Capillary Electrophoresis (CE)
Introduction:
Capillary electrophoresis (CE), also known as high performance capillary electrophoresis (HPCE), is a new type of liquid phase separation technology driven by a high-voltage DC electric field with capillary separation channels.
Capillary electrophoresis actually involves electrophoresis, chromatography, and its crossover, which allows analytical chemistry to go from nanoliter level to nanoliter level and enables single cell analysis and even single-molecule analysis. The long-term separation of biological macromolecules such as proteins has also taken a new turn for the better.
Basic theory:
1 double layer
Electric double layer refers to the surface between the two phases separated by a relatively fixed and free two-component ions and the surface of the ionosphere, all submerged in the liquid interface will produce double layer. In capillary electrophoresis, either the surface of charged particles or the surface of the capillary tube has an electrical double layer.
2 Zeta potential
In a dielectric solution, any charged particle can be considered as part of a double-layer system in which the ion's own charge is neutralized by the charged ion of the opposite sign, some of which are irreversibly adsorbed onto the ion, Others are free in the vicinity and diffuse into the dielectric for ion exchange. The "fixed" ion has a tangent plane and the potential between the nearest ion and its nearest ion is called the Zeta potential of the ion.
Quartz-based capillaries are the most commonly used capillaries for capillary electrophoresis. The inner surface of the tube is negatively charged at pH> 3. When it comes in contact with the solution, it forms two internal ions and free ions. The two ion composition and the surface of the charge-coupled layer of ions, namely the electric double layer, the first part of which is also known as the Stern layer. The second layer is the diffusion layer. The charge density of the free part of the diffusion layer decreases sharply with increasing distance from the surface. The potential between the boundary layers at the start of the Stern layer and the free part of the electric double layer is referred to as the Zeta potential of the tube wall. Typical values are generally between 0 and 100 mV. The value of the Zeta potential decays exponentially with increasing distance, and the distance it takes to decay an exponential unit is called the thickness (δ) of the electrical double layer. The zeta potential of the molten silicon surface is related to the number of charges on its surface and the thickness of the electric double layer, which in turn is influenced by the nature of the ions, the pH of the buffer solution, the balance between the cation and the silicon melt surface in the buffer solution, and the like.
3 Mobility, Absolute Mobility and Effective Mobility
Charged particles in the DC electric field in a certain medium (solvent) in the directional motion occurs called electrophoresis. Electrophoresis unit speed under the field known as the mobility. Mobility measured in an infinite dilution solution (extrapolated from dilute solution data) is called absolute mobility.
Charged ions in the electric field in addition to the role of electric field force, but also by the role of solvent resistance. After a certain period of time, the effect of the two forces will reach a balance, this time for uniform ion motion, electrophoresis into the steady state. The actual activity of the different solutions, especially the different pH, so the dissociation of the sample molecules of different charge will change, then the mobility can be called effective electrophoretic mobility. In general, the more charged ion, the greater the dissociation, the smaller the volume, the faster the electrophoresis.
4 electroosmosis, electroosmotic flow and apparent mobility
Electro-osmosis is another important motivation for sample migration. The so-called electrosurgery refers to the directional flow of solvent in the capillary due to the axial DC electric field. Electrosmosis is caused by localized charges. Local charge refers to firmly attached to the tube wall, under the action of the electric field can not migrate ions or charged groups. The so-called double electric layer is formed by the attraction of the counter-ions to the counter ions in the solution, so that the solvent moves in an overall direction under the effect of the electric field (and the collision) to form an electroosmotic flow (the electroosmotic flow in the capillary is a flat plug) .
Under capillary zone electrophoresis, the electroosmotic flow flows from the anode to the cathode. The size of the electroosmotic flow is affected by the Zeta potential, the thickness of the electric double layer and the viscosity of the medium. In general, the larger the zeta potential is, the thinner the electric double layer is and the smaller the viscosity is, the larger the electroosmotic flow is.
