Capillary electrophoresis (CE), also known as capillary zone electrophoresis (CZE), can be used to separate ionic species by their charge and frictional forces. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field. Introduced in the 1960s, the technique of capillary electrophoresis (CE) was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte. It has been suggested that Electrophoretic mobility be merged into this article or section. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... The term chiral (pronounced ) is used to describe an object which is non-superimposable on its mirror image. ... Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid, ribonucleic acid, or protein molecules through an electric charge. ... Two-dimensional gel electrophoresis, commonly abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. ... It has been suggested that Electrophoretic mobility be merged into this article or section. ... En [ [ ciencia ] ] y [ [ ingeniería ] ], los conductores son los materiales de los cuales contenga las cargas movibles [ [ electricidad ] ]. Cuando una diferencia potencial eléctrica se impresiona a través de puntos separados en un conductor, las cargas móviles dentro del conductor se fuerzan para moverse, y una corriente el... For other uses, see Liquid (disambiguation). ... In physics, the space surrounding an electric charge or in the presence of a time-varying magnetic field has a property called an electric field. ... The 1960s decade refers to the years from 1960 to 1969. ... Blood flows from digestive system heart to arteries, which narrow into arterioles, and then narrow further still into capillaries. ... An electrolyte is a substance containing free ions that behaves as an electrically conductive medium. ...

Instrumentation

The instrumentation needed to perform capillary electrophoresis is relatively simple. A basic schematic of a capillary electrophoresis system is shown in figure 1. The system's main components are a sample vial, source and destination vials, a capillary, electrodes, a high-voltage power supply, a detector, and a data output and handling device. The source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution. To introduce the sample, the capillary inlet is placed into a vial containing the sample and then returned to the source vial (sample is introduced into the capillary via capillary action, pressure, or siphoning). The migration of the analytes is then initiated by an electric field that is applied between the source and destination vials and is supplied to the electrodes by the high-voltage power supply. It is important to note that all ions, positive or negative, are pulled through the capillary in the same direction by electroosmotic flow, as will be explained. The analytes separate as they migrate due to their electrophoretic mobility, as will be explained, and are detected near the outlet end of the capillary. The output of the detector is sent to a data output and handling device such as an integrator or computer. The data is then displayed as an electropherogram, which reports detector response as a function of time. Separated chemical compounds appear as peaks with different retention times in an electropherogram.^[1] A schematic of the Washington Metro. ... An electrode is an electrical conductor used to make contact with a metallic part of a circuit (e. ... International safety symbol Caution, risk of electric shock (ISO 3864), colloquially known as high voltage symbol. ... A wall wart style variable DC power supply with its cover removed. ... Capillary action, capillarity, or capillary motion is the ability of a substance (the standard reference is to a tube in plants but can be seen readily with porous paper) to draw a substance up against gravity. ... An integrator is a device to perform the mathematical operation known as integration, a fundamental operation in calculus. ... This article is about the machine. ... Look up time in Wiktionary, the free dictionary. ... A chemical compound is a chemical substance of two or more different chemically bonded chemical elements, with a fixed ratio determining the composition. ...

Figure 1: Diagram of capillary electrophoresis system

This image has been released into the public domain by the copyright holder, its copyright has expired, or it is ineligible for copyright. ...

