydroxide (10 min), 0.1mol/L sodium hydroxide (10 min), ultra pure water (5 min), and background buffer solution (5 min) in this order.

3. And selecting a pressure injection mode, injecting a complexing agent solution and a metal ion reference substance solution in sequence under the condition of 50mbar, wherein the sample injection time of the complexing agent solution is 8s, the sample injection time of the metal ion reference substance solution is 60s, and the positive pressure of 16kV is applied to the complexing agent at the inlet end. The operation starts.

As shown in FIG. 6, it can be seen from FIG. 6 that the peak areas of the five metal ions are increased when the SDS concentration is increased from 4.2mmol/L to 7.2mmol/L, wherein Ni²⁺，Co²⁺And Cu²⁺The increase in peak area of (a) is not significant. However, as the concentration of SDS increased from 7.2mmol/L to 16.2mmol/L, the peak areas all decreased gradually. Furthermore, as the SDS content in the sample solution increases, Hg²⁺And Cd²⁺The tailing peak of (a) is also improved. The above phenomenon may be due to Hg²⁺And Cd²⁺Has low detection sensitivity, and leads the micelle to be accumulated in a solvent to Hg²⁺And Cd²⁺The enrichment effect is more obvious. However, once the amount of SDS is large enough, it is difficult to dilute with the organic solvent in the background buffer, thereby hindering the release of the metal ions from the micelles. Therefore, 7.2mmol/LSDS was selected for subsequent experiments.

Fifthly, investigating the concentration of sodium acetate:

1. taking 9 clean capillary electrophoresis sample bottles, adding 1mol/L sodium hydroxide, 0.1mol/L sodium hydroxide, ultrapure water, background buffer solutions (4, 5 and 6), complexing agent solutions and metal ion reference substance solutions (which are filtered by 0.22 mu m microporous filter membranes before sample loading) into No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-8 bottles respectively, and taking the No. 9 bottle as a waste liquid bottle.

1mol/L of sodium hydroxide added into the bottle No. 1 is from an Agilent standard solution; the 0.1mol/L sodium hydroxide added into the No. 2 bottle is diluted by 1mol/L sodium hydroxide and ultrapure water; ultrapure water added into the bottle No. 3 and water used for preparing the solution in the experimental process are both in HPLC level; 4. the background buffer solution added into the bottles 5 and 6 is 180mmol/L sodium acetate solution containing 50% methanol, and the liquid level heights of the bottles 5 and 6 are kept consistent; the complexing agent in the complexing agent solution added into the bottle No. 7 is 30 mmol/L1, 10-phenanthroline; metal ion reference solution added into No. 8 bottle contains Ni²⁺，Co²⁺，Cu²⁺，Hg²⁺And Cd²⁺4. mu.g/mL, 8. mu.g/mL and 8. mu.g/mL, respectively, the concentration of SDS was 7.2mmol/L, and the concentrations of sodium acetate were 140mmol/L, 160mmol/L, 180mmol/L, 200mmol/L and 220mmol/L, respectively.

2. Before the start of the run, the capillary column was activated with 1.0mol/L sodium hydroxide (10 min), 0.1mol/L sodium hydroxide (10 min), ultra pure water (5 min), and background buffer solution (5 min) in this order.

3. And selecting a pressure injection mode, injecting a complexing agent solution and a metal ion reference substance solution in sequence under the condition of 50mbar, wherein the sample injection time of the complexing agent solution is 8s, the sample injection time of the metal ion reference substance solution is 60s, and the positive pressure of 16kV is applied to the complexing agent at the inlet end. The operation starts.

As shown in FIGS. 7 and 8, it can be seen from FIG. 7 that the increase in the sodium acetate concentration in the range of 140-180mmol/L facilitates the detection of metal ions. However, as the concentration of sodium acetate in the sample solution increases to be equal to or greater than the concentration of sodium acetate in the buffer, the detection sensitivity of the metal ions decreases. Furthermore, Ni²⁺And Hg²⁺The peak shape of (A) becomes worse with increasing sodium acetate concentration, particularly at 220 mmol/L. This phenomenon is due to the increased difference in conductivity between the sample solution and the background buffer zone as the concentration of sodium acetate in the sample solution increases. This results in de-stacking of metal ions and unexpected distortion peaks in the sample solution region. Therefore, 180mmol/L sodium acetate was the optimum condition.

Sixthly, investigating the content of the methanol:

1. taking 9 clean capillary electrophoresis sample bottles, adding 1mol/L sodium hydroxide, 0.1mol/L sodium hydroxide, ultrapure water, background buffer solutions (4, 5 and 6), complexing agent solutions and metal ion reference substance solutions (which are filtered by 0.22 mu m microporous filter membranes before sample loading) into No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-8 bottles respectively, and taking the No. 9 bottle as a waste liquid bottle.