In capillary electrophoresis, the migration of sample molecules is a combination of effective electrophoretic mobility and electroosmotic mobility, when the mobility is called apparent mobility. In most aqueous solutions, the surface of quartz (or glass) capillaries dissociate by silanolysis to produce a negative localized charge, creating an electroosmotic flow to the negative electrode. Electroosmotic velocity in capillaries can be an order of magnitude faster than electrophoresis, so that the sample components can migrate in the same direction. Positive ions move in the same direction as the electroosmosis, so it should flow out first. Neutral molecules and electroosmotic flow at the same speed, with the infiltration and line. Anion due to its movement direction and electro-osmosis, out after the neutral particles. The relationship between electroosmotic flow and pH is very close. Electrosmosis is affected by the Zeta potential, which is determined by the charge on the surface of the capillary wall, which in turn is affected by the pH of the buffer solution. Therefore, the value of the electroosmotic flow is a function of the pH of the buffer solution. Generally, as the pH increases Increases, to neutral or alkaline, its value will become very large. In addition, any factors that affect the dissociation of the tube wall, such as the capillary wash process, the composition of the electrophoresis buffer, viscosity, temperature, etc., can affect or alter the electroosmotic flow. Electromagnetic fields and many substances that interact with the surface of the capillary, such as surfactants and proteins, can have a major impact on electroosmotic flow.
Electroosmosis plays an important role in electrophoretic separation and is an electromotive phenomenon that accompanies electrophoresis. In most cases, the electroosmotic flow rate is 5-7 times faster than electrophoresis. Therefore, the capillary electrophoresis (CE) in the use of electroosmotic flow can be positive and negative ions and neutral molecules together to produce a differential migration in one direction, in a CE operation at the same time to complete separation of positive and negative ions. Since the size and direction of the electroosmotic flow can affect the efficiency, selectivity, and resolution of the CE separation, it becomes an important parameter for optimizing the separation conditions. Small changes in the electroosmotic flow will seriously affect the reproducibility of CE separation (migration time and peak area). Therefore, the control of electroosmotic flow is an important task in CE. The methods used to control the electroosmotic flow are mainly to change the composition and concentration of the buffer solution; to change the pH value of the buffer solution; to add the additives; to modify the inner wall of the capillary tube - physical or chemical coating and dynamic deactivation; to apply a radial electric field; to change the temperature Wait.
Neutrals can be used as a marker for determining electroosmosis. For example, dimethylformamide (DMF), dimethylsulfoxide (DMSO), β-naphthol, acetone, methanol and ethanol, etc., can be used as an electro-osmotic marker.
classification:
1. Separation mode
Capillary electrophoresis can be divided into different types of capillary electrophoresis and capillary electrophoresis according to the different separation modes, providing a variety of separation options for sample separation, which is very important for the separation and analysis of complex samples.
Single capillary, single packed tube CGE, array capillary electrophoresis, chip capillary electrophoresis, capillary electrophoresis / mass spectrometry, capillary electrophoresis / nuclear magnetic resonance, capillary electrophoresis / laser-induced fluorescence,
2 mode of operation
Capillary electrophoresis can be subdivided into manual, semi-automatic and fully automatic capillary electrophoresis according to the operation mode.
3. Separate channel shape
According to the separation channel shape is divided into round, flat, square capillary electrophoresis.
4. Buffer medium
CE can be divided into aqueous capillary electrophoresis and non-aqueous capillary electrophoresis (NACE) depending on the medium used to formulate the buffer. NACE is an organic solvent-based electrophoresis buffer solution instead of water as a buffer solution to increase the solubility of hydrophobic substances, especially for insoluble in the aqueous solution can not be CE separation of substances or similar in aqueous solution difficult Isolated homologues broaden the field of CE analysis.
Features:
Capillary electrophoresis usually uses a flexible (polyimide) coated fused silica tube with an internal diameter of 25-100 μm. The standard capillary has an outer diameter of 375 μm and some tubes have an outer diameter of 160 μm. Capillary features are: small volume (a 100 cm × 75 μm tube volume of only 4.4 μL); side / cross-sectional area ratio, and therefore heat fast, can withstand high electric fields (100-1000 V / cm); free to use Solution, gel, etc. as support medium; in the solution medium can produce a planar shape of electroosmotic flow.
