CN100570354C - Method for determination of aristolochic acid Ⅰ and aristolochic acid Ⅱ in Chinese herbal medicine by capillary electrophoresis-electrochemical analysis technique - Google Patents
Method for determination of aristolochic acid Ⅰ and aristolochic acid Ⅱ in Chinese herbal medicine by capillary electrophoresis-electrochemical analysis technique Download PDFInfo
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- CN100570354C CN100570354C CNB200610016927XA CN200610016927A CN100570354C CN 100570354 C CN100570354 C CN 100570354C CN B200610016927X A CNB200610016927X A CN B200610016927XA CN 200610016927 A CN200610016927 A CN 200610016927A CN 100570354 C CN100570354 C CN 100570354C
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- 241000411851 herbal medicine Species 0.000 title claims abstract description 51
- BBFQZRXNYIEMAW-UHFFFAOYSA-N aristolochic acid I Chemical compound C1=C([N+]([O-])=O)C2=C(C(O)=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 34
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- 238000000840 electrochemical analysis Methods 0.000 title claims abstract 3
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- 239000012086 standard solution Substances 0.000 claims abstract description 15
- 239000000243 solution Substances 0.000 claims abstract description 11
- 238000002484 cyclic voltammetry Methods 0.000 claims abstract description 10
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- 239000004480 active ingredient Substances 0.000 claims abstract description 7
- 238000005516 engineering process Methods 0.000 claims abstract description 6
- 239000000523 sample Substances 0.000 claims description 38
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 22
- 238000004458 analytical method Methods 0.000 claims description 22
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 241000196324 Embryophyta Species 0.000 claims description 7
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Abstract
本发明涉及通过毛细管电泳-电化学分析技术测定中草药中马兜铃酸I和马兜铃酸II含量的方法。步骤为：配制缓冲溶液；进行中草药中活性成分标准品溶液的配制及其循环伏安实验；对检测条件进行优化，并在此优化条件下进行重现性、检测限和线性范围考察；中草药样品溶液的制备；中草药CE-ED指纹图谱的获得；通过对比标准混合溶液和实际样品中活性成分的峰高确定实际样品中活性成分的含量。本发明能够建立中草药的标准CE-ED指纹图谱，同时进行活性成分的定量检测，具有高的分离效率、低的检测限、成本低、重现性好等优点，是控制及评价中草药质量的先进方法。The invention relates to a method for measuring the contents of aristolochic acid I and aristolochic acid II in Chinese herbal medicines by capillary electrophoresis-electrochemical analysis technology. The steps are: preparing a buffer solution; preparing a standard solution of active ingredients in Chinese herbal medicine and its cyclic voltammetry experiment; optimizing the detection conditions, and investigating the reproducibility, detection limit and linear range under the optimized conditions; Chinese herbal medicine samples The preparation of the solution; the acquisition of CE-ED fingerprint of Chinese herbal medicine; the content of the active ingredient in the actual sample is determined by comparing the peak height of the active ingredient in the standard mixed solution and the actual sample. The invention can establish standard CE-ED fingerprints of Chinese herbal medicines, and at the same time perform quantitative detection of active ingredients. It has the advantages of high separation efficiency, low detection limit, low cost and good reproducibility, and is an advanced method for controlling and evaluating the quality of Chinese herbal medicines. method.
Description
Technical field
The present invention relates to method by aristolochic acid I and aristolochic acid II content in the Capillary Zone Electrophoresis with Electrochemical analytical technology mensuration Chinese herbal medicine.
Background technology
The history that is applied in the existing more than one thousand years of China of Chinese herbal medicine is the crystallization of Chinese nation's excellent culture.The analysis of Chinese herbal medicine is crucial, but also is simultaneously very complicated.At first, the active component difference that contains of diverse herbal medicine; Secondly, the active component of herbal medicine of the same race also is subjected to factor affecting such as soil, weather and growth phase, or even same herbal medicine, and the content of different parts active component also is different; Moreover Chinese herbal medicine also has certain toxic and side effect.Therefore, how fast, efficiently and easily the active component in the Chinese herbal medicine is carried out qualitative and quantitative analysis, be the target that Chinese medicine analysis and research person makes great efforts always.