Detection

Separation by capillary electrophoresis can be detected by several detection devices. The majority of commercial systems use UV or UV-Vis absorbance as their primary mode of detection. In these systems, a section of the capillary itself is used as the detection cell. The use of on-tube detection enables detection of separated analytes with no loss of resolution. In general, capillaries used in capillary electrophoresis are coated with a polymer for increased stability. The portion of the capillary used for UV detection, however, must be optically transparent. Bare capillaries can break relatively easily and, as a result, capillaries with transparent coatings are available to increase the stability of the cell window. The path length of the detection cell in capillary electrophoresis (~ 50 micrometers) is far less than that of a traditional UV cell (~ 1 cm). According to the Beer-Lambert law, the sensitivity of the detector is proportional to the path length of the cell. To improve the sensitivity, the path length can be increased, though this results in a loss of resolution. The capillary tube itself can be expanded at the detection point, creating a "bubble cell" with a longer path length or additional tubing can be added at the detection point as shown in figure 2. Both of these methods, however, will decrease the resolution of the separation.^[2] Note: Ultraviolet is also the name of a 1998 UK television miniseries about vampires. ... In spectroscopy, the absorbance A is defined as , where I is the intensity of light at a specified wavelength λ that has passed through a sample (transmitted light intensity) and is the intensity of the light before it enters the sample (or incident light intensity). ... A polymer (from Greek: πολυ, polu, many; and μέρος, meros, part) is a substance composed of molecules with large molecular mass composed of repeating structural units, or monomers, connected by covalent chemical bonds. ... In chemistry, the path length is defined as the distance that light (UV/VIS) travels through a sample in an analytical cell. ... A micrometre (American spelling: micrometer, symbol µm) is an SI unit of length equal to one millionth of a metre, or about a tenth of the diameter of a droplet of mist or fog. ... This article is about the unit of length. ... In optics, the Beer-Lambert law, also known as Beers law or the Lambert-Beer law or the Beer-Lambert-Bouguer law is an empirical relationship that relates the absorption of light to the properties of the material through which the light is traveling. ...

Figure 2: Techniques for increasing the pathlength of the capillary: a.) a bubble cell and b.) a z-cell (additional tubing).^[1]

Fluorescence detection can also be used in capillary electrophoresis for samples that naturally fluoresce or are chemically modified to contain fluorescent tags. This mode of detection offers high sensitivity and improved selectivity for these samples, but cannot be utilized for samples that do not fluoresce. The set-up for fluorescence detection in a capillary electrophoresis system can be complicated. The method requires that the light beam be focused on the capillary, which can be difficult for many light sources.^[2] Laser-induced fluorescence has been used in CE systems with detection limits as low as 10^-18 to 10^-21 mol. The sensitivity of the technique is attributed to the high intensity of the incident light and the ability to accurately focus the light on the capillary.^[1] This image has been released into the public domain by the copyright holder, its copyright has expired, or it is ineligible for copyright. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ... In molecular biology and biotechnology, a fluorescent tag is a part of a molecule that researchers have attached chemically to aid in detection of the molecule to which it has been attached. ... For other uses, see Laser (disambiguation). ... In physics, intensity is a measure of the time-averaged energy flux. ...

In order to obtain the identity of sample components, capillary electrophoresis can be directly coupled with mass spectrometers or Surface-Enhanced Raman Spectroscopy (SERS). In most systems, the capillary outlet is introduced into an ion source that utilizes electrospray ionization (ESI). The resulting ions are then analyzed by the mass spectrometer. This set-up requires volatile buffer solutions, which will affect the range of separation modes that can be employed and the degree of resolution that can be achieved.^[2] The measurement and analysis are mostly done with a specialized gel analysis software. Mass spectrometry is a technique for separating ions by their mass-to-charge (m/z) ratios. ... SERS can mean:- Selective En bloc Redevelopment Scheme, a housing strategy in Singapore. ... Electrospray (nanoSpray) ionization source Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions. ... The ability of a liquid to evaporate quickly and at relatively low temperatures. ...

For CE-SERS, capillary electrophoresis eluants can be deposited onto a SERS-active substrate. Analyte retention times can be translated into spatial distance by moving the SERS-active substrate at a constant rate during capillary electrophoresis. This allows the subsequent spectroscopic technique to be applied to specific eluants for identification with high sensitivity. SERS-active substrates can be chosen that do not interfere with the spectrum of the analytes.^[3]

Modes of separation

The separation of compounds by capillary electrophoresis is dependent on the differential migration of analytes in an applied electric field. The electrophoretic migration velocity (u_p) of an analyte toward the electrode of opposite charge is: This article is about velocity in physics. ...