As a result, capillary electrophoresis can have the following advantages:
(1) The number of high-performance trays is between 105 and 106 pieces / m. When using CGE, the number of trays can reach more than 107 pieces / m;
(2) quick general in ten minutes to complete the separation;
(3) Sample volume required for microinjection is nL level;
(4) multi-mode can be selected according to need different separation mode and only one instrument;
(5) Economical experiments consume but a few milliliters of buffer solution, the maintenance cost is very low;
(6) Automatic CE is a high degree of automation of the separation method.
The disadvantage of capillary electrophoresis is:
(1) poor preparation due to the small injection volume;
(2) due to the small capillary diameter, the optical path is too short, with some detection methods (such as UV absorption spectroscopy), the sensitivity is low;
(3) Electro-osmosis will change due to the sample composition, thereby affecting the separation of reproducibility.
Instrument system:
The basic structure of a capillary electrophoresis system includes an injection system, two buffer tanks, a high voltage power supply, a detector, a control system, and a data processing system.
Due to the limitations of the capillary diameter, the detection signal is the most prominent issue for the CE system. UV-Vis method (UV) is a commonly used detection method of CE, but is limited by instruments, single wavelength and other factors. Currently the most widely used is a diode array (PDA) detector. Conventional detectors include highly sensitive laser photothermal (LIP) and fluorescence (FL) detectors. In recent years, Laser Induced Fluorescence (LIF), well-selective amperometry (EC), well-versatile conductivity (CD) aids and mass spectrometry (MS) structures with structural information have also been produced in practical applications Species detector. To date, other methods of detection have been used in conjunction with CE except for inductively coupled plasma (ICP) and infrared (IR) techniques and are mostly commercialized. CE should be used according to the characteristics of the analysis of the material, select the appropriate separation mode and detector to avoid weaknesses, get the best analysis.
Affect the separation factor:
1 buffer
The choice of buffer reagent is mainly determined by the desired pH, and the separation effects of different buffer reagents are not the same at the same pH, and some may be very different. CE commonly used buffer reagents: phosphate, borax or boric acid, acetate and so on.
Buffer salt concentration directly affects the ionic strength of the electrophoretic medium, thus affecting the Zeta potential, and Zeta potential changes will affect the electroosmotic flow. The increase of the buffer concentration, the increase of the ionic strength, the reduction of the electric double layer thickness, the decrease of Zeta potential and the decrease of the electroosmotic flow and the longer residence time of the sample in the capillary facilitate the separation of components with shorter migration time and higher efficiency of analysis. Meanwhile, with the increase of electrolyte concentration, the conductivity of the electrolyte will be much higher than the conductivity of the sample solution to make the sample have a stacking effect on the capillary column to enhance the sample enrichment and increase the sample capacity, thereby improving the analysis sensitivity . However, when the electrolyte concentration is too high and the current is increased, the sample component is bee-shaped due to the thermal effect, and the separation effect is rather poor. In addition, the ions can also affect the electroosmosis through the interaction with the tube wall and influencing the viscosity, dielectric constant, etc. of the solution, and the ionic strength is too high or too low to improve the separation efficiency.
2 pH
The pH of the buffer system, depending on the nature of the sample and the efficiency of the separation, is a key factor in determining the success or failure of a separation. Different samples require different conditions of pH separation, pH control of the buffer system, generally only change the size of the electroosmotic flow. pH can affect the dissociation ability of the sample, the sample in a highly polar medium dissociation increases, the electrophoresis rate also increases, thus affecting separation selectivity and separation sensitivity. pH also affects the degree of protonation of the silanol groups on the inner wall of the capillary and the chemical stability of the solute. The pH is between 4 and 10, and the dissociation of the silanol group increases with increasing pH. The electroosmotic flow also increases high. Therefore, pH is a factor that can not be ignored when the separation conditions are optimized.
3 separate the voltage
In CE, the separation of voltage is also an important parameter to control electroosmosis. High voltage is a precondition to achieve fast and efficient CE. With the increase of voltage, sample migration increases, analysis time shortens, but Joule heating in capillary increases, baseline stability decreases and sensitivity decreases. The lower the separation voltage, the better the separation. , The analysis time is prolonged and the peak shape is widened, leading to the decrease of separation efficiency. Therefore, a relatively high separation voltage will increase the resolution and shorten the analysis time, but the voltage is too high will make the band widen and reduce the separation efficiency. When the electrolyte concentrations are the same, current values and Joule heat in non-aqueous media are much smaller than those in aqueous media, thus allowing the use of higher separation voltages in non-aqueous media.
4 temperature
Temperature affects separation reproducibility and separation efficiency, and temperature control can regulate the size of the electroosmotic flow. With the increase of temperature, the viscosity of the buffer decreases, the ability of silica wall to dissociate is enhanced, the electroosmotic speed is increased, the analysis time is shortened and the analysis efficiency is improved. However, when the temperature is too high, the temperature difference inside the capillary column will increase, the Joule heating effect will be enhanced, the column efficiency will decrease, and the separation efficiency will also decrease.
5 additives
Additives such as neutral salts, zwitterions, surfactants and organic solvents to the electrolyte solution cause significant changes in the electroosmotic flow. Surfactants are commonly used as modifiers for electroosmotic flow. The size and direction of the electroosmotic flow are controlled by varying the concentration, but when the concentration of surfactant is above the critical micelle concentration, micelles form. Adding organic solvents will reduce the ionic strength, Zeta potential increases, the solution viscosity decreases, changing the surface charge distribution on the wall, so that electroosmotic flow decreases. In electrophoresis, buffers are usually formulated with water, but often with water-organic solvents can improve resolution or separation selectivity.
6 injection
There are two general CE injection methods: hydrodynamics and electromigration injection. Electromigration injection is under the action of an electric field, depending on the sample ions electromigration and (or) electroosmotic flow of the sample injection, it will produce the phenomenon of electric discrimination, will reduce the accuracy and reliability of the analysis, but this method is especially suitable for viscosity Large buffer and CGE conditions. Fluid mechanics sampling is a universal method, which can be achieved by siphon, pressurization at the injection end, or evacuation at the detector side. However, the selectivity is poor and the sample and its background are introduced into the capillary simultaneously, which may have an impact on the subsequent separation. Through the injection time can also improve the separation effect, injection time is too short, the peak area is too small, the analysis error. Injection time is too large, the sample is overloaded, the injection zone diffusion, will cause overlap between the peaks, and improve the separation voltage, the separation effect worsened.
In addition, capillary electrophoresis has many advantages such as high separation performance and low reagent consumption, but its sensitivity of routine analysis can not meet the requirement of trace analysis, which limits its application and promotion. Sample preparation techniques can increase sample throughput or pre-enrich trace amounts of analytes, removing sample matrices and using them with capillary electrophoresis technology not only increases the sensitivity of the assay but also eliminates most of the potential matrix interferences, Is an ideal enrichment separation detection technology. Commonly used with CE-flow injection technology, solid-phase extraction-CE combined with solid-phase microextraction-CE technology, liquid microextraction-CE combined technology, microdialysis-CE combined with membrane extraction -CE combined technology.
application:
CE has a wide range of separation modes (a wide range of separation media and principles) and is therefore versatile enough to be used in a wide range of applications. CEs (usually volatile and insoluble) that can be formulated as solutions or suspensions are CE For separation and analysis, as small as inorganic ions, large biological macromolecules and supramolecules, and even the entire cell can be separated and tested. It is widely used in the fields of life science, medical science, clinical medicine, molecular biology, forensic and detection, chemical, environmental, customs, agronomy, production process monitoring, product quality control and single cell and single molecule analysis.