At present, the method that is used for traditional Chinese medicine research mainly is high performance liquid chromatography (HPLC) method, and it is used for the existing patent documentation report of the fingerprint analysis (patent No.: 200410014271) of Chinese herbal medicine.Nuclear magnetic resonance method fingerprint discriminating Chinese herbal medicine has also been applied for the patent (patent No.: 200510044396) in addition.HPLC is in analysis during pharmaceutically active ingredient, that its advantage is that post is imitated is higher, selectivity good, widely applicable etc., but also have following unfavorable factor: (1) traditional Chinese medicine ingredients is complicated and very unclear, has increased the difficulty of sample pre-treatments, and easily pollute, influence column life; (2) post of every post is imitated actual less than 10,000 column plates; (3) the consumption quantity of solvent is big; (4) analysis time is generally longer.Nuclear magnetic resonance method not only can carry out qualitative and quantitative analysis, and can also obtain more structural information, but its operation than complexity and instrument costliness, thereby limited its widespread use.
Compare with them, capillary electrophoresis (CE) is a kind of new separate analytical technique, but development is rapidly, and separation efficiency is used for having many advantages when Chinese medicine is analyzed than HPLC height: (1) capillary column easy cleaning, needn't worry that post pollutes and scraps; (2) sample pre-treatments is simple; (3) analysis time is short; (4), might make same separation condition be fit to multi-component analysis in the several samples because post is imitated height; (5) used chemical reagent is few, and is inexpensive, and analysis cost is low; (6) clastotype is many, is suitable for all kinds of chemical composition analysis in the Chinese medicine.
The main method that is used to detect medicinal herb components with the CE coupling is ultraviolet spectrum (UltravioletSpectrophotometry, UV), UV detects because its versatility is better, simple in structure, it is the most widely used detection method during present medicinal herb components is analyzed, but it is not high that its major defect is sensitivity, and mainly to be that light path is too short cause for this.And electrochemical assay (ED) has many advantages with respect to the UV method,, equipment good, highly sensitive as selectivity and simple to operate, be convenient to promote the use of etc.Like this, will have the CE of high separating efficiency and highly sensitive ED combines and is used for fingerprint and differentiates that Chinese herbal medicine is very favorable for the true and false, quality, safety and the drug effect of the used herbal medicine that guarantees to be used as medicine.But up to now, also there is not CE-ED method fingerprint to differentiate the document and the patent report of Chinese herbal medicine.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art, the invention provides method by aristolochic acid I and aristolochic acid II content in the Capillary Zone Electrophoresis with Electrochemical analytical technology mensuration Chinese herbal medicine.This is a kind of instrument cheapness, simple to operate and quick and sensitive analytical approach, is the Chinese herbal medicine fingerprint map analyzing is measured the method that combines with active component content.
Method of the present invention is divided following step:
1), the processing of separation capillary and electrode in order to guarantee to separate the reappearance with testing result, the processing of separation capillary and electrode is crucial:
(1) kapillary is handled:
Kapillary leads to 0.1mol L before using for the first time
-1The NaOH aqueous solution is spent the night.In experimentation,, between each sample introduction, wash kapillary with 1-3min secondary water, 4-6min buffer solution in order to guarantee the reappearance of analysis result;
(2) processing of electrode:
When analytical standard product solution, with the ultrasonic 0.5-1.5min of carbon fiber microdisk electrode (CFE), it can use at least 1 week before the operation; Before each operation with electrode ultrasonic outside, also electrode scan round 10-30 week in buffer solution (0.2V-1.2V) to be activated;
2), the preparation of buffer solution
The phosphate buffered solution of pH 〉=9.5 (PBS) is used 1.0mol L
-1The NaOH adjust pH, the preparation of the PBS buffer solution of pH value＜9.5 is directly to mix Na
2HPO
4And NaH
2PO
4Aqueous solution get final product;
3), the preparation of active component standard solution and cyclic voltammetry experiment thereof in the Chinese herbal medicine
The preparation of active component standard solution:
The active component standard items are mixed with 3-8mol L with dissolve with methanol
-1Solution;
The cyclic voltammetry experiment of active component standard solution:
