Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other electrophoretic techniques including capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis and micellar electrokinetic chromatography (MEKC) belong also to this class of methods. In CE methods, analytes migrate through electrolyte solutions under the influence of an electric field. Analytes can be separated according to ionic mobility and/or partitioning into an alternate phase via non-covalent interactions. Additionally, analytes may be concentrated or "focused" by means of gradients in conductivity and pH.
1 Instrumentation 2 Detection 3 Modes of separation 4 Efficiency and resolution 5 Applications 6 References 7 Bibliography 8 External links
Figure 1: Diagram of capillary electrophoresis system
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. Sample is introduced into the capillary via capillary action, pressure, siphoning, or electrokinetically, and the capillary is then returned to the source vial. The migration of the analytes is 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. In the most common mode of CE, all ions, positive or negative, are pulled through the capillary in the same direction by electroosmotic flow. The analytes separate as they migrate due to their electrophoretic mobility, 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. Capillary electrophoresis was first combined with mass spectrometry by Richard D. Smith and coworkers, and provides extremely high sensitivity for the analysis of very small sample sizes. Despite the very small sample sizes (typically only a few nanoliters of liquid are introduced into the capillary), high sensitivity and sharp peaks are achieved in part due to injection strategies that result in concentration of analytes into a narrow zone near the inlet of the capillary. This is achieved in either pressure or electrokinetic injections simply by suspending the sample in a buffer of lower conductivity (e.g. lower salt concentration) than the running buffer. A process called field-amplified sample stacking (a form of isotachophoresis) results in concentration of analyte in a narrow zone at the boundary between the low-conductivity sample and the higher-conductivity running buffer. To achieve greater sample throughput, instruments with arrays of capillaries are used to analyze many samples simultaneously. Such capillary array electrophoresis (CAE) instruments with 16 or 96 capillaries are used for medium- to high-throughput capillary DNA sequencing, and the inlet ends of the capillaries are arrayed spatially to accept samples directly from SBS-standard footprint 96-well plates. Certain aspects of the instrumentation (such as detection) are necessarily more complex than for a single-capillary system, but the fundamental principles of design and operation are similar to those shown in Figure 1. 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 (frequently polyimide or Teflon) for increased flexibility. The portion of the capillary used for UV detection, however, must be optically transparent. For polyimide-coated capillaries, a segment of the coating is typically burned or scraped off to provide a bare window several millimeters long. This bare section of capillary can break easily, and 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. Post-column detection utilizing a sheath flow configuration has also been described.
Figure 2: Techniques for increasing the pathlength of the capillary: a) a bubble cell and b) a z-cell (additional tubing).
displaystyle u_ p
) of an analyte toward the electrode of opposite charge is:
displaystyle u_ p =mu _ p E,
The electrophoretic mobility can be determined experimentally from the migration time and the field strength:
displaystyle mu _ p =left( frac L t_ r right)left( frac L_ t V right)
is the distance from the inlet to the detection point,
displaystyle t_ r
is the time required for the analyte to reach the detection point (migration time),
is the applied voltage (field strength), and
displaystyle L_ t
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 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. 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.
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,
displaystyle u_ o
can be written as:
displaystyle u_ o =mu _ o E
displaystyle mu _ o
is the electroosmotic mobility, which is defined as:
displaystyle mu _ o = frac epsilon zeta eta
is the zeta 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:
displaystyle u_ p +u_ o =(mu _ p +mu _ o )E
Since the electroosmotic flow of the buffer solution is generally
greater than that of the electrophoretic mobility 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. 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
Figure 4: Depiction of the interior of a fused-silica gel capillary in the presence of a buffer solution.