u_p = μ_pE

where μ_p is the electrophoretic mobility and E is the electric field strength. The electrophoretic mobility is proportional to the ionic charge of a sample and inversely proportional to any frictional forces present in the buffer. When two species in a sample have different charges or experience different frictional forces, they will separate from one another as they migrate through a buffer solution. The frictional forces experienced by an analyte ion depend on the viscosity (η) of the medium and the size and shape of the ion.^[2] Accordingly, the electrophoretic mobility of an analyte at a given pH is given by: This article is about proportionality, the mathematical relation. ... For other uses, see Friction (disambiguation). ... For other uses, see Force (disambiguation). ... For other uses, see Viscosity (disambiguation). ... This article is about the electrically charged particle. ... For other uses, see PH (disambiguation). ...

where z is the net charge of the analyte and r is the Stokes radius of the analyte. The Stokes radius is given by: The Stokes radius or hydrodynamic diameter is the radius of a hydrated atom, but can also be applied to molecules. ...

where k_B is the Boltzmann constant, and T is the temperature, D is the diffusion coefficient. These equations indicate that the electrophoretic mobility of the analyte is proportional to the charge of the analyte and inversely proportional to its radius. The electrophoretic mobility can be determined experimentally from the migration time and the field strength: The Boltzmann constant (k or kB) is the physical constant relating temperature to energy. ... For other uses, see Temperature (disambiguation). ... The Diffusion Coefficient is given by where D is the diffusion coefficient in dimensions of [length2 time-1] T is the temperature in dimensions of Kelvin R is the gas constant in dimensions of [energy temperature-1 parts-1] See also Ficks law of diffusion Categories: Science stubs ... This article is about an authentication, authorization, and accounting protocol. ...

where L is the distance from the inlet to the detection point, t_r is the time required for the analyte to reach the detection point (migration time), V is the applied voltage (field strength), and L_t is the total length of the capillary.^[2] Since only charged ions are affected by the electric field, neutral analytes are poorly separated by capillary electrophoresis.

The velocity of migration of an analyte in capillary electrophoresis will also depend upon the rate of electroosmotic flow (EOF) of the buffer solution. In a typical system, the electroosmotic flow is directed toward the negatively charged cathode so that the buffer flows through the capillary from the source vial to the destination vial. Separated by differing electrophoretic mobilities, analytes migrate toward the electrode of opposite charge.^[1] As a result, negatively charged analytes are attracted to the positively charged anode, counter to the EOF, while positively charged analytes are attracted to the cathode, in agreement with the EOF as depicted in figure 3. Electroosmotic (or Electro-osmotic) flow is the motion of water through very narrow channels generated when an electrical voltage is applied across the channel. ... Diagram of a copper cathode in a Daniells cell. ... Diagram of a zinc anode in a galvanic cell. ... Diagram of a copper cathode in a Daniells cell. ...

Figure 3: Diagram of the separation of charged and neutral analytes (A) according to their respective electrophoretic and electroosmotic flow mobilities

The velocity of the electroosmotic flow, u_o can be written as: This image has been released into the public domain by the copyright holder, its copyright has expired, or it is ineligible for copyright. ...

u_o = μ_oE

where μ_o is the electroosmotic mobility, which is defined as:

where ζ is the zeta potential of the capillary wall, and ε is the relative permittivity of the buffer solution. Experimentally, the electroosmotic mobility can be determined by measuring the retention time of a neutral analyte.^[2] The velocity (u) of an analyte in an electric field can then be defined as: The zeta potential in physical chemistry is measured by electrophoresis. ... The permittivity of a medium is an intensive physical quantity that describes how an electric field affects and is affected by the medium. ...

u_p + u_o = (μ_p + μ_o)E

Since the electroosmotic flow of the buffer solution is generally greater than that of the electrophoretic flow of the analytes, all analytes are carried along with the buffer solution toward the cathode. Even small, triply charged anions can be redirected to the cathode by the relatively powerful EOF of the buffer solution. Negatively charged analytes are retained longer in the capilliary due to their conflicting electrophoretic mobilities.^[1] The order of migration seen by the detector is shown in figure 3: small multiply charged cations migrate quickly and small multiply charged anions are retained strongly.^[2] A cation is an ion with positive charge. ... An anion is an ion with negative charge. ...

Electroosmotic flow is observed when an electric field is applied to a solution in a capillary that has fixed charges on its interior wall. Charge is accumulated on the inner surface of a capillary when a buffer solution is placed inside the capillary. In a fused-silica capillary, silanol (Si-OH) groups attached to the interior wall of the capillary are ionized to negatively charged silanoate (Si-O^-) groups at pH values greater than three. The ionization of the capillary wall can be enhanced by first running a basic solution, such as NaOH or KOH through the capillary prior to introducing the buffer solution. Attracted to the negatively charged silanoate groups, the positively charged cations of the buffer solution will form two inner layers of cations (called the diffuse double layer or the electrical double layer) on the capillary wall as shown in figure 4. The first layer is referred to as the fixed layer because it is held tightly to the silanoate groups. The outer layer, called the mobile layer, is farther from the silanoate groups. The mobile cation layer is pulled in the direction of the negatively charged cathode when an electric field is applied. Since these cations are solvated, the bulk buffer solution migrates with the mobile layer, causing the electroosmotic flow of the buffer solution. Other capillaries including Teflon capillaries also exhibit electroosmotic flow. The EOF of these capillaries is probably the result of adsorption of the electrically charged ions of the buffer onto the capillary walls.^[1] The rate of EOF is dependent on the field strength and the charge density of the capillary wall. The wall's charge density is proportional to the pH of the buffer solution. The electroosmotic flow will increase with pH until all of the available silanols lining the wall of the capillary are fully ionized.^[2] The chemical compound silicon dioxide, also known as silica, is the oxide of silicon, chemical formula SiO2. ... General Silanols are compounds containing silicon atoms to which hydroxy substituents bond directly. ... Sodium hydroxide (NaOH), also known as caustic soda or lye in North America, is a caustic metallic base used in industry (mostly as a strong chemical base) in the manufacture of paper, textiles, and detergents. ... The chemical compound potassium hydroxide, (KOH) sometimes known as caustic potash, potassa, potash lye, and potassium hydrate, is a metallic base. ... Solvation is the attraction and association of molecules of a solvent with molecules or ions of a solute. ... Teflon is a trademark of DuPont and is commonly used for the chemical compound polytetrafluoroethylene. ... Adsorption is a process that occurs when a gas or liquid solute accumulates on the surface of a solid or, more rarely, a liquid (adsorbent), forming a molecular or atomic film (the adsorbate). ...

Figure 4: Depiction of the interior of a fused-silica gel capillary in the presence of a buffer solution.

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Efficiency and resolution

The number of theoretical plates, or separation efficiency, in capillary electrophoresis is given by:

where N is the number of theoretical plates, μ is the apparent mobility in the separation medium and D_m is the diffusion coefficient of the analyte. According to this equation, the efficiency of separation is only limited by diffusion and is proportional to the strength of the electric field. The efficiency of capillary electrophoresis separations is typically much higher than the efficiency of other separation techniques like HPLC. Unlike HPLC, in capillary electrophoresis there is no mass transfer between phases.^[2] In addition, the flow profile in EOF-driven systems is flat, rather than the rounded laminar flow profile characteristic of the pressure-driven flow in chromatography columns as shown in figure 5. As a result, EOF does not significantly contribute to band broadening as in pressure-driven chromatography. Capillary electrophoresis separations can have several hundred thousand theoretical plates.^[4] A theoretical plate in separation processes is a hypothetical zone in which two phases establish an equilibrium, also referred to as an equilibrium stage or a theoretical tray. ... diffusion (disambiguation). ... Chromatography is a family of analytical chemistry techniques for the separation of mixtures. ... Mass transfer is the phrase commonly used in engineering for physical processes that involve molecular and convective transport of atoms and molecules within physical systems. ... Laminar flow (bottom) and turbulent flow (top) over a submarine hull. ... This article is about pressure in the physical sciences. ...

Figure 5: Flow profiles of laminar and electroosmotic flow.

The resolution (R_s) of capillary electrophoresis separations can be written as: This image has been released into the public domain by the copyright holder, its copyright has expired, or it is ineligible for copyright. ...