With 33 μ m CFE scan rounds in containing the PBS solution of standard items, investigate its cyclic voltammetric behavior;
4), the optimization of testing conditions, and under this optimal conditions, carry out the investigation of reappearance, detectability and the range of linearity
The optimization of testing conditions:
Change-detection current potential, 5-50mmol L between 1.0-1.3V
-1Between change to change between buffer concentration, 7-11 between pH value of buffer solution, 5-40s to change and change separation voltage between sample injection time and 5-22.5kV and investigate influence peak current, transit time, degree of separation and separation efficiency;
5), the preparation of Chinese herbal medicine sample solution
Pulverize the herbal medicine sample with the plant comminutor that has 20-80 mesh sieve, the herbal medicine sample that takes by weighing 0.3-0.8g soaks 10-18h with methyl alcohol, the ultrasonic 30-60min of water-bath then, the centrifugal about 5-10min of 2000-3500rpm afterwards, this extraction process repeats 1-3 time again, at last, the extract constant volume is in the methyl alcohol of 5-10mL; Before the sample introduction, all sample solutions are all with the cellulose esters membrane filtration of 0.22 μ m and be diluted to needed concentration and analyze;
6), the acquisition of Chinese herbal medicine CE-ED finger-print
Carrying out electrophoretic analysis under the top condition that the acquisition of Chinese herbal medicine CE-ED finger-print is in step 4) to be obtained obtains;
7), the peak height of active component is determined the content of active component in the actual sample in contrast standard mixed solution and the actual sample;
8), the CE-ED finger-print that obtained of contrast, differentiate different parts of kindred plant and kindred plant not from several different fingerprint fragments respectively, thereby differentiate Chinese herbal medicine and adulterant thereof.
Method of the present invention is compared with other Chinese herbal medicine analytical approach, and its advantage is:
(1) distinguishing feature of CE is simple, efficient, quick and micro-: only need the kapillary of a cheapness, little dish working electrode, high direct voltage instrument (± 30kV), electrochemical analyser and some common agents get final product; Usually as long as CE finishes a testing process a few minutes to tens minute, and can obtain the theoretical cam curve up to hundreds of thousands/rice; Sampling volume has only several liters of receiving usually, and is particularly when analyzing poisonous sample, very little to environmental impact.
(2) kapillary is normally made by quartz glass, can stand the cleaning of highly basic, strong acid, in the mensuration of the Chinese herbal medicine sample of complexity, helps the removing of sample impurity in the post.
(3) Electrochemical Detection is compared with other detection method, because it has response to electroactive component, so its selectivity is good.In addition, the detectability of Amperometric Detection Coupled is low, can reach 10 usually
-8Mol L
-1, the range of linearity reaches 2-3 the order of magnitude.
Description of drawings
Fig. 1 is root, footpath leaf and fruit (that is: a dutchmanspipe root of birthwort plant; The b birthwort; The c herba aristolochiae) CE-ED finger-print contrast; I and II are respectively fingerprint fragment I and II among the figure.
Fig. 2 is three kinds of akebis (that is: d caulis aristologhiae manshuriensis; The e caulis clematidis armandii; The f akebi) CE-ED finger-print contrast; I, II and III are respectively fingerprint fragment I, II and III among the figure.
Embodiment
The present invention is an example with birthwort plant and three kinds of akebis, is described further in conjunction with the embodiments.
The aristolochic acid of finding in Aristolochia and asarum plant (AAs) (comprising aristolochic acid I and II, that is: AA-I and AA-II) has the structure of nitro phenanthrene carboxylic acid, has the effect of diuresis and pain relieving in Chinese herbal medicine.Yet excessive absorption can cause serious injury of kidney by AAs in the made health products of birthwort, slimming drugs and the Chinese herbal medicine.In recent years, because the edible made slimming drugs of Aristolochia fangchi that contain AAs have caused a lot of poisonings, therefore cause the great attention of countries in the world.U.S. food and drug administration have issued that the herbal medicinal product information that contains AAs is in case the further generation of poisoning.Caulis aristologhiae manshuriensis, Aristolochia fangchi and dutchmanspipe root are deleted from the Pharmacopoeia of the People's Republic of China of version in 2005 owing to contain a large amount of AAs, and be no longer medicinal.Therefore, the content of AAs is very important in quantitative test medicinal herbs and products thereof.
