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Capillary electrophoresis (CE) systems are powerful instruments that can separate proteins, nucleic acids, or other species from a mixture quickly, with great accuracy and a relatively small investment of researcher time. CE and its cousin, gel electrophoresis, both separate molecules according to charge and size, sometimes referred to as a molecule’s electrophoretic mobility. But in contrast to conventional gel electrophoresis, which separates molecules as they migrate through a slab gel matrix, capillary electrophoresis separates molecules as they travel along the inside of a small capillary tube that is filled with a conductive, liquid buffer, rather than a gel. CE separation is faster and gives higher resolution because the thin tubes have a higher surface-to-volume ratio than slab gels, so they dissipate heat faster, and thus run at high voltages without overheating, in contrast to slab gels.
Capillary electrophoresis applications
Applications of capillary electrophoresis include protein and nucleic acid characterization, and detection of molecules without stains or labels (though sometimes labels like fluorescent tags can lend greater clarity or speed with automation; see below). Because CE requires only small samples, it is advantageous for analyzing rare or expensive substances. It is used in drug development to separate both basic and chiral pharmaceuticals. Furthermore, CE is highly amenable to automation, which greatly augments the power of this technique, particularly in genetic sequencing, single-nucleotide polymorphism (SNP) analysis, and human identification by DNA fingerprinting. The importance of using CE for the latter application, rather than standard gel electrophoresis, is that the higher voltage possible with capillary tubes allows for single-nucleotide resolution.
Types of capillary electrophoresis
Many types of CE have evolved as researchers creatively tweak the basic protocols to solve their particular conundrums. Some of the main forms of CE include:
- Capillary zone electrophoresis (CZE)
- Capillary gel electrophoresis (CGE)
- Capillary isoelectric focusing (CIEF)
- Micellar electrokinetic capillary chromatography (MEKC or MECC)
- Capillary electrochromatography (CEC).
CZE is the simplest form of CE, in which species are separated according to their charge-to-mass ratio. CGE is a variation of CZE using gel rather than liquid within the tube, in order to also separate species by size. CIEF separates species according to their isoelectric points (pIs). MEKC (or MECC) uses micelles made from surfactants added to the buffer to separate mixtures of ionic and neutral species, such as separating hydrophobic pharmaceutical compounds from their polar metabolites. CEC is a hybrid of CZE and HPLC that uses an electric field instead of pressure to drive buffer through a packed column.
Capillary electrophoresis analysis and methods
Most CE systems are comprised of a few key components. A capillary, through which the sample flows during separation, has each end positioned in a buffer reservoir—one is a source reservoir containing an anode submersed in buffer, and the other is a destination reservoir containing a cathode submersed in buffer. Near the source reservoir is a sample vial that will be used at the beginning of the process to load or inject the sample into the source end of the capillary. The anode and cathode are both connected to a high-voltage power supply. Positioned along the capillary near its end is a detector, which is connected to a computer for data acquisition and analysis.
Sample is loaded or injected into the source end of the capillary by vacuum, pressure, or electrokinetic injection. The power supply provides charge to the anode and cathode, establishing an electric field between the source and destination reservoirs, which initiates the migration of both positively and negatively charged ions in the sample by electroosmotic flow (see below). The detector detects the ions near the end of the capillary, which have separated during their migration along the capillary according to their electrophoretic mobilities. Most systems pass light through the end of the tube and then measure the amount of light absorbed by the molecules (see below). Typically the raw data are observed in electropherograms, which display peaks representing different molecules as a function of migration time.
Table 1 – Considerations for purchasing a capillary electrophoresis system
Regardless of their charge, all molecules move toward the negatively charged cathode due to the force of the buffer’s electroosmotic flow. Electroosmotic flow results when an electric field is applied across a capillary filled with electrolytes. However, differences in charge and size cause ions to move at different velocities despite being carried along by the electroosmotic flow. For example, cations in the flow are drawn toward the cathode, while traveling in the electroosmotic flow, so they are first to elute (beginning with the highest-charged ions). Anions traveling in the flow are drawn in the opposite direction, toward the anode behind them, which slows them down and makes their effective velocity slower than the electroosmotic flow. Thus, anions elute later than cations, with the highest-charged anions eluting last. Highly charged anions require higher voltages to move them toward the cathode. In theory, the maximum resolution of CE is realized when the electrophoretic and electroosmotic mobilities of molecules are of comparable magnitude but opposite sign. Greater resolving power is also achieved with lower velocities, though this also increases the run time. The importance of resolution versus experiment time must be weighed relative to each other.
Considerations for purchasing a capillary electrophoresis system
If you are in the market for an automated system for protein or nucleic acid separation, you are in luck. Table 1 lists purchasing considerations for those in the market for a CE system.
Most CE systems offer at least some degree of automation, if not total automation. For example, systems such as the Beckman Coulter (Indianapolis, IN; www.beckmancoulter.com) PA 800 plus Pharmaceutical Analysis System™, particularly designed for analyzing pharmaceutical compounds, include multiple avenues for automating the study of protein purity and heterogeneity. The system is based on sodium dodecyl sulfate (SDS)-gel CE for separating proteins by size, and includes automatic applications for common protein analyses, including SDS-gel separation for determining protein purity, quantitative analyses of IgG antibody purity, analyses of charge heterogeneity by capillary isoelectric focusing, and analyses of glycoprotein microheterogeneity by carbohydrate profiling.
Another powerful system is the 7100 CE System from Agilent Technologies (Santa Clara, CA; www.agilent.com), which offers extended light path capillaries to augment sensitivity. If you are looking for the option of separating proteins prior to Western blotting, instruments for automated Western blotting from ProteinSimple (Santa Clara, CA; www.proteinsimple.com) are based on CE technology, with the added step of using antibodies for identification following separation. The instruments can run up to 96 samples simultaneously in a run of about 19 hr, convenient for overnight use.
Besides automation, several other features should be considered when perusing CE systems. One example is temperature controls for the samples and buffers (especially during long, automated runs). On a related note, automated systems that run unattended must also have reliable replenishment systems for buffers. Another point to consider is the maximum number of capillaries that the system can run simultaneously, which depends on your unique needs. It may also be advantageous to ask whether the system must run with a fixed number of capillaries; for example, if you only need to run 10 capillaries, must you use 96 glass tubes (and the buffer that runs through them) in order for the system to function?
In addition, you might need to choose between different detectors, depending on your mode(s) of detection and species of interest. Many systems use absorbance detectors in the ultraviolet-visible range, in either fixed or variable wavelength configurations. Greater sensitivity can be had with laser-induced fluorescence detectors, which have lower limits of detection compared to UV-VIS detectors. The newer contactless conductivity detectors, based on an electrical current passed between two metal electrodes positioned around the capillary tube, also give greater sensitivity.
Latest advances in capillary electrophoresis
CE is one of the few scientific techniques that has quickly affected society. The impact of CE on criminal justice cannot be overstated, with sequencing and DNA fingerprinting of the criminally accused now routine and even automated. CE’s higher voltage and better resolution give clear separation of DNA molecules differing by only one nucleotide, which sometimes can mean the difference between conviction or exoneration when the DNA evidence is strong. In fact, sequencing vendors such as Illumina (San Diego, CA; www.illumina.com), Life Technologies (Grand Island, NY; www.lifetechnologies.com), and Oxford Nanopore Technologies (Oxford, U.K.; www.nanoporetech.com) have recently (or nearly, in the case of Oxford Nanopore Technologies) introduced small, mobile DNA sequencing units that can be easily transported to crime scenes, and operated by nonscientific personnel. With CE systems in the hands of nonresearchers, CE enters the realm of truly accessible and indispensable scientific technologies.
Caitlin Smith is a freelance science writer who has a Ph.D. in Neuroscience from Yale University and postdoctoral work in Electrophysiology and Synaptic Plasticity; e-mail: [email protected].
Please check out our Capillary Electrophoresis Instrument section to find manufacturers that sell these products