1mol/L of sodium hydroxide added into the bottle No. 1 is from an Agilent standard solution; the 0.1mol/L sodium hydroxide added into the No. 2 bottle is diluted by 1mol/L sodium hydroxide and ultrapure water; ultrapure water added into the bottle No. 3 and water used for preparing the solution in the experimental process are both in HPLC level; 4. the background buffer solution added into the bottles 5 and 6 is 180mmol/L sodium acetate solution containing 30%, 40%, 50%, 60% and 70% methanol respectively, and the liquid level heights of the bottles 5 and 6 are kept consistent; the complexing agent in the complexing agent solution added into the bottle No. 7 is 30 mmol/L1, 10-phenanthroline; metal ion reference solution added into No. 8 bottle contains Ni²⁺，Co²⁺，Cu^{2 +}，Hg²⁺And Cd²⁺4. mu.g/mL, 8. mu.g/mL and 8. mu.g/mL, respectively, the concentration of SDS was 7.2mmol/L, and the concentration of sodium acetate was 180mmol/L, respectively.

2. Before the start of the run, the capillary column was activated with 1.0mol/L sodium hydroxide (10 min), 0.1mol/L sodium hydroxide (10 min), ultra pure water (5 min), and background buffer solution (5 min) in this order.

3. And selecting a pressure injection mode, injecting a complexing agent solution and a metal ion reference substance solution in sequence under the condition of 50mbar, wherein the sample injection time of the complexing agent solution is 8s, the sample injection time of the metal ion reference substance solution is 60s, and the positive pressure of 16kV is applied to the complexing agent at the inlet end. The operation starts.

As shown in fig. 9 and 10, it can be seen from fig. 9 that the peak areas of the five metal ions increase and then gradually decrease as the methanol content increases; as can be seen from FIG. 10, when the methanol content is 30%, Ni²⁺，Co²⁺And Cu²⁺Shows a lower peak, Hg²⁺，Cd²⁺Or even not detected. When the methanol content exceeds 50%, the separation between the metal ions and 1, 10-phenanthroline becomes poor, thereby affecting Hg²⁺And Cd²⁺Detection of (3). The above phenomenon can be explained by the fact that: the low methanol content is not sufficient to reduce the affinity between the micelles and the metal ions, and does not release the metal ions. However, as the organic solvent content in the background buffer increases, the electrolyte concentration of the background buffer is diluted, affecting the local electric field strength and resulting in a decrease in the signal strength of the dotted metal ions. Therefore, 50% methanol is the optimum condition.

And seventhly, investigating the injection time of the sample to be detected:

1. taking 9 clean capillary electrophoresis sample bottles, adding 1mol/L sodium hydroxide, 0.1mol/L sodium hydroxide, ultrapure water, background buffer solutions (4, 5 and 6), complexing agent solutions and metal ion reference substance solutions (which are filtered by 0.22 mu m microporous filter membranes before sample loading) into No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-8 bottles respectively, and taking the No. 9 bottle as a waste liquid bottle.

1mol/L of sodium hydroxide added into the bottle No. 1 is from an Agilent standard solution; the 0.1mol/L sodium hydroxide added into the No. 2 bottle is diluted by 1mol/L sodium hydroxide and ultrapure water; ultrapure water added into the bottle No. 3 and water used for preparing the solution in the experimental process are both in HPLC level; 4. the background buffer solution added into bottles No. 5 and No. 6 is 180mmol/L sodium acetate solution containing 50 percent methanol,the liquid level heights of the bottles 5 and 6 are kept consistent; the complexing agent in the complexing agent solution added into the bottle No. 7 is 30 mmol/L1, 10-phenanthroline; metal ion reference solution added into No. 8 bottle contains Ni²⁺，Co²⁺，Cu²⁺，Hg²⁺And Cd²⁺4. mu.g/mL, 8. mu.g/mL and 8. mu.g/mL, respectively, the concentration of SDS was 7.2mmol/L, and the concentration of sodium acetate was 180mmol/L, respectively.

2. Before the start of the run, the capillary column was activated with 1.0mol/L sodium hydroxide (10 min), 0.1mol/L sodium hydroxide (10 min), ultra pure water (5 min), and background buffer solution (5 min) in this order.

3. And selecting a pressure injection mode, injecting a complexing agent solution and a metal ion reference substance solution in sequence under the condition of 50mbar, wherein the sample injection time of the complexing agent solution is 8s, the sample injection time of the metal ion reference substance solution is 40s, 50s, 60s, 80s and 100s respectively, and the positive pressure of 16kV is applied to the inlet end of the complexing reagent. The operation starts.

As a result, as shown in fig. 11 and 12, it can be seen from fig. 11 that the peak areas of the five metal ions increase as the injection time increases from 40s to 80 s. However, when the injection time is longer than 80s, the peak area is almost constant or decreases. In addition, fig. 12 shows that the peak heights of five metal ions increase with the increase of the injection time, but the separation efficiency is poor when the sample is injected for 100 s. The explanation for the above phenomenon is that a higher enrichment effect can be obtained by extending the injection time of the sample solution, and the overloaded sample amount cannot be completely complexed by the complexing agent, resulting in a deteriorated separation effect. Depending on the enrichment effect and the degree of separation, 80s was chosen as the optimal injection time for the sample solution.