Embodiment 1: the detection by quantitative of birthwort plant different parts and fingerprint discriminatory analysis
The fruit of plant birthwort, footpath leaf and root all are herbal medicine, and they are called birthwort, herba aristolochiae and dutchmanspipe root respectively.
(1) preparation of buffer solution, its concrete grammar is:
The pH value is that 9.5,10.0,10.5 and 11.0 phosphate buffered solution (PBS) is used 1.0mol L
-1NaOH regulates, and the pH value is: 7.0, the preparation of 8.0 and 9.0 buffer solution is directly to mix Na
2HPO
4And NaH
2PO
4Aqueous solution get final product.
(2) preparation of AAs standard solution and cyclic voltammetry experiment thereof, process is as follows:
The preparation of AAs standard solution: contain 7.6 * 10
-4Mol L
-1AA-I and 2.0 * 10
-3Mol L
-1The mixed solution of AA-II is prepared with methyl alcohol, and lays in 4 ℃ refrigerator, is diluted to desired concn with secondary water before using.
The cyclic voltammetry experiment of AAs standard solution: at first one week of scan round is as a setting in PBS solution with 33 μ m CFE.Then this electrode is being contained 5 * 10
-5Mol L
-1AA-I and 5.0 * 10
-5MolL
-1AA-I and 1.3 * 10
-4Mol L
-1In difference one week of scan round in the PBS solution of AA-II potpourri, investigate the cyclic voltammetric behavior of AA-I and AA-II.
(3) optimization of testing conditions, and under this optimal conditions, carry out the investigation of reappearance, detectability and the range of linearity;
The optimization detailed process of testing conditions is:
Change-detection current potential, 5-50mmol L between 1.0-1.3V
-1Between change to change between buffer concentration, 7-11 between pH value of buffer solution, 5-40s to change and change separation voltage between sample injection time and 5-22.5kV and investigate influence peak current, transit time, degree of separation and separation efficiency.
Under the separation and testing conditions of the best: the detection current potential is 1.20V; Buffer solution is 2.0 * 10
-2MolL
-1PBS (pH=10.0); 17cm height sample introduction 25s; Separation voltage is 12.5kV, and AA-I and AA-II reach baseline separation and have high sensitivity in 5min.
Under the described top condition, tested the series of standards mixed solution in the above, concentration range is from 4.0 * 10
-8To 1.9 * 10
-5Mol L
-1AA-I and 1.0 * 10
-7To 7.0 * 10
-5Mol L
-1AA-II.Obtained crossing over the range of linearity of two orders of magnitude, related coefficient surpasses 0.999.Adopt this CE-ED method actual detected to AA-I and the detectability of AA-II be respectively 4.0 * 10
-8Mol L
-1With 1.0 * 10
-7MolL
-1(signal to noise ratio (S/N ratio) is 3).
Under top condition, advance the standard biased sample continuously: 9.5 * 10
-6Mol L
-1AA-I and 2.5 * 10
-5MolL
-1AA-II, the reappearance in a few days of investigation the method.Continuous 7 sample introductions, the peak current of AA-I and AA-II and the RSD of transit time (n=7) value are respectively 2.7,0.5% and 2.2,0.4%.We have also investigated the reappearance in the daytime of this method, and the peak current of AA-I and AA-II and the RSD of transit time (n=6) value are respectively 4.9,0.7% and 2.9,0.7%.
(4) preparation of Chinese herbal medicine sample solution, detailed process is:
Pulverize the herbal medicine sample with the plant comminutor that has 40 mesh sieve, take by weighing 0.5g herbal medicine sample and soak 16h, then the ultrasonic 30min of water-bath with methyl alcohol, the centrifugal 8min of 3000rpm afterwards, this extraction process repeats (1.0mL * 2) again twice, and last, the extract constant volume is in 7mL methyl alcohol.Before the experiment, all sample solutions are all with the cellulose esters membrane filtration of 0.22 μ m and be diluted to needed concentration direct injection analysis.
