Produsf Review
Migrating toward Capillary
Capillary electrophoresis, as its name suggests, incorporates the separation mechanisms of traditional electrophoresis into a capillary format. CE has much to offer biochemists who use conventional slabgel electrophoresis as well as analytical chemists who use various chromatographic techniques. It offers the ease and speed of HPLC and can be used for a wide variety of applications-from peptides and proteins to inorganic ions and industrial polymers. It also has the ability to handle very small samples (on the order of microliters), uses only a small amount of solvent (buffer), results in high column efficiencies, and lends itself easily to quantitation and automation. Although the use of capillaries for performing electrophoretic separations was first suggested by Everaerts in the mid1960s, the first demonstration of the potential of CE for highly efficient separations did not appear until 1981, when James Jorgenson and his research group at the University of North Carolina used zone electrophoresis in open-tubular glass capillaries to separate amino acids, dipeptides, and amines. As the advantages of CE became apparent over the next several years, CE drew more and more attention from researchers in various fields. For many years, investigators interested in using CE had to build their own systems from a controllable high-voltage
CE combines the ease and speed of HPLC with extremely high eficiency and the ability to handle very
Although half of the companies represented here manufacture only one complete CE system, five (AT1 Unicam, Beckman, Bio-Rad, Applied Biosystems, and Thermo Separation Products) offer a series of instruments with varying capabilities. In addition, two manufacturers (AT1 Unicam and Waters) offer instruments specifically intended for capillary ion analysis, and Thermo Separations offers a chemical kit to allow ion analysis.
small samples
In its early stages, CE was limited to freezone CE, known commonly as capillary zone electrophoresis (CZE). As understanding of the theory affecting electrophoretic separations in capillaries increased, researchers expanded CE into other modes: micellar electrokinetic chromatography (MEKC), capillary gel electrophoresis (CGE) , capillary isoelectric focusing (CIEF), and capillary isotachophoresis (CITP) . CZE, CZE is still the most commonly used mode of CE, in part because it can separate a wide variety of charged molecules. CZE has been used to determine proteins, nucleotides, organic acids, water-soluble vitamins, and free metal ions, among others. Separation in CZE is based on the combination of electroosmotic flow (the bulk flow resulting from the interaction
power supply, two electrode assemblies, two buffer reservoirs, a fused-silica capillary with a viewing window, and a UV detector. These early homemade CE systems, however, were inconvenient for routine analysis and too imprecise for quantitative analysis. By 1988,instrument companies became interested in producing commercial instruments. Since then, the market for CE has virtually exploded, and at least 10 companies currently include a complete CE system in their instrument line. Table l, although not intended to be comprehensive, compares the features of 10 currently available CE instruments.
CE modes
Analytical Chemistry, Vol. 66, No. 22,November 15, 1994 1137 A
between the cations along the negatively charged inner capillary wall and the applied field) and electrophoretic migration (the movement of ions toward the electrode of opposite polarity). The ends of a narrow-bore (10-200 pm i.d.) capillary are immersed in buffer, and high voltages are used to separate molecules based on differences in charge-to-
size ratio. The electroosmotic flow depends on the field strength; temperature; the buffer composition, pH, ionic strength, and viscosity; and the capillary’s surface characteristics. All of these factors can influence the separation. In CE, the electroosmotic flow carries all molecules, including negatively charged and neutral ones, toward the an-
Table I. Summary of representative products
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1138 A Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
ode. Because nothing is retained, the “retention time” commonly used in chromatography becomes the “migration time,” which is the time it takes for an analyte to migrate from the beginning of the capillary to the detector window. In most CZE systems, cations elute first because they are carried by both the electroosmotic flow and electrophoretic migration. Neu-
trals elute next, although they are not s e p arated from each other because they are moved only by the electroosmotic flow. Anions elute last because they migrate in the opposite direction to the electroosmotic flow. MEKC. MEKC, sometimes known as MECC (micellar electrokinetic capillary chromatography) was developed by
Product impany
Shigeru Terabe and his colleagues at Kyoto University (Japan) in the early 1980s. Unlike the other CE modes, which are based on traditional electrophoresis techniques, MEKC uses a unique separation mechanism. And unlike CZE, MEKC can separate a wide range of neutral species as well as anions and cations. Applications include derivatized amino acids, or-
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ganophosphates, food dyes, and antibiotics and other drugs. In MEKC, ionic surfactants are added to the buffer to form micelles, spherical aggregates with hydrophilic heads and hydrophobic tails. Different analyte molecules interact to different extents with the micelles, which in turn move through the capillary at a different rate than the elec-
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troosmotic flow-induced bulk buffer flow. Molecules are thus separated on the basis of the strength of their interaction with the micelles-the stronger the interaction, the longer the molecule will remain associated with the micelle and the longer it will take to migrate through the capillary to the detector window. CGE. CGE is the capillary version of traditional slabgel electrophoresis. It is particularly useful for the separation of biological macromolecules, such as oligonucleotides, DNA restriction fragments, and proteins. In CGE, the capillary is filled with a sieve-based gel, such as cross-linked polyacrylamide or linear polymers. The gel composition is used to manipulate the separation; as in conventional slab-gel electrophoresis, migration times decrease as the pore size increases. UEF. Isoelectric focusing is used primarily to separate large biomolecules, such as proteins, as a function of their isoelectric point. The capillary is filled with a mixture of carrier ampholytes, and once voltage is applied, the ampholytes separate, with those that are positively charged migrating to the cathode and those that are negatively charged migrating toward the anode. When analytes are added to the capillary, they migrate within the pH gradient until they reach a point at which their overall charge is neutral (their isoelectric point). During this process the current drops as the analyte molecules are focused at their isoelectric points. After the focusing is complete, the bands must be mobilized so that they can migrate past the detector window. Electroosmotic flow must be minimized, usually by coating the capillary wall with methylcellulose or polyacrylamide. U T . . Isotachophoresis uses two different buffer systems to “sandwich” the analytes between a leading electrolyte and a trailing electrolyte. Separation occurs in the area between the two electrolytes as a function of analyte mobility, and each of the focused bands moves at the same velocity past the detector. Like isoelectric focusing, isotachophoresis depends on having zero electroosmotic flow. lnstrumentation
A commercial CE system consists of a means of injection (usually an autosampler) , a high-voltage power supply, a compartment containing the capillary connected to two buffer reservoirs and highvoltage electrodes, and a detector. 1140 A
Most systems allow the user to inject the sample in a variety of ways, depending on the type of CE being performed. During electromigration injection, a high voltage is applied while the capillary is immersed in the sample solution, and sample ions migrate into the capillary according to their electrophoretic mobilities. In electrokinetic injection, sample ions are loaded into the capillary by a combination of electroosmotic pumping and electromigration. Various forms of displacement injection are also available: A sample can be injected by applying pressure to the sample vial with an inert gas (pressure injection), by applying a vacuum to the capillary outlet (vacuum injection), or by changing the relative heights of the sample and outlet buffer vials (gravity injection).