Capillary electrophoresis (CE), also known as high performance capillary electrophoresis (HPCE), is a new type of liquid phase separation technology driven by a high-voltage DC electric field with capillary separation channels.
Capillary electrophoresis actually involves electrophoresis, chromatography, and its crossover, which allows analytical chemistry to go from nanoliter level to nanoliter level and enables single cell analysis and even single-molecule analysis. The long-term separation of biological macromolecules such as proteins has also taken a new turn for the better.
Basic theory:
1 double layer
Electric double layer refers to the surface between the two phases separated by a relatively fixed and free two-component ions and the surface of the ionosphere, all submerged in the liquid interface will produce double layer. In capillary electrophoresis, either the surface of charged particles or the surface of the capillary tube has an electrical double layer.
2 Zeta potential
In a dielectric solution, any charged particle can be considered as part of a double-layer system in which the ion's own charge is neutralized by the charged ion of the opposite sign, some of which are irreversibly adsorbed onto the ion, Others are free in the vicinity and diffuse into the dielectric for ion exchange. The "fixed" ion has a tangent plane and the potential between the nearest ion and its nearest ion is called the Zeta potential of the ion.
Quartz-based capillaries are the most commonly used capillaries for capillary electrophoresis. The inner surface of the tube is negatively charged at pH> 3. When it comes in contact with the solution, it forms two internal ions and free ions. The two ion composition and the surface of the charge-coupled layer of ions, namely the electric double layer, the first part of which is also known as the Stern layer. The second layer is the diffusion layer. The charge density of the free part of the diffusion layer decreases sharply with increasing distance from the surface. The potential between the boundary layers at the start of the Stern layer and the free part of the electric double layer is referred to as the Zeta potential of the tube wall. Typical values are generally between 0 and 100 mV. The value of the Zeta potential decays exponentially with increasing distance, and the distance it takes to decay an exponential unit is called the thickness (δ) of the electrical double layer. The zeta potential of the molten silicon surface is related to the number of charges on its surface and the thickness of the electric double layer, which in turn is influenced by the nature of the ions, the pH of the buffer solution, the balance between the cation and the silicon melt surface in the buffer solution, and the like.
3 Mobility, Absolute Mobility and Effective Mobility
Charged particles in the DC electric field in a certain medium (solvent) in the directional motion occurs called electrophoresis. Electrophoresis unit speed under the field known as the mobility. Mobility measured in an infinite dilution solution (extrapolated from dilute solution data) is called absolute mobility.
Charged ions in the electric field in addition to the role of electric field force, but also by the role of solvent resistance. After a certain period of time, the effect of the two forces will reach a balance, this time for uniform ion motion, electrophoresis into the steady state. The actual activity of the different solutions, especially the different pH, so the dissociation of the sample molecules of different charge will change, then the mobility can be called effective electrophoretic mobility. In general, the more charged ion, the greater the dissociation, the smaller the volume, the faster the electrophoresis.
4 electroosmosis, electroosmotic flow and apparent mobility
Electro-osmosis is another important motivation for sample migration. The so-called electrosurgery refers to the directional flow of solvent in the capillary due to the axial DC electric field. Electrosmosis is caused by localized charges. Local charge refers to firmly attached to the tube wall, under the action of the electric field can not migrate ions or charged groups. The so-called double electric layer is formed by the attraction of the counter-ions to the counter ions in the solution, so that the solvent moves in an overall direction under the effect of the electric field (and the collision) to form an electroosmotic flow (the electroosmotic flow in the capillary is a flat plug) .
Under capillary zone electrophoresis, the electroosmotic flow flows from the anode to the cathode. The size of the electroosmotic flow is affected by the Zeta potential, the thickness of the electric double layer and the viscosity of the medium. In general, the larger the zeta potential is, the thinner the electric double layer is and the smaller the viscosity is, the larger the electroosmotic flow is.
In capillary electrophoresis, the migration of sample molecules is a combination of effective electrophoretic mobility and electroosmotic mobility, when the mobility is called apparent mobility. In most aqueous solutions, the surface of quartz (or glass) capillaries dissociate by silanolysis to produce a negative localized charge, creating an electroosmotic flow to the negative electrode. Electroosmotic velocity in capillaries can be an order of magnitude faster than electrophoresis, so that the sample components can migrate in the same direction. Positive ions move in the same direction as the electroosmosis, so it should flow out first. Neutral molecules and electroosmotic flow at the same speed, with the infiltration and line. Anion due to its movement direction and electro-osmosis, out after the neutral particles. The relationship between electroosmotic flow and pH is very close. Electrosmosis is affected by the Zeta potential, which is determined by the charge on the surface of the capillary wall, which in turn is affected by the pH of the buffer solution. Therefore, the value of the electroosmotic flow is a function of the pH of the buffer solution. Generally, as the pH increases Increases, to neutral or alkaline, its value will become very large. In addition, any factors that affect the dissociation of the tube wall, such as the capillary wash process, the composition of the electrophoresis buffer, viscosity, temperature, etc., can affect or alter the electroosmotic flow. Electromagnetic fields and many substances that interact with the surface of the capillary, such as surfactants and proteins, can have a major impact on electroosmotic flow.
Electroosmosis plays an important role in electrophoretic separation and is an electromotive phenomenon that accompanies electrophoresis. In most cases, the electroosmotic flow rate is 5-7 times faster than electrophoresis. Therefore, the capillary electrophoresis (CE) in the use of electroosmotic flow can be positive and negative ions and neutral molecules together to produce a differential migration in one direction, in a CE operation at the same time to complete separation of positive and negative ions. Since the size and direction of the electroosmotic flow can affect the efficiency, selectivity, and resolution of the CE separation, it becomes an important parameter for optimizing the separation conditions. Small changes in the electroosmotic flow will seriously affect the reproducibility of CE separation (migration time and peak area). Therefore, the control of electroosmotic flow is an important task in CE. The methods used to control the electroosmotic flow are mainly to change the composition and concentration of the buffer solution; to change the pH value of the buffer solution; to add the additives; to modify the inner wall of the capillary tube - physical or chemical coating and dynamic deactivation; to apply a radial electric field; to change the temperature Wait.
Neutrals can be used as a marker for determining electroosmosis. For example, dimethylformamide (DMF), dimethylsulfoxide (DMSO), β-naphthol, acetone, methanol and ethanol, etc., can be used as an electro-osmotic marker.
classification:
1. Separation mode
Capillary electrophoresis can be divided into different types of capillary electrophoresis and capillary electrophoresis according to the different separation modes, providing a variety of separation options for sample separation, which is very important for the separation and analysis of complex samples.
Single capillary, single packed tube CGE, array capillary electrophoresis, chip capillary electrophoresis, capillary electrophoresis / mass spectrometry, capillary electrophoresis / nuclear magnetic resonance, capillary electrophoresis / laser-induced fluorescence,
2 mode of operation
Capillary electrophoresis can be subdivided into manual, semi-automatic and fully automatic capillary electrophoresis according to the operation mode.
3. Separate channel shape
According to the separation channel shape is divided into round, flat, square capillary electrophoresis.
4. Buffer medium
CE can be divided into aqueous capillary electrophoresis and non-aqueous capillary electrophoresis (NACE) depending on the medium used to formulate the buffer. NACE is an organic solvent-based electrophoresis buffer solution instead of water as a buffer solution to increase the solubility of hydrophobic substances, especially for insoluble in the aqueous solution can not be CE separation of substances or similar in aqueous solution difficult Isolated homologues broaden the field of CE analysis.
Features:
Capillary electrophoresis usually uses a flexible (polyimide) coated fused silica tube with an internal diameter of 25-100 μm. The standard capillary has an outer diameter of 375 μm and some tubes have an outer diameter of 160 μm. Capillary features are: small volume (a 100 cm × 75 μm tube volume of only 4.4 μL); side / cross-sectional area ratio, and therefore heat fast, can withstand high electric fields (100-1000 V / cm); free to use Solution, gel, etc. as support medium; in the solution medium can produce a planar shape of electroosmotic flow.
As a result, capillary electrophoresis can have the following advantages:
(1) The number of high-performance trays is between 105 and 106 pieces / m. When using CGE, the number of trays can reach more than 107 pieces / m;
(2) quick general in ten minutes to complete the separation;
(3) Sample volume required for microinjection is nL level;
(4) multi-mode can be selected according to need different separation mode and only one instrument;
(5) Economical experiments consume but a few milliliters of buffer solution, the maintenance cost is very low;
(6) Automatic CE is a high degree of automation of the separation method.
The disadvantage of capillary electrophoresis is:
(1) poor preparation due to the small injection volume;
(2) due to the small capillary diameter, the optical path is too short, with some detection methods (such as UV absorption spectroscopy), the sensitivity is low;
(3) Electro-osmosis will change due to the sample composition, thereby affecting the separation of reproducibility.
Instrument system:
The basic structure of a capillary electrophoresis system includes an injection system, two buffer tanks, a high voltage power supply, a detector, a control system, and a data processing system.
Due to the limitations of the capillary diameter, the detection signal is the most prominent issue for the CE system. UV-Vis method (UV) is a commonly used detection method of CE, but is limited by instruments, single wavelength and other factors. Currently the most widely used is a diode array (PDA) detector. Conventional detectors include highly sensitive laser photothermal (LIP) and fluorescence (FL) detectors. In recent years, Laser Induced Fluorescence (LIF), well-selective amperometry (EC), well-versatile conductivity (CD) aids and mass spectrometry (MS) structures with structural information have also been produced in practical applications Species detector. To date, other methods of detection have been used in conjunction with CE except for inductively coupled plasma (ICP) and infrared (IR) techniques and are mostly commercialized. CE should be used according to the characteristics of the analysis of the material, select the appropriate separation mode and detector to avoid weaknesses, get the best analysis.
Affect the separation factor:
1 buffer
The choice of buffer reagent is mainly determined by the desired pH, and the separation effects of different buffer reagents are not the same at the same pH, and some may be very different. CE commonly used buffer reagents: phosphate, borax or boric acid, acetate and so on.
Buffer salt concentration directly affects the ionic strength of the electrophoretic medium, thus affecting the Zeta potential, and Zeta potential changes will affect the electroosmotic flow. The increase of the buffer concentration, the increase of the ionic strength, the reduction of the electric double layer thickness, the decrease of Zeta potential and the decrease of the electroosmotic flow and the longer residence time of the sample in the capillary facilitate the separation of components with shorter migration time and higher efficiency of analysis. Meanwhile, with the increase of electrolyte concentration, the conductivity of the electrolyte will be much higher than the conductivity of the sample solution to make the sample have a stacking effect on the capillary column to enhance the sample enrichment and increase the sample capacity, thereby improving the analysis sensitivity . However, when the electrolyte concentration is too high and the current is increased, the sample component is bee-shaped due to the thermal effect, and the separation effect is rather poor. In addition, the ions can also affect the electroosmosis through the interaction with the tube wall and influencing the viscosity, dielectric constant, etc. of the solution, and the ionic strength is too high or too low to improve the separation efficiency.
2 pH
The pH of the buffer system, depending on the nature of the sample and the efficiency of the separation, is a key factor in determining the success or failure of a separation. Different samples require different conditions of pH separation, pH control of the buffer system, generally only change the size of the electroosmotic flow. pH can affect the dissociation ability of the sample, the sample in a highly polar medium dissociation increases, the electrophoresis rate also increases, thus affecting separation selectivity and separation sensitivity. pH also affects the degree of protonation of the silanol groups on the inner wall of the capillary and the chemical stability of the solute. The pH is between 4 and 10, and the dissociation of the silanol group increases with increasing pH. The electroosmotic flow also increases high. Therefore, pH is a factor that can not be ignored when the separation conditions are optimized.
3 separate the voltage
In CE, the separation of voltage is also an important parameter to control electroosmosis. High voltage is a precondition to achieve fast and efficient CE. With the increase of voltage, sample migration increases, analysis time shortens, but Joule heating in capillary increases, baseline stability decreases and sensitivity decreases. The lower the separation voltage, the better the separation. , The analysis time is prolonged and the peak shape is widened, leading to the decrease of separation efficiency. Therefore, a relatively high separation voltage will increase the resolution and shorten the analysis time, but the voltage is too high will make the band widen and reduce the separation efficiency. When the electrolyte concentrations are the same, current values and Joule heat in non-aqueous media are much smaller than those in aqueous media, thus allowing the use of higher separation voltages in non-aqueous media.
4 temperature
Temperature affects separation reproducibility and separation efficiency, and temperature control can regulate the size of the electroosmotic flow. With the increase of temperature, the viscosity of the buffer decreases, the ability of silica wall to dissociate is enhanced, the electroosmotic speed is increased, the analysis time is shortened and the analysis efficiency is improved. However, when the temperature is too high, the temperature difference inside the capillary column will increase, the Joule heating effect will be enhanced, the column efficiency will decrease, and the separation efficiency will also decrease.
5 additives
Additives such as neutral salts, zwitterions, surfactants and organic solvents to the electrolyte solution cause significant changes in the electroosmotic flow. Surfactants are commonly used as modifiers for electroosmotic flow. The size and direction of the electroosmotic flow are controlled by varying the concentration, but when the concentration of surfactant is above the critical micelle concentration, micelles form. Adding organic solvents will reduce the ionic strength, Zeta potential increases, the solution viscosity decreases, changing the surface charge distribution on the wall, so that electroosmotic flow decreases. In electrophoresis, buffers are usually formulated with water, but often with water-organic solvents can improve resolution or separation selectivity.
6 injection
There are two general CE injection methods: hydrodynamics and electromigration injection. Electromigration injection is under the action of an electric field, depending on the sample ions electromigration and (or) electroosmotic flow of the sample injection, it will produce the phenomenon of electric discrimination, will reduce the accuracy and reliability of the analysis, but this method is especially suitable for viscosity Large buffer and CGE conditions. Fluid mechanics sampling is a universal method, which can be achieved by siphon, pressurization at the injection end, or evacuation at the detector side. However, the selectivity is poor and the sample and its background are introduced into the capillary simultaneously, which may have an impact on the subsequent separation. Through the injection time can also improve the separation effect, injection time is too short, the peak area is too small, the analysis error. Injection time is too large, the sample is overloaded, the injection zone diffusion, will cause overlap between the peaks, and improve the separation voltage, the separation effect worsened.
In addition, capillary electrophoresis has many advantages such as high separation performance and low reagent consumption, but its sensitivity of routine analysis can not meet the requirement of trace analysis, which limits its application and promotion. Sample preparation techniques can increase sample throughput or pre-enrich trace amounts of analytes, removing sample matrices and using them with capillary electrophoresis technology not only increases the sensitivity of the assay but also eliminates most of the potential matrix interferences, Is an ideal enrichment separation detection technology. Commonly used with CE-flow injection technology, solid-phase extraction-CE combined with solid-phase microextraction-CE technology, liquid microextraction-CE combined technology, microdialysis-CE combined with membrane extraction -CE combined technology.
application:
CE has a wide range of separation modes (a wide range of separation media and principles) and is therefore versatile enough to be used in a wide range of applications. CEs (usually volatile and insoluble) that can be formulated as solutions or suspensions are CE For separation and analysis, as small as inorganic ions, large biological macromolecules and supramolecules, and even the entire cell can be separated and tested. It is widely used in the fields of life science, medical science, clinical medicine, molecular biology, forensic and detection, chemical, environmental, customs, agronomy, production process monitoring, product quality control and single cell and single molecule analysis.