(5) acquisition of Chinese herbal medicine CE-ED finger-print;
The acquisition of Chinese herbal medicine CE-ED finger-print is to carry out electrophoretic separation under the top condition that is obtained to detect (as shown in Figure 1) obtain in (3), can be respectively from 2 different fingerprint fragments the spectrogram that is obtained is carried out visual analysis, directly debates knowledge.
(6) peak height of active components A As is determined the content of active components A As in the actual sample in contrast standard mixed solution and the actual sample.The content of AAs is all listed in the table 1 in birthwort plant different parts (that is: root, footpath leaf and fruit).
Active components A As assay result in the table 1 birthwort plant different parts
NF: expression does not detect.
By table 1 as seen, the content of AAs is visibly different in the different parts of birthwort plant.The content of AAs is maximum in the root, secondly is fruit, yet does not but detect AAs in the leaf of footpath.
In order to guarantee to separate the reappearance with testing result, the processing of separation capillary and electrode is crucial:
(1) detailed process of kapillary processing is:
Kapillary leads to 0.1mol L before using for the first time
-1The NaOH aqueous solution is spent the night.In experimentation,, between each sample introduction, wash kapillary with 2min secondary water, 4-6min buffer solution in order to guarantee the reappearance of analysis result.
(2) processing procedure of electrode is:
When analytical standard product solution, before every day experiment, can use at least 1 week after with the ultrasonic 1min of carbon fiber microdisk electrode.And when analyzing actual sample, bigger because the composition in the actual sample is complicated to the state influence of electrode, therefore, before experiment every day,, also electrode 20 weeks of scan round in buffer solution (0.2V-1.2V) to be activated the ultrasonic 1min of electrode.
Detection by quantitative of 2: three kinds of akebis of embodiment and fingerprint discriminatory analysis
Three kinds of herbal medicine: caulis aristologhiae manshuriensis, caulis clematidis armandii and akebi are often obscured by people, because their appearance is similar, and all are referred to as " akebi " in Chinese.Yet they belong to different types of plant.In actual applications, people's constant error replaces nontoxic caulis clematidis armandii and akebi with the caulis aristologhiae manshuriensis of renal toxicity and carcinogenicity.The main active that has renal toxicity and carcinogenicity in the caulis aristologhiae manshuriensis is AAs, and therefore, the detection by quantitative of AAs and fingerprint differentiate that it is very useful that these three kinds of akebis prepare Chinese medicine for these medicinal herbs of safe handling.
(1) preparation of buffer solution, its concrete grammar is:
The pH value is that 9.5,10.0,10.5 and 11.0 phosphate buffered solution (PBS) is used 1.0mol L
-1NaOH regulates, and the pH value is: 7.0, the preparation of 8.0 and 9.0 buffer solution is directly to mix Na
2HPO
4And NaH
2PO
4Aqueous solution get final product.
(2) preparation of AAs standard solution and cyclic voltammetry experiment thereof, process is as follows:
The preparation of AAs standard solution: contain 7.6 * 10
-4Mol L
-1AA-I and 2.0 * 10
-3Mol L
-1The mixed solution of AA-II is prepared with methyl alcohol, and lays in 4 ℃ refrigerator, is diluted to desired concn with secondary water before using.
The cyclic voltammetry experiment of AAs standard solution: at first one week of scan round is as a setting in PBS solution with 33 μ m CFE.Then this electrode is being contained 5 * 10
-5Mol L
-1AA-I and 5.0 * 10
-5MolL
-1AA-I and 1.3 * 10
-4Mol L
-1In difference one week of scan round in the PBS solution of AA-II potpourri, investigate the cyclic voltammetric behavior of AA-I and AA-II.
(3) optimization of testing conditions, and under this optimal conditions, carry out the investigation of reappearance, detectability and the range of linearity;
The optimization detailed process of testing conditions is:
Change-detection current potential, 5-50mmol L between 1.0-1.3V
-1Between change to change between buffer concentration, 7-11 between pH value of buffer solution, 5-40s to change and change separation voltage between sample injection time and 5-22.5kV and investigate influence peak current, transit time, degree of separation and separation efficiency.
Under the separation and testing conditions of the best: the detection current potential is 1.20V; Buffer solution is 2.0 * 10
-2MolL
-1PBS (pH=10.0); 17cm height sample introduction 25s; Separation voltage is 12.5kV, and AA-I and AA-II reach baseline separation and have high sensitivity in 5min.
Under the described top condition, tested the series of standards mixed solution in the above, concentration range is from 4.0 * 10
-8To 1.9 * 10
-5Mol L
-1AA-I and 1.0 * 10
-7To 7.0 * 10
-5Mol L
-1AA-II.Obtained crossing over the range of linearity of two orders of magnitude, related coefficient surpasses 0.999.Adopt this CE-ED method actual detected to AA-I and the detectability of AA-II be respectively 4.0 * 10
-8Mol L
-1With 1.0 * 10
-7MolL
-1(signal to noise ratio (S/N ratio) is 3).
Under top condition, advance the standard biased sample continuously: 9.5 * 10
-6Mol L
-1AA-I and 2.5 * 10
-5MolL
-1AA-II, the reappearance in a few days of investigation the method.Continuous 7 sample introductions, the peak current of AA-I and AA-II and the RSD of transit time (n=7) value are respectively 2.7,0.5% and 2.2,0.4%.We have also investigated the reappearance in the daytime of this method, and the peak current of AA-I and AA-II and the RSD of transit time (n=6) value are respectively 4.9,0.7% and 2.9,0.7%.
(4) preparation of Chinese herbal medicine sample solution, detailed process is:
Pulverize the herbal medicine sample with the plant comminutor that has 40 mesh sieve, take by weighing 0.5g herbal medicine sample and soak 16h, then the ultrasonic 30min of water-bath with methyl alcohol, the centrifugal 8min of 3000rpm afterwards, this extraction process repeats (1.0mL * 2) again twice, and last, the extract constant volume is in 7mL methyl alcohol.Before the experiment, all sample solutions are all with the cellulose esters membrane filtration of 0.22 μ m and be diluted to needed concentration direct injection analysis.
(5) acquisition of Chinese herbal medicine CE-ED finger-print;
The acquisition of Chinese herbal medicine CE-ED finger-print is to carry out electrophoretic separation under the top condition that is obtained to detect (as shown in Figure 2) obtain in (3), can be respectively from 3 different fingerprint fragments the spectrogram that is obtained is carried out visual analysis, directly debates knowledge.
(6) peak height of active components A As is determined the content of active components A As in the actual sample in contrast standard mixed solution and the actual sample.The content of AAs is all listed in the table 2 in three kinds of akebis (that is: caulis aristologhiae manshuriensis, caulis clematidis armandii and akebi).
Active components A As assay result in three kinds of akebis of table 2
NF: expression does not detect.
By table 2 as seen, in caulis aristologhiae manshuriensis, contain AA-I and AA-II, and in caulis clematidis armandii and akebi, do not detect AAs.Therefore, misuse can be introduced the AAs of renal toxicity and carcinogenicity in the body and damage.
In order to guarantee to separate the reappearance with testing result, the processing of separation capillary and electrode is crucial:
(1) detailed process of kapillary processing is:
Kapillary leads to 0.1mol L before using for the first time
-1The NaOH aqueous solution is spent the night.In experimentation,, between each sample introduction, wash kapillary with 2min secondary water, 4-6min buffer solution in order to guarantee the reappearance of analysis result.
(2) processing procedure of electrode is:
When analytical standard product solution, before every day experiment, can use at least 1 week after with the ultrasonic 1min of CFE electrode.And when analyzing actual sample, because the composition in the actual sample is complicated, bigger to the state influence of electrode, therefore, before experiment every day,, also electrode 20 weeks of scan round in buffer solution (0.2V-1.2V) to be activated the ultrasonic 1min of CFE electrode.
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