Capillary electrophoresis

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Electrophoretic separation and analysis method based on the difference in mobility and distribution behavior of each component in the sample

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capillary electrophoresis : With elasticity quartz capillary To separate channels HVDC The electric field is drive According to the difference in mobility and distribution behavior of each component in the sample Electrophoretic separation Analytical methods.

Chinese name: Capillary electrophoresis

Foreign name: capillary electrophoresis
Features: Widely used, efficient, fast and micro

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▪ Chip two-dimensional electrophoresis separation
▪ Chip free flow electrophoresis
▪ expectation
six basic operation

Basic Introduction

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Equipment used

Main components are 0~30kV Adjustable voltage and current stabilizing power supply Quartz with inner diameter less than 100 μ m (commonly 50~75 μ m) and length generally 30~100cm capillary , electrode trough, detector and injection device. The detector has ultraviolet/visible spectrum Photodetector 、 laser induced fluorescence Detector and Electrochemical detector The former is most commonly used. Sample injection method includes electrokinetic method（ Electromigration ）Pressure method（ Positive pressure 、 Negative pressure ）And Siphon method 。 The complete instrument is also equipped with automatic flushing, automatic sample injection temperature control 、 Data acquisition and processing And other components.

capillary electrophoresis Quartz used capillary column In case of pH value ＞ 3 Surface band negative electricity , formed when contacting with buffer solution Electric double layer Under the action of high-voltage electric field, the buffer solution at the side of the double electric layer is formed due to the positive electricity And moves towards the negative pole, thus forming Electroosmotic flow 。 At the same time buffer solution Medium, charged particle Under the action of electric field, they move to the opposite direction of their charge polarity at different speeds, forming electrophoresis. Charged particles capillary In buffer Migration speed It is equal to the vector sum of electrophoresis and electroosmosis. Various particles can be separated due to different migration speeds caused by different charge, mass, volume and shape.

Separation mode

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Capillary zone electrophoresis （Capillary Zone Electrophoresis，CZE）

The most common model used to analyze charged solutes. Each component in the sample is mobility Different and divided into different zones. To reduce Electroosmotic flow and Adsorption phenomenon , you can set capillary Interior wall fabrication chemical modification 。

Capillary gel electrophoresis （Capillary Gel Electrophoresis，CGE）

Capillary gel electrophoresis, in which monomer is loaded into the capillary to initiate polymerization to form a gel, which is mainly used for determination protein 、 DNA etc. Macromolecular compound 。 In addition Polymer solution And other substances with screening function, such as glucan 、 Polyethylene oxide , loaded into capillary for analysis, called capillary gel free sieving electrophoresis, so this mode is sometimes referred to as capillary sieving electrophoresis, which is divided into gel and gel free sieving.

Micellar electrokinetic capillary chromatography （Micellar Electrokinetic Capillary Electrophoresis，MECE）

Micellar electrokinetic capillary chromatography, adding Ionic surfactant as Sodium lauryl sulfate , forming micelle , the separated substance aqueous phase And micellar phase（ Quasi stationary phase ）Distribution between and Electroosmotic flow stay capillary Internal migration to achieve separation. This mode can be used for Neutral substance Separation of.

Affinity capillary electrophoresis （Affinity Capillary Electrophoresis， ACE）

Affinity capillary electrophoresis, coating on the inner wall of the capillary or adding affinity to the gel Ligands To achieve the separation purpose with different affinity.

capillary Electrochromatography （Capillary Electrochromatography， CEC）

Capillary electrochromatography HPLC Of stationary phase Fill the capillary tube or coat the stationary phase on the inner wall of the capillary tube to Electroosmotic flow by mobile phase drive This mode combines electrophoresis and Liquid chromatography The separation mechanism of.

Capillary isoelectric focusing Capillary Ionic Focusing (CIEF)

Capillary isoelectric focusing electrophoresis is to minimize the electroosmotic flow through the inner wall coating, and then Amphoteric electrolyte Mixed injection, acid and alkali in two electrode tanks, add high voltage After, on capillary PH gradient is established in the capillary tube, and solutes migrate to their respective Isoelectric point After focusing, use pressure or change the pH value of the solution stored in the electrode tank at the end of the detector to make the solute pass through the detector.

Capillary isotachophoresis （Capillary Isotachophoresis， CITP）

Capillary isotachophoresis with lead electrolyte And subsequent electrolytes, so that solutes can be separated according to their different electrophoretic degrees.