optional MS, conductivity, or fluorescence detectors. System control for CE instruments varies from front panel LCD displays to sophisticated PC-based controllers. Most systems are compatible with stripchart recorders, integrators, or PC-based data acquisition. The choice
As is the case with any analytical instrument, the key to choosing the right CE system is having a clear idea of what it will be used for, both now and in the next five years. If the system will be used only for a specific type of analysis, a relatively simple system geared toward that application is probably all that you need. If, on the other hand, the system will be used for a variety of different types of analyses implementing several CE modes, a more sophisticated system with more than one type of available detector would be a wise investment. Start by considering the type of injection you’ll need. If your CE system will be used to analyze a large number of samples under the same conditions, an autosampler will make this task much more manageable. This is especially important, says Jorgenson, because CE runs can often be completed in only 5 min, and manual injection gets tedious very quickly. For flexibility, Jorgenson recommends a CE system that can accommodate electromigration or electrokinetic injecton and at least one form of displacement injection (preferably pressure injection). Because electromigration and electrokinetic injection are affected by the mobility of Most CE systems allow either constant the analyte, discrimination based on movoltage, current, or power operation, albility is possible with these types of injecthough programmable voltage and curtion. Displacement injection doesn’t cause rent are sometimes available. A voltage discrimination problems but can’t be range of 0-30 kV and a current range of used for CGE. Gravity injection can cause 0-250 or 300 pA are standard. its own problems, says Jorgenson, beBecause electroosmotic flow and eleccause of the length of capillary required to trophoretic mobility are both affected by allow changing the relative heights of the temperature, and the high voltages used in buffer vials. CE can raise the temperature of the capilNext, consider the power supply. Most lary significantly, most CE systems insystems have a reversible-voltage power clude a means of controlling the temperasupply, and all allow at least constant curture of the capillary compartment. Some rent and constant voltage operation. For systems also offer a way to control the tem- isotachophoresis, you’ll need to operate perature of the autosampler carousel so under constant current; whether constant that temperature-sensitive samples can be current or constant voltage is better for easily analyzed. the other modes depends on the sample. Although a variety of detection methProgrammable voltage is useful, says ods have been used in the CE research Jorgenson, to prevent band distortions community, most commercial systems are caused by a high initial voltage. The voltequipped with only with a UV-vis detecage can be set to be low initially and then tor. Some companies, however, do offer raised to complete the separation. For iso-
CE has gone fiom a topic of j”icndamelzta1 research OR homemade systems to a technique widely used for routhe analysts and basic research. b
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
electric focusing, Jorgenson continues, constant power allows you to achieve maximum speed yet avoid overheating. Temperature control is so important to reproducibility in CE, says Jorgenson, that you should strongly consider a system that incorporates temperature control for the capillary compartment. Both liquid flow and forced-air temperature control have pros and cons. Liquid flow allows precise temperature control but necessitates the use of cartridge-based capillaries s u p plied by the manufacturer, Forced-air temperature control is more difficult to achieve but is more flexible, allowing you to use your own capillaries more easily. Finally, consider the type and sensitivity of the detector. The small optical pathlength in CE makes W detector sensitivity crucial, says Jorgenson. “All W detectors are not equal, and if you don’t pay sufficient attention up front, you’re sure to regret it later.” He recommends asking the manufacturer to run some representative samples from your lab on its system before you make a purchase. Although conductivity and fluorescence detectors are useful for achieving selectivity when the particular samples are amenable, they are available from only a few companies. MS detectors, although offered by a few manufacturers as an option, are difficult to use because of interfacing problems.
User support abounds Because methods development in CE is new to most scientists, manufacturers of CE systems offer a wide range of user s u p port products and activities. Several (including Applied Biosystems, Beckman, Bio-Rad, Hewlett Packard, and Waters) offer primers on CE theory and methods development, and all offer an extensive range of technical bulletins and application notes. Training classes for users provide CE basics and methodology, as well as information on specific applications. Many manufacturers also offer software designed to facilitate CE system control and data acquisition. CE manufacturers have also made changing capillaries easