Capillary electrophoresis

Capillary electrophoresis
Acronym	CE
Classification	Electrophoresis
Analytes	Chiral molecules
Other Techniques
Related	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 electrolyte.

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, chemical compounds appear as peaks with different retention times in an electropherogram.^[1]

Detection

Separation by capillary electrophoresis can be detected by several detection devices. The majority of commercial systems use 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]

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]

In order to obtain the identity of sample components, capillary electrophoresis can be directly coupled with 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.

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:

$u p = μ p E$

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 pH is given by:

$\mu_p = \frac{z}{6\pi \eta r}$

where $z$ is the net charge of the analyte and $r$ is the Stokes radius of the analyte. The Stokes radius is given by:

$r=\frac{k_B T}{6 \pi \eta\ D}$

where $k B$ 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:

$\mu_p = \left ( \frac{L}{t_r} \right )\left ( \frac{L_t}{V} \right )$

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 cathode, in agreement with the EOF as depicted in figure 3.

The velocity of the electroosmotic flow, $u o$ can be written as:

$u o = μ o E$

where $μ o$ is the electroosmotic mobility, which is defined as:

$\mu_o= \frac{\epsilon \zeta}{\eta}$

where $ζ$ 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:

$u p + u o = (μ p + μ o) E$

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]

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-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]

Efficiency and resolution

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

$N=\frac{\mu V}{2 D_m}$

where $N$ is the number of 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]

The resolution ( $R s$ ) of capillary electrophoresis separations can be written as:

$R_s = \frac{1}{4}\left ( \frac{\triangle \mu_p \sqrt{N} }{\mu_p +\mu_o} \right )$

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 time.^[2]

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 isoelectric focusing.

References

^ ^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 ^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.
^ Lin H.; Natan, M.; Keating, C. Anal. Chem. 2000, 72, 5348-5355.
^ 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]