Capillary electrophoresis (
CE), also known as capillary zone electrophoresis (CZE), can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional
electrophoresisElectrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
, electrically charged analytes move in a conductive
liquidLiquid is one of the three classical states of matter . Like a gas, a liquid is able to flow and take the shape of a container. Some liquids resist compression, while others can be compressed. Unlike a gas, a liquid does not disperse to fill every space of a container, and maintains a fairly...
medium under the influence of an
electric fieldIn physics, an electric field surrounds electrically charged particles and time-varying magnetic fields. The electric field depicts the force exerted on other electrically charged objects by the electrically charged particle the field is surrounding...
. 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
electrolyteIn chemistry, an electrolyte is any substance containing free ions that make the substance electrically conductive. The most typical electrolyte is an ionic solution, but molten electrolytes and solid electrolytes are also possible....
.
Instrumentation
The instrumentation needed to perform capillary electrophoresis is relatively simple. A basic
schematicA schematic diagram represents the elements of a system using abstract, graphic symbols rather than realistic pictures. A schematic usually omits all details that are not relevant to the information the schematic is intended to convey, and may add unrealistic elements that aid comprehension...
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,
electrodeAn electrode is an electrical conductor used to make contact with a nonmetallic part of a circuit...
s, a high-
voltageVoltage, otherwise known as electrical potential difference or electric tension is the difference in electric potential between two points — or the difference in electric potential energy per unit charge between two points...
power supplyA power supply is a device that supplies electrical energy to one or more electric loads. The term is most commonly applied to devices that convert one form of electrical energy to another, though it may also refer to devices that convert another form of energy to electrical energy...
, 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 actionCapillary action, or capilarity, is the ability of a liquid to flow against gravity where liquid spontanously rise in a narrow space such as between the hair of a paint-brush, in a thin tube, or in porous material such as paper or in some non-porous material such as liquified carbon fiber, or in a...
, 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
integratorAn integrator is a device to perform the mathematical operation known as integration, a fundamental operation in calculus.The integration function is often part of engineering, physics, mechanical, chemical and scientific calculations....
or
computerA computer is a programmable machine designed to sequentially and automatically carry out a sequence of arithmetic or logical operations. The particular sequence of operations can be changed readily, allowing the computer to solve more than one kind of problem...
. The data is then displayed as an electropherogram, which reports detector response as a function of
timeTime is a part of the measuring system used to sequence events, to compare the durations of events and the intervals between them, and to quantify rates of change such as the motions of objects....
. Separated
chemical compoundA chemical compound is a pure chemical substance consisting of two or more different chemical elements that can be separated into simpler substances by chemical reactions. Chemical compounds have a unique and defined chemical structure; they consist of a fixed ratio of atoms that are held together...
s appear as peaks with different migration times in an electropherogram.
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
polymerA polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...
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 lengthIn chemistry, the path length is defined as the distance that light travels through a sample in an analytical cell. Typically, a sample cell is made of quartz, glass, or a plastic rhombic cuvette with a volume typically ranging from 0.1 mL to 10 mL or larger used in a spectrophotometer. For the...
of the detection cell in capillary electrophoresis (~ 50
micrometersA micrometer , is by definition 1×10-6 of a meter .In plain English, it means one-millionth of a meter . Its unit symbol in the International System of Units is μm...
) is far less than that of a traditional UV cell (~ 1 cm). According to the
Beer-Lambert lawIn optics, the Beer–Lambert law, also known as Beer's law or the Lambert–Beer law or the Beer–Lambert–Bouguer law relates the absorption of light to the properties of the material through which the light is travelling.-Equations:The law states that there is a logarithmic dependence between the...
, 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.
FluorescenceFluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
detection can also be used in capillary electrophoresis for samples that naturally fluoresce or are chemically modified to contain
fluorescent tagIn 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. The tag is some kind of fluorescent molecule...
s. 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.
LaserA laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...
-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
intensityIrradiance is the power of electromagnetic radiation per unit area incident on a surface. Radiant emittance or radiant exitance is the power per unit area radiated by a surface. The SI units for all of these quantities are watts per square meter , while the cgs units are ergs per square centimeter...
of the incident light and the ability to accurately focus the light on the capillary.
In order to obtain the identity of sample components, capillary electrophoresis can be directly coupled with mass spectrometers or
Surface Enhanced Raman SpectroscopySurface enhanced Raman spectroscopy or surface enhanced Raman scattering is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces...
(SERS). In most systems, the capillary outlet is introduced into an ion source that utilizes
electrospray ionizationElectrospray ionization is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized...
(ESI). The resulting ions are then analyzed by the mass spectrometer. This set-up requires
volatileIn chemistry and physics, volatility is the tendency of a substance to vaporize. Volatility is directly related to a substance's vapor pressure. At a given temperature, a substance with higher vapor pressure vaporizes more readily than a substance with a lower vapor pressure.The term is primarily...
buffer solutions, which will affect the range of separation modes that can be employed and the degree of resolution that can be achieved.
The measurement and analysis are mostly done with a specialized
gel analysis software.
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.
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
velocityIn physics, velocity is speed in a given direction. Speed describes only how fast an object is moving, whereas velocity gives both the speed and direction of the object's motion. To have a constant velocity, an object must have a constant speed and motion in a constant direction. Constant ...
(
) of an analyte toward the electrode of opposite charge is:
where
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
frictionFriction is the force resisting the relative motion of solid surfaces, fluid layers, and/or material elements sliding against each other. There are several types of friction:...
al
forceIn physics, a force is any influence that causes an object to undergo a change in speed, a change in direction, or a change in shape. In other words, a force is that which can cause an object with mass to change its velocity , i.e., to accelerate, or which can cause a flexible object to deform...
s 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
viscosityViscosity is a measure of the resistance of a fluid which is being deformed by either shear or tensile stress. In everyday terms , viscosity is "thickness" or "internal friction". Thus, water is "thin", having a lower viscosity, while honey is "thick", having a higher viscosity...
(η) of the medium and the size and shape of the
ionAn ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
. Accordingly, the electrophoretic mobility of an analyte at a given
pHIn chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
is given by:
where
is the net charge of the analyte and
is the
Stokes radiusThe Stokes radius, Stokes-Einstein radius, or hydrodynamic radius RH, named after George Gabriel Stokes, is not the effective radius of a hydrated molecule in solution as often mentioned. Rather it is the radius of a hard sphere that diffuses at the same rate as the molecule. The behavior of this...
of the analyte. The Stokes radius is given by:
where
is the
Boltzmann constant, and
is the
temperatureTemperature is a physical property of matter that quantitatively expresses the common notions of hot and cold. Objects of low temperature are cold, while various degrees of higher temperatures are referred to as warm or hot...
, 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
radiusIn classical geometry, a radius of a circle or sphere is any line segment from its center to its perimeter. By extension, the radius of a circle or sphere is the length of any such segment, which is half the diameter. If the object does not have an obvious center, the term may refer to its...
. The electrophoretic mobility can be determined experimentally from the migration time and the field strength:
where
is the distance from the inlet to the detection point,
is the time required for the analyte to reach the detection point (migration time),
is the applied voltage (field strength), and
is the total length of the capillary. 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
cathodeA cathode is an electrode through which electric current flows out of a polarized electrical device. Mnemonic: CCD .Cathode polarity is not always negative...
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. As a result, negatively charged analytes are attracted to the positively charged
anodeAn anode is an electrode through which electric current flows into a polarized electrical device. Mnemonic: ACID ....
, counter to the EOF, while positively charged analytes are attracted to the
cathodeA cathode is an electrode through which electric current flows out of a polarized electrical device. Mnemonic: CCD .Cathode polarity is not always negative...
, in agreement with the EOF as depicted in
figure 3.
The velocity of the electroosmotic flow,
can be written as:
where
is the electroosmotic mobility, which is defined as:
where
is the
zeta potentialZeta potential is a scientific term for electrokinetic potential in colloidal systems. In the colloidal chemistry literature, it is usually denoted using the Greek letter zeta, hence ζ-potential...
of the capillary wall, and
is the relative permittivity of the buffer solution. Experimentally, the electroosmotic mobility can be determined by measuring the retention time of a neutral analyte. The velocity (
) of an analyte in an electric field can then be defined as:
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 capillary due to their conflicting electrophoretic mobilities. 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.
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,
silanolSilanol, also known as silyl alcohol, is a chemical with formula SiH3OH. It is the simplest silicon alcohol, and is a heavy, volatile, colorless, flammable liquid. At room temperature it is a polar liquid...
(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
KOHPotassium hydroxide is an inorganic compound with the formula KOH, commonly called caustic potash.Along with sodium hydroxide , this colorless solid is a prototypical strong base. It has many industrial and niche applications. Most applications exploit its reactivity toward acids and its corrosive...
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
solvatedSolvation, also sometimes called dissolution, is the process of attraction and association of molecules of a solvent with molecules or ions of a solute...
, 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
adsorptionAdsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...
of the electrically charged ions of the buffer onto the capillary walls. 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.
Efficiency and resolution
The number of theoretical plates, or separation efficiency, in capillary electrophoresis is given by:
where
is the number of
theoretical plateA theoretical plate in many separation processes is a hypothetical zone or stage in which two phases, such as the liquid and vapor phases of a substance, establish an equilibrium with each other. Such equilibrium stages may also be referred to as an equilibrium stage, ideal stage or a theoretical...
s,
is the apparent mobility in the separation medium and
is the
diffusionMolecular diffusion, often called simply diffusion, is the thermal motion of all particles at temperatures above absolute zero. The rate of this movement is a function of temperature, viscosity of the fluid and the size of the particles...
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
HPLCHigh-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
. Unlike HPLC, in capillary electrophoresis there is no
mass transferMass transfer is the net movement of mass from one location, usually meaning a stream, phase, fraction or component, to another. Mass transfer occurs in many processes, such as absorption, evaporation, adsorption, drying, precipitation, membrane filtration, and distillation. Mass transfer is used...
between phases. In addition, the flow profile in EOF-driven systems is flat, rather than the rounded
laminar flowLaminar flow, sometimes known as streamline flow, occurs when a fluid flows in parallel layers, with no disruption between the layers. At low velocities the fluid tends to flow without lateral mixing, and adjacent layers slide past one another like playing cards. There are no cross currents...
profile characteristic of the
pressurePressure is the force per unit area applied in a direction perpendicular to the surface of an object. Gauge pressure is the pressure relative to the local atmospheric or ambient pressure.- Definition :...
-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.
The resolution (
) of capillary electrophoresis separations can be written as:
According to this equation, maximum resolution is reached when the electrophoretic and electroosmotic mobilities are similar in
magnitudeThe magnitude of an object in mathematics is its size: a property by which it can be compared as larger or smaller than other objects of the same kind; in technical terms, an ordering of the class of objects to which it belongs....
and opposite in sign. In addition, it can be seen that high resolution requires lower velocity and, correspondingly, increased analysis time.
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 chromatographyMicellar electrokinetic chromatography , is a chromatography technique, used in analytical chemistry. It is a modification of capillary electrophoresis , where the samples are separated by differential partitioning between micelles and a surrounding aqueous buffer solution .The basic set-up and...
. Charged polymers such as
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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 electrophoresisGel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
. Some capillary electrophoresis systems can also be used for microscale liquid chromatography or capillary
electrochromatographyElectrochromatography is a chemical separation technique in analytical chemistry, biochemistry and molecular biology used to resolve and separate mostly large biomolecules such as proteins. It is a combination of size exclusion chromatography and gel electrophoresis...
. A capillary electrophoresis system can also be used for
isotachophoresisIsotachophoresis is a technique in analytical chemistry used to separate charged particles. It is a further development of electrophoresis. It is a powerful separation technique using a discontinuous electrical field to create sharp boundaries between the sample constituents.In conventional...
,
isoelectric focusingIsoelectric focusing , also known as electrofocusing, is a technique for separating different molecules by their electric charge differences...
, and
affinity electrophoresisAffinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. The methods include the so-called mobility shift electrophoresis, charge shift...
. In the case of amino acid separations, the ion charge ranges from -1 to -3 electrons but the size of the amino acid is dominated by the dye label; therefore changes in charge have a significant effect on mobility relative to changes in size.
See also
- Affinity electrophoresis
Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. The methods include the so-called mobility shift electrophoresis, charge shift...
- 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. The principle behind this type of separation relies on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH...
- Label-free HPCE Systems
- Kinetic capillary electrophoresis
Kinetic capillary electrophoresis or KCE is capillary electrophoresis of molecules that interact during electrophoresis. KCE was introduced and developed by Professor Sergey Krylov and his research group at York University, Toronto, Canada...
External links