According to this equation, maximum resolution is reached when the electrophoretic and electroosmotic mobilities are similar in magnitude and opposite in sign. In addition, it can be seen that high resolution requires lower velocity and, correspondingly, increased analysis time.^[2] The largest and the smallest element of a set are called extreme values, or extreme records. ... The magnitude of a mathematical object is its size: a property by which it can be larger or smaller than other objects of the same kind; in technical terms, an ordering of the class of objects to which it belongs. ...

Related techniques

As discussed above, separations in a capillary electrophoresis system are typically dependent on the analytes having different electrophoretic mobilities. However, some classes of analyte cannot be separated by this effect because they are neutral (uncharged) or because they may not differ significantly in electrophoretic mobility. However, there are several techniques that can help separate such analytes with a capillary electrophoresis system. Adding a surfactant to the electrolyte can facilitate the separation of neutral compounds by micellar electrokinetic chromatography. Charged polymers such as DNA can be separated by filling the capillary with a gel matrix that retards longer strands more than shorter strands. This is called capillary gel electrophoresis. This is a high-resolution alternative to slab gel electrophoresis. Some capillary electrophoresis systems can also be used for microscale liquid chromatography or capillary electrochromatography. A capillary electrophoresis system can also be used for isotachophoresis and isoelectric focusing. In 1984, the Terabe group reported a technique that enabled capillary electrophoresis (CE) instrumentation to be used in the separation of neutral (as well as ionic) species. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid, ribonucleic acid, or protein molecules through an electric charge. ... Chromatography is a family of analytical chemistry techniques for the separation of mixtures. ... Electrochromatography is a chemical separation technique in analytical chemistry, biochemistry and molecular biology used to resolve and separate mostly large biomolecules such as proteins. ... Isotachophoresis (Greek: iso = equal, tachos = speed, phoresis = migration) is a technique in analytical chemistry used to separate charged particles. ... Isoelectric focusing, also known as electrofocusing, is a technique for separating different molecules by their electric charge differences. ...

References

1. ^ ^{a b c d e f} Skoog, D.A.; Holler, F.J.; Crouch, S.R "Principles of Instrumental Analysis" 6th ed. Thomson Brooks/Cole Publishing: Belmont, CA 2007.
2. ^ ^{a b c d e f g h i j} Skoog, D.A.; Holler, F.J.; Crouch, S.R "Principles of Instrumental Analysis" 6th ed. Chapter 30 Thomson Brooks/Cole Publishing: Belmont, CA 2007.
3. ^ Lin H.; Natan, M.; Keating, C. Anal. Chem. 2000, 72, 5348-5355.
4. ^ Skoog, D.A.; Holler, F.J.; Nieman, T.A. "Principles of Instrumental Analysis, 5th ed." Saunders college Publishing: Philadelphia, 1998.

References not cited in-line:

• Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111.
• Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 113.
• Foley, J.P. Anal. Chem. 1990, 62, 1302.
• Carretero, A.S.; Cruces-Blanco, C.; Ramirez, S.C.; Pancorbo, A.C.; Gutierrez, A.F. J. Agric. Food. Chem. 2004, 52, 5791.
• Cavazza, A.; Corradini, C.; Lauria, A.; Nicoletti, I. J. Agric. Food Chem. 2000, 48, 3324.
• Rodrigues, M.R.A.; Caramao, E.B.; Arce, L.; Rios, A.; Valcarcel, M. J. Agric. Food Chem. 2002, 50, 425.
• CE animations [1]

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• Micronit Microfluidics - Glass chips (lab-on-a-chip) and microfluidic tools for capillary electrophoresis

• Beckman Coulter [2] Instruments for Pharmaceutical Quality Control, CdT analysis, small molecule analysis and protein characterisation.

• CE Resources [3] CE Instruments for biomedical and chemical analysis. The CE-P2 is currently the only portable automated CE system in the world.

See also

DNA Separation by Silica Adsorption DNA Separation by Silica Adsorption is an important method of DNA separation that is used in novel technologies that use micro-channels. ...