The above modes are mainly applied in CZE, CGE and MECE.

Instrument requirements

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The instruments used are Capillary electrophoresis apparatus 。 For the varieties measured by capillary electrophoresis in the text, the specified measurement parameters, in addition to the analysis mode and detection method (such as ultraviolet Absorption or Fluorescence detector Wavelength Electrochemical detector (applied potential, etc.) should be in accordance with the provisions under this item, other parameters such as capillary Inner diameter, length, pH value and concentration of buffer solution Modifier Amount added Operating voltage Or the size of the current, the length of operation time, the temperature of the capillary tube, etc., can refer to the data specified under this item, according to the conditions and Pretesting And make necessary adjustments.

The solution in the electrode bath and capillary is buffer solution, which can be added Organic solvent As modifier and added surface active agent , called Running buffer 。 The running buffer should be degassed before use. The peak time scale of each component in the electrophoresis spectrum Migration time 。 Micellar electrokinetic capillary chromatography In micelle amount to Liquid chromatography The stationary phase of the capillary Internal adjoint Electroosmotic flow Migration, so Capacity factor by Infinity Finally, the components of. Other parameters are the same as those used for liquid chromatography.

test method

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capillary electrophoresis The commonly used detection methods are absorption spectrum ， fluorescence spectrum , thermal mirror, Raman spectra , MS and electrochemical method 。

Separation system

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Chip based high performance high speed capillary electrophoresis（ HPCE ）Separation system

In recent years technological development Fast, in protein Deoxyribonucleic acid (DNA), etc Biomacromolecule The results showed that the separation analysis showed significant advantages. In the early 1990s, Manz, Widmer and others proposed for the first time that micro electromechanical systems (MEMS, MEMS ）And analytical chemistry Based Micro total analysis system (miniaturized total analysis systems, mTAS). Currently, with Proteomic research With the deepening and development of protein separation, how to achieve rapid and efficient protein separation has become an important problem to be solved. Protein on chip capillary electrophoresis Separation can significantly improve the analysis speed and separation efficiency Therefore, this research direction has attracted wide attention, and many papers have been published.

Microfluidic chip Characteristics of capillary electrophoresis

chip capillary electrophoresis The conventional capillary electrophoresis operation is carried out on the chip by using glass, quartz or various Polymer materials Process micron channels to HVDC Electric field is the driving force for sample injection, separation and detection. It is similar to that of conventional capillary electrophoresis Separation principle Same, so when separating Biomacromolecule The samples have advantages. In addition, compared with conventional Capillary electrophoresis system In contrast, the chip based capillary electrophoresis system also has the advantages of short separation time separation efficiency High, small system volume and easy integration of different operation units. The above advantages of chip capillary electrophoresis make it Protein separation One of the important means in analysis. Rodriguez et al capillary , short capillary and glass chip to Zone electrophoresis The separation mode of human immunoglobulin G (IgG) and fluorescein Isothiocyanate ( FITC ）To compare the separation performance of the three systems. When long capillary tubes with an effective separation length of 35 am and short capillary tubes with an effective separation length of 6 am are used, the separation time is 335 S and 84 S respectively, Number of theoretical trays 27 750 and 41 816, respectively. When using a glass chip with an effective separation length of only 2.8 cm, the separation time is shortened to 15 S, and the number of theoretical plates reaches 49 000. It can be seen that glass chips are used for Capillary zone electrophoresis At the separation speed and Column efficiency Obviously superior to conventional Capillary electrophoresis system 。

The Mode of Microfluidic Chip Capillary Electrophoresis for Protein Separation

Chip reported in literature capillary electrophoresis Protein separation mainly uses Zone electrophoresis 、 gel electrophoresis 、 Isoelectric focusing 、 micelle Electrokinetic chromatography and Two-dimensional electrophoresis Isomode 。

Chip capillary zone electrophoresis

Capillary zone electrophoresis is a chip capillary electrophoresis A basic separation mode for protein separation. It is based on different protein molecules Migration rate It is a simple and fast way to realize separation separation method 。 use Zone electrophoresis The separation model has successfully separated a variety of protein samples.

