Capillary electrophoresis

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.

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 migration times in an electropherogram. The technique is often attributed to James W. Jorgensen and Krynn DeArman Lukacs, who first demonstrated the capabilities of this technique. 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 a 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.

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. This decrease is almost unnoticeable if a smooth aneurysm is produced in the wall of a capillary by heating and pressurization, as plug flow can be preserved. This invention by Gary Gordon, US Patent 5061361, typically triples the absorbance path length. When used with a UV absorbance detector, the wider cross-section of the analyte in the cell allows for an illuminating beam twice as large, which reduces shot noise by a factor of two. Together these two factors increase the sensitivity of Agilent Technologies's Bubble Cell CE Detector six times over that of one using a straight capillary. This cell and its manufacture are described on page 62 of the June 1995 issue of the Hewlett-Packard Journal.

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. Numerous labeling strategies are used to create fluorescent derivatives or conjugates of non-fluorescent molecules, including proteins and DNA. 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. 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. Multi-color fluorescence detection can be achieved by including multiple dichroic mirrors and bandpass filters to separate the fluorescence emission amongst multiple detectors (e.g., photomultiplier tubes), or by using a prism or grating to project spectrally resolved fluorescence emission onto a position-sensitive detector such as a CCD array. CE systems with 4- and 5-color LIF detection systems are used routinely for capillary DNA sequencing and genotyping ("DNA fingerprinting") applications.

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 setup 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. The measurement and analysis are mostly done with a specialized.

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.

The separation of compounds by capillary electrophoresis is dependent on the differential migration of analytes in an applied electric field. The electrophoretic migration velocity (${\displaystyle u_{p}}$) of an analyte toward the electrode of opposite charge is: