Capillary electrophoresis
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2008-06-21T15:44:58Z
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{{Infobox chemical analysis
| name = Capillary electrophoresis
| image =
| caption =
| acronym = CE
| classification =[[Electrophoresis]]
| analytes = [[Biomolecule]]s<br>[[Chirality (chemistry)|Chiral molecules]]
| manufacturers =
| related = [[gel electrophoresis]]<br>[[Two-dimensional gel electrophoresis]]
| hyphenated = [[Capillary electrophoresis mass spectrometry]]
}}
'''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]]. While its use has been sporadic, CE offers unparalleled resolution and selectivity allowing for separation of analytes with very little physical difference. Efficiencies of millions of plates are routinely reported. Once thought impossible, separation of large proteins differing in only one amino acid (ie. D-Lysine substituted for L-Lysine) and even an isotopic separation of <sup>14</sup>N and <sup>15</sup>N ammonium hydroxide have been reported.<ref name="Yeung">Yeung, K.K.-C.; Lucy, C.A. ''Electrophoresis''. '''1999''', ''20'', 2554-2559.</ref> No other technique has shown such powerful selectivity with the ability for extremely high sensitivity. As few as 6 molecules of a substance have been separated and detected with the help of laser-induced fluorescence (LIF).
=== 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, [[electrode]]s, 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 compound]]s appear as peaks with different retention times in an electropherogram.<ref name="Baker">Skoog, D.A.; Holler, F.J.; Crouch, S.R "Principles of Instrumental Analysis" 6th ed. Thomson Brooks/Cole Publishing: Belmont, CA '''2007'''.</ref>
[[Image:Capillaryelectrophoresis.gif|frame|Figure 1: Diagram of capillary electrophoresis system]]
===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 [[micrometre|micrometers]]) is far less than that of a traditional UV cell (~ 1 [[Metre#SI multiples|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.<ref name="Cucino">Skoog, D.A.; Holler, F.J.; Crouch, S.R "Principles of Instrumental Analysis" 6th ed. Chapter 30 Thomson Brooks/Cole Publishing: Belmont, CA '''2007'''.</ref>
[[Image:Extendedpathlength.gif|frame|Figure 2: Techniques for increasing the pathlength of the capillary: a.) a bubble cell and b.) a z-cell (additional tubing).<ref name="Baker"/> </small>]]
[[Fluorescence]] detection can also be used in capillary electrophoresis for samples that naturally fluoresce or are chemically modified to contain [[fluorescent tag]]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.<ref name="Cucino"/> [[Laser]]-induced fluorescence has been used in CE systems with detection limits as low as 10<sup>-18</sup> to 10<sup>-21</sup> mol. The sensitivity of the technique is attributed to the high [[intensity]] of the [[wiktionary:incident|incident]] light and the ability to accurately focus the light on the capillary.<ref name="Baker"/>
In order to obtain the identity of sample components, capillary electrophoresis can be directly coupled with [[mass spectrometer]]s 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 [[Volatility (chemistry)|volatile]] buffer solutions, which will affect the range of separation modes that can be employed and the degree of resolution that can be achieved.<ref name="Cucino"/>
The measurement and analysis are mostly done with a specialized [http://www.labimage.net 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.<ref name="Lin">Lin H.; Natan, M.; Keating, C. ''Anal. Chem''. '''2000''', ''72'', 5348-5355.</ref>
=== 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]] (<math>u_p</math>) of an analyte toward the electrode of opposite charge is:
<math> u_p = \mu_p E \,</math>
where <math>\mu_p</math> 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 [[friction]]al [[force]]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 [[viscosity]] (η) of the medium and the size and shape of the [[ion]].<ref name="Cucino"/> Accordingly, the electrophoretic mobility of an analyte at a given [[pH]] is given by:
<math>\mu_p = \frac{z}{6\pi \eta r}</math>
where <math>z</math> is the net charge of the analyte and <math>r</math> is the [[Stokes radius]] of the analyte. The Stokes radius is given by:
<math> r=\frac{k_B T}{6 \pi \eta\ D}</math>
where <math>k_B</math> is the [[Boltzmann constant]], and <math>T</math> 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:
<math>\mu_p = \left ( \frac{L}{t_r} \right )\left ( \frac{L_t}{V} \right )</math>
where <math>L</math> is the distance from the inlet to the detection point, <math>t_r</math> is the time required for the analyte to reach the detection point (migration time), <math>V</math> is the applied voltage (field strength), and <math>L_t</math> is the total length of the capillary.<ref name="Cucino"/> 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.<ref name="Baker"/> 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''.
[[Image:Electroosmoticflow.gif|frame|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, <math>u_o</math> can be written as:
<math> u_o= \mu_o E </math>
where <math>\mu_o</math> is the electroosmotic mobility, which is defined as:
<math>\mu_o= \frac{\epsilon \zeta}{\eta}</math>
where <math>\zeta</math> is the [[zeta potential]] of the capillary wall, and <math>\epsilon</math> is the [[relative permittivity]] of the buffer solution. Experimentally, the electroosmotic mobility can be determined by measuring the retention time of a neutral analyte.<ref name="Cucino"/> The velocity (<math>u</math>) of an analyte in an electric field can then be defined as:
<math> u_p + u_o = (\mu_p +\mu_o) E</math>
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.<ref name="Baker"/> The order of migration seen by the detector is shown in ''figure 3'': small multiply charged [[cation]]s migrate quickly and small multiply charged [[anion]]s are retained strongly.<ref name="Cucino"/>
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<sup>-</sup>) 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 [[Potassium hydroxide|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 [[solvation|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.<ref name="Baker"/> 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.<ref name="Cucino"/>
[[Image:Capillarywall.gif|frame|Figure 4: Depiction of the interior of a fused-silica gel capillary in the presence of a buffer solution.]]
===Efficiency and resolution===
The number of theoretical plates, or separation efficiency, in capillary electrophoresis is given by:
<math> N=\frac{\mu V}{2 D_m}</math>
where <math>N</math> is the number of [[theoretical plate]]s, <math>\mu</math> is the apparent mobility in the separation medium and <math>D_m</math> 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.<ref name="Cucino"/> 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.<ref name="Skoog">Skoog, D.A.; Holler, F.J.; Nieman, T.A. "Principles of Instrumental Analysis, 5th ed." Saunders college Publishing: Philadelphia, '''1998'''.</ref>
[[Image:Flowprofile.gif|frame|Figure 5: Flow profiles of laminar and electroosmotic flow.]]
The resolution (<math>R_s</math>) of capillary electrophoresis separations can be written as:
<math>R_s = \frac{1}{4}\left ( \frac{\triangle \mu_p \sqrt{N} }{\mu_p +\mu_o} \right )</math>
According to this equation, [[maximum]] resolution is reached when the electrophoretic and electroosmotic mobilities are similar in [[magnitude (mathematics)|magnitude]] and opposite in sign. In addition, it can be seen that high resolution requires lower velocity and, correspondingly, increased analysis time.<ref name="Cucino"/>
==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]].
==References==
<references/>
''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 [http://www.shsu.edu/%7Echm_tgc/sounds/sound.html]
==See also==
[[DNA Separation by Silica Adsorption]]
CE Primer for Beginners & Experts [http://www.mtc-usa.com/ce.asp]
[[Category:Chromatography]]
[[Category:Electrophoresis]]
[[bs:Kapilarna elektroforeza]]
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[[es:Electroforesis capilar]]
[[fr:Électrophorèse capillaire]]
[[it:Elettroforesi capillare]]
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