Colyer et al Capillary electrophoresis chip , using the zone electrophoresis mode for human serum albumin The sample was separated and four protein regions (namely IgG transferrin 、a-1- Antitrypsin and albumin Band, respectively, to simulate the 7, p, dl and albumin bands in serum protein samples). Of which protein Fluorescent labeling After separation, due to Fluorescent dye The sensitivity of TNS (2-toluidinonaphtha. 1-ene-5-sulfonate) labeled serum protein was low, so the separation of five bands of actual human serum protein samples was not achieved. Xiao et al Zone electrophoresis Mode at 50 mmoVL Phosphate buffer (pH 2.15) as the working buffer, when the channel width is 30um Polydimethylsiloxane (PDMS) chip, realized in 35s Cytochrome C and lysozyme Rapid separation of. Dodge et al. designed a PDMS chip integrated with 8 micro valves and 1 micro pump, and realized the liquid flow control Effective control 。 They first use the separation mode of zone electrophoresis to separate bovine serum albumin and myoglobin Then, the separated protein components were introduced into the micro mixer for enzymolysis through the function of valves, and the products were analyzed by mass spectrometry. The job Display chip Technology can be used Mass Spectrometry Pretreatment of pre complex protein samples. Zhuang et al mmol /L borate Buffer (pH 10.3) as the buffer system of chip electrophoresis immunoglobulin O/1-1 Antitrypsin , bovine serum albumin and Ferritin And the clinical diagnosis of pregnancy induced hypertension Rheumatic heart disease 、 Multiple myeloma Urine samples of patients Electrophoretic analysis , within 2 min, we got Electrophoretic system coincident Analysis results 。

On chip capillary electrophoresis protein isolate Qualitative research An important problem to be solved in is the channel surface macromolecule Protein adsorption. The tiny adsorption effect between the protein and the inner wall of the chip channel will reduce the protein's separation efficiency , causing the peak shape to become wider and trailing, affecting the reproducibility of separation. stay Capillary zone electrophoresis In the separation mode, the permanent modification of the inner wall of the channel and the dynamic modification of the buffer by adding additives are generally used to inhibit protein adsorption.

Wu et al. adopt multi-layer 88% hydrolysis polypropylene Alcohol（ PVA ）Modify PDMS chip to Zone electrophoresis The mode effectively separates the two Basic protein ( lysozyme and Ribonuclease ）And two typical acidic Protein（ bovine serum albumin And mouth Lactoglobulin )。 The coating can be inhibited within the range of pH 3~11 Electroosmotic flow Production and protein Adsorption , and the effect is stable, Continuous operation The separation effect is still good after 70 times. The research team then used self-assembly method to process epoxy modified polymer coating on the surface of PDMS chip channel to inhibit protein adsorption, and successfully separated lysozyme and Ribonuclease A 。 Chief, wait Running buffer Inorganic electrolyte is added in NaCl And neutral surface active agent Twain 20 To inhibit protein adsorption, using chips Capillary zone electrophoresis Carried out monoclonal antibody Separation analysis of‘

Chip capillary gel electrophoresis

stay Proteomics and Protein separation Gel electrophoresis is widely used in research Separation technology 。 It uses gel and other polymers as separation media network structure It is a separation mode according to the different molecular volumes of the tested components. Using gel electrophoresis mode to separate proteins on chip is more conducive to achieving high speed and efficiency of separation operation. Yao, etc Sodium dodecyl sulfonate ( SDS ）The separation mode of gel electrophoresis was compared between chip SDS capillary gel electrophoresis and conventional capillary gel electrophoresis. The results showed that the former separation efficiency It is obviously better than the latter, and the separation time is also obviously lower than the latter.

And general Capillary gel electrophoresis Similarly, the screening media commonly used in microchip capillary gel electrophoresis are also divided into gel and non gel polymer solution. Cross linked polyacrylamide gel is a widely used gel screening medium. Herr et al- polyacrylamide gel electrophoresis (SDS · PAGE) protein separation method was transplanted to the chip by Photopolymerization The cross-linked polyacrylamide gel with a concentration of 6% is prepared in the chip channel as the screening medium relative molecular mass (M,) 5 proteins were separated between 5500 and 39000, and the separation distance was only 4 mm, separation efficiency achieve Number of theoretical trays 4．41×105。 The research group's' 1 introduction later Microchannel A crosslinked polyacrylamide membrane with a concentration of 22% was prepared for the preconcentration of protein samples. The protein molecules with a relative molecular weight of 12000~205000 were effectively enriched. The crosslinked polyacrylamide gel with a concentration of 8% was used as the screening medium for separation.

Agirregabiria, etc Polymethyl methacrylate (PM-MA) chip using SU-8 Photoresist make Microchannel 12% polypropylene amide Gel is used as screening medium to separate proteins. Then the research group integrated on the chip Metal electrode , successfully separated with the same separation mode relative molecular mass 20 000 and 97 000 respectively Trypsin inhibitor and phosphorylase Two proteins. However, the preparation of cross-linked polyalenamide gels is complex and difficult to use. In contrast, linear polyacrylamide（ PLA )、 Polyvinyl alcohol ( PEG )、 Polyoxyethylene ( PEO ）Non gel screening media such as can be easily prepared and used, and can be polymerized first and then injected into the channel without being carried out in the channel polymerization It is suitable for use in complex channel systems Capillary gel electrophoresis The medium non rubber screening medium has been widely used. Yao et al. used SDS 14.200 gel buffer (Beckman Coulter company product) to separate six proteins with relative molecular weights between 9000 and 16000 on glass chips within 35 seconds. Giordano et al dyestuff Add samples and Buffer The dynamic labeling of protein was carried out in, and the separation buffer liquid system was optimized. Finally, 5% PEO (M,=100000) was selected as the screening medium. The system is effective for cattle serum albumin Of detection limit 500ng/mL, and completed the separation and analysis of actual human serum samples.

On chip Capillary gel electrophoresis The adsorption of protein on the inner wall of the channel is still an important problem to be solved. Bousse et al. used polybutadiene Methyl acrylamide (PDMA) physically coated the inner wall of the microchannel of the glass chip, reducing the electroosmotic flow to 0.5 × 10 ～ m zV s gel electrophoresis The protein of Bio Rad Company was separated in 40 s by the separation mode of Standard sample ’， separation efficiency Up to 107 Tray /m。 Nagata is waiting PMMA Used in the chip PEG The coating, with 5% linear polyacrylamide as screening medium, is realized in the channel with separation length of 3 mm Trypsin inhibitor 、 bovine serum albumin The high speed separation of three proteins with Lugalactosidase was only 8 seconds.

Chip isoelectric focusing separation

chip Isoelectric focusing The principle of protein separation is basically the same as that of conventional capillary isoelectric focusing Isoelectric point of protein (pI). Hofmann et al capillary It is applied to protein analysis.

Li et al polycarbonate On the (PC) chip, the separation plant adopts isoelectric focusing mode bovine serum albumin And enhanced green fluorescent protein ( EGFP )。 Das et al. 26 3 Adoption high polymer Chip, in Isoelectric focusing electrophoresis Under the mode, the separation length and voltage conditions were optimized, and finally the separation was completed within 1.5 min in a 1.9 cm long channel Green fluorescent protein And R Phycoerythrin The separation voltage is 500 V. Cui et al successfully separated histone green fluorescent protein, heterologous Phycocyanin And phycoerythrin. The author also reported that by changing the additives in samples and separation media Methyl cellulose The concentration of can be changed Protein separation The required channel distance, Tsai, etc Hexamethyldisiloxane Plasma polymerization The method of film modification of glass chip channel inhibits protein adsorption Isoelectric focusing Phycocyanin (pI: 4.65) hemoglobin (pI: 7.0) and Cytochrome C (pI: 9.6) The separation of three protein mixtures was completed within 3 minutes, separation efficiency 19 600 tray/m. Huang et al Amphoteric electrolyte Add to the solution Hydroxymethyl cellulose As an additive, it can inhibit protein adsorption.

Microchip micellar electrokinetic capillary electrophoresis

micelle Electric capillary electrophoresis It is a separation technology combining capillary electrophoresis and micellar solubilization chromatography. Its principle is that in the channel containing micellar solution, the solute component is Electric field force Under the action of, the separation occurs according to the different distribution between the micellar phase and the aqueous phase. Jin et al. used the micellar electrokinetic chromatography separation mode on the glass chip CE ·SDS Buffer As a separation medium, successfully realized relative molecular mass Separation of eight proteins between 14400 and 200000. Dou et al. modified the PDMS chip channel with Brij35 (polyoxyethylene [23] dodecan01) and realized the separation mode of micellar electrokinetic chromatography glucose oxidase and myoglobin Effective separation of. The chip coating can significantly reduce protein in the pH range of 2~6 macromolecule And reduce the washing time required for protein detection, thereby improving the separation efficiency. Huang et al. used 0.1% dodecyl in PDMS chip malt dust ( DDM ）Mixed with 0.03% SDS micelle , dynamically modify the channel, effectively inhibit protein adsorption, and control Electroosmotic flow So that the protein can be effectively separated under non denaturing conditions.

Chip two-dimensional electrophoresis separation

chip capillary electrophoresis The successful application has promoted the development of high-speed and efficient chip two-dimensional electrophoresis technology. For multi-component complex protein samples, the traditional one-dimensional separation method is usually unable to meet the requirements, and two-dimensional separation technology is needed to improve separation efficiency , add Peak capacity 。 And traditional Capillary electrophoresis system In contrast, on the chip Two-dimensional electrophoresis Separation can be realized through the design of chip channel structure to realize the direct cross or connection of channels, without the need to make complex two-dimensional capillary electrophoresis interface, thus avoiding the existence of Dead volume Caused by Spectral band Expansion phenomenon.

In the research of chip two-dimensional electrophoresis for protein separation, the first dimension separation mode is often used Isoelectric focusing pattern. Chen et al. made a two-dimensional capillary electrophoresis PDMS chip, using the first dimensional isoelectric focusing and the second dimensional gel electrophoresis Fluorescent labeled bovine serum albumin and carbonic anhydrase And Texas Red Ovalbumin Perform separation analysis. Li et al. designed a two-dimensional separation polymer core 4t sheet using isoelectric focusing and gel electrophoresis. After the first dimensional isoelectric focusing separation of protein samples is completed, the second dimensional gel electrophoresis separation can be completed in multiple parallel channels. whole separation process Complete within 10 min, Peak capacity Up to 1700. Herr et al.: "1 Development r Isoelectric focusing I Freedom Zone electrophoresis Two dimensional chip system, chip channel is 200 bucket m wide and 20 bucket m deep. The sample to be measured is separated by isoelectric focusing in the horizontal channel, and the separated sample zone is driven by the electric field into the longitudinal zone electrophoresis channel for the second dimensional separation. System adoption fluorescence microscope The separation performance was evaluated by imaging method, and the peak capacity of separation reached 1300 within 5 minutes. Wang et al. prevent one-dimensional isoelectric focusing and Two-dimensional gel electrophoresis The separation buffer between the systems was mixed, and four standard proteins were effectively separated within 20 minutes. It is also reported that SDS gel electrophoresis and micelle Electric capillary electrophoresis Binding protein Two-dimensional electrophoresis Separation. The system can complete the separation of 10 proteins within 12 minutes, Peak capacity About 1000.

In addition, there is a class of two-dimensional separation systems based on chips, which are mainly used for the separation and analysis of protein hydrolysates. Usually, micellar electrokinetic capillary is used for the first dimension separation electrophoresis Or capillary electrochromatography mode, and the second dimension separation uses Zone electrophoresis Model 2000, Ramsey Research Group. "1 For the first time, a two-dimensional separation system combining micellar electrokinetic capillary electrophoresis (first dimension) and zone electrophoresis (second dimension) was established on a glass chip, and was applied to Cytochrome C 、 Ribonuclease , d, L, albumin, etc Trypsin Degradation product Separation. Later, the research team improved the system, lengthened the length of the first dimensional electrophoresis channel, and used a small diameter corner channel to reduce diffusion. The bovine serum white protease hydrolysate was separated in about 15 minutes, Peak capacity Up to 4200. In 2001, they also developed the open tube Electrochromatography Chip combined with zone electrophoresis Two-dimensional electrophoresis System, its electricity chromatographic separation Some 25 cm long Octadecyl trimethoxy silane Coated annular Channel, Zone electrophoresis Some of them use straight channels with a length of 1.2 am, realizing the first time in 13 minutes casein Separation of trypsin hydrolysates.

Compared with one-dimensional separation chip, two-dimensional chip separation system has a high separation efficiency And peak capacity are expected to play a greater role in the separation of complex protein samples.

Chip free flow electrophoresis

In addition to the above separation mode, the chip Free flow Electrophoresis is also a chip Electrophoretic separation The important method of protein. Chip free flow electrophoresis refers to the process in which the sample flows continuously with the buffer solution in the chip through an applied electric field Electric field direction conduct Electromigration , so that according to Electrophoretic mobility Different electrophoresis separation modes for separation. Raymond et al. used chip free flow electrophoresis mode to separate human serum albumin 、 bradykinin and Ribonuclease A , its separation length is 3.1 cm, Outflow time 62 S. Kobayashi et al. used the separation mode of free flow electrophoresis to achieve continuous Protein separation , and use Hydroxypropyl methyl cellulose Coated to inhibit protein adsorption, effectively separated within 25 minutes Cytochrome C And myoglobin. Recently, Kohl.heyer et al. H 3. Made a free flow Isoelectric focusing The glass chip for protein separation successfully human serum albumin (pI=4.4) and isoelectric focusing Markers (pH 3 and 9) separation.

expectation

Microfluidic chip Capillary electrophoresis system It has outstanding advantages in protein separation and analysis, especially in Clinical test And field monitoring have good prospects for development. At the same time, it is also of great significance for the development of integration, miniaturization and portability of analytical instruments. According to literature reports, Renzi and others have developed handheld microfluidic chips Electrophoretic separation Protein device. The device consists of electrophoresis chip, small laser induced fluorescence detecting system as well as high-voltage power supply Its volume is only 11.5 cm × 11.5 cm × 19.0 cm, which can be used for Field analysis Bedside medical diagnosis and evidence collection analysis. In recent years, there have been reports on the use of chips capillary electrophoresis Perform clinical Urine protein and Lipoprotein Report of the test. Recently, Pandey et al Agilent P200 of the company Protein chip To detect trace amounts of Albuminuria , convert the protein's Electrophoretic separation and fluorescence detection Integrated, automated, and realized its Clinical laboratory Application of.

Many scientific researchers are committed to Microfluidic chip Study on the technology of capillary electrophoresis coupled with mass spectrometry to further improve the separation of complex samples Analytical capability 。 The above systems are available in Protein separation Analysis and Proteomic research It has broad application prospects. Especially for multidimensional separation and analysis of complex protein samples, chips capillary electrophoresis With its fast and efficient characteristics, it can be used as one of the one-dimensional separation methods to significantly improve the analytical flux of protein. I believe that with the deepening of research and Related technologies With the continuous development of, the microfluidic chip capillary electrophoresis technology for protein separation will become increasingly mature clinical diagnosis And proteome research.^[1]

basic operation

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(1) Start the machine according to the instrument operation manual, preheat and input various parameters, such as capillary temperature, operating voltage, detection wavelength and flushing procedure. operation Buffer Filtration and degassing are required. Place the rinsing solution, buffer solution, etc. in the sample bottle, and put them in order Sample injector 。

(2) The quality of capillary treatment has a great influence on the determination results. New uncoated capillary tubes should be thicker Lye At higher temperatures (e.g. with 1 mol/L sodium hydroxide Rinse the solution at 60 ℃) to make the inner wall of the capillary generate silicon hydroxyl, and then rinse with 0.1mol/L sodium hydroxide solution, water and operating buffer solution for several minutes respectively. Only buffer solution can be used for flushing between two injections, but if the separation performance changes, 0.1 mol/L sodium hydroxide solution must be used for flushing at first, or even concentrated sodium hydroxide solution must be used for warming flushing. The washing of gel capillary, coated capillary and filling capillary should be carried out according to the attached instructions. When washing, place the sample bottles containing the solution in the sample injector in turn, and set the sequence and time for washing.

(3) The type, pH value and concentration of operating buffer solution, and additives [used to increase the solubility of solute and/or control solute Dissociation degree ， Chiral resolution The selection of [, etc.] also has a great impact on the determination results. It should be prepared according to the provisions under each variety, and adjusted and optimized according to the results of the preliminary test.

(4) To be tested Test solution Put the bottle in the sampler and set the operating parameters, such as injection pressure（ Electric injection Voltage), injection time, positive or negative end injection, operating voltage or current detector Parameters, etc., start the test. According to the preliminary electrophoresis Spectrogram Adjust instrument parameters and operating buffer to obtain optimized results. Then the optimized conditions are used for formal testing.

(5) After the test, flush the capillary with water, and keep both ends of the capillary in water. If the capillary is not used for a long time, dry it with nitrogen, and finally shut down the machine.

(6) Quantitative determination to adopt Internal standard method Is appropriate. When the pressure or pressure reduction method is used for sample injection, the viscosity of the test solution will affect the injection volume, and attention should be paid to keeping the sample solution and Reference solution Consistency of viscosity; When the electrokinetic method is used for sample injection, the tested component is subject to electrical discrimination and solution ionic strength Will affect the components to be tested Migration volume , also pay attention to its impact.^[2]

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