In certain situations where strong electroosomotic flow toward the cathode is undesirable, the inner surface of the capillary can be coated with polymers, surfactants, or small molecules to reduce electroosmosis to very low levels, restoring the normal direction of migration (anions toward the anode, cations toward the cathode). CE instrumentation typically includes power supplies with reversible polarity, allowing the same instrument to be used in "normal" mode (with EOF and detection near the cathodic end of the capillary) and "reverse" mode (with EOF suppressed or reversed, and detection near the anodic end of the capillary). One of the most common approaches to suppressing EOF, reported by Stellan Hjertén in 1985, is to create a covalently attached layer of linear polyacrylamide. The silica surface of the capillary is first modified with a silane reagent bearing a polymerizable vinyl group (e.g. 3-methacryloxypropyltrimethoxysilane), followed by introduction of acrylamide monomer and a free radical initiator. The acrylamide is polymerized in situ, forming long linear chains, some of which are covalently attached to the wall-bound silane reagent. Numerous other strategies for covalent modification of capillary surfaces exist. Dynamic or adsorbed coatings (which can include polymers or small molecules) are also common. For example, in capillary sequencing of DNA, the sieving polymer (typically polydimethylacrylamide) suppresses electroosmotic flow to very low levels. A variety of dynamic capillary coating agents are commercially available to modify, suppress, or reverse the direction of electroosmotic flow. Besides modulating electroosmotic flow, capillary wall coatings can also serve the purpose of reducing interactions between "sticky" analytes (such as proteins) and the capillary wall. Such wall-analyte interactions, if severe, manifest as reduced peak efficiency, asymmetric (tailing) peaks, or even complete loss of analyte to the capillary wall. Efficiency and resolution The number of theoretical plates, or separation efficiency, in capillary electrophoresis is given by:
displaystyle N= frac mu V 2D_ m
is the number of theoretical plates,
is the apparent mobility in the separation medium and
displaystyle 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, although
practical considerations limit the strength of the electric field to
several hundred volts per centimeter. Application of very high
potentials (>20-30 kV) may lead to arcing or breakdown of the
capillary. Further, application of strong electric fields leads to
resistive heating (Joule heating) of the buffer in the capillary. At
sufficiently high field strengths, this heating is strong enough that
radial temperature gradients can develop within the capillary. Since
electrophoretic mobility of ions is generally temperature-dependent
(due to both temperature-dependent ionization and solvent viscosity
effects), a non-uniform temperature profile results in variation of
electrophoretic mobility across the capillary, and a loss of
resolution. The onset of significant Joule heating can be determined
by constructing an "
Figure 5: Flow profiles of laminar and electroosmotic flow.
The resolution (
displaystyle R_ s
) of capillary electrophoresis separations can be written as:
displaystyle R_ s = frac 1 4 left( frac triangle mu _ p sqrt N mu _ p +mu _ o right)
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
Besides diffusion and Joule heating (discussed above), factors that
may decrease the resolution in capillary electrophoresis from the
theoretical limits in the above equation include, but are not limited
to, the finite widths of the injection plug and detection window;
interactions between the analyte and the capillary wall; instrumental
non-idealities such as a slight difference in height of the fluid
reservoirs leading to siphoning; irregularities in the electric field
due to, e.g., imperfectly cut capillary ends; depletion of buffering
capacity in the reservoirs; and electrodispersion (when an analyte has
higher conductivity than the background electrolyte). Identifying
and minimizing the numerous sources of band broadening is key to
successful method development in capillary electrophoresis, with the
objective of approaching as close as possible to the ideal of
Capillary electrophoresis may be used for the simultaneous
determination of the ions NH4+,, Na+, K+, Mg2+ and Ca2+ in saliva.
One of the main application of CE in forensic science is the
development of methods for amplification and detection of DNA
fragments using polymerase chain reaction (PCR) which has to lead to
rapid and dramatic advances in
^ Graham Kemp, Capillary electrophoresis: a versatile family of
analytical techniques Archived April 27, 2006, at the Wayback Machine.
Biotechnology and Applied Biochemistry (1998) 27, (9–17)
^ 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.
^ a b c d e f g h i Skoog, D.A.; Holler, F.J.; Crouch, S.R "Principles
of Instrumental Analysis" 6th ed. Chapter 30 Thomson Brooks/Cole
Publishing: Belmont, CA 2007.
^ Dovichi, Norman (2000). "How capillary electrophoresis sequenced the
human genome" (PDF). Angewandte Chemie International Edition. 39:
^ Butler, John (2004). "Forensic
Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113. 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, 4215.
CE animations 
v t e
Agarose gel electrophoresis
Difference gel electrophoresis
Electrophoretic mobility shift assay
Electrical mobility Isoelectric focusing
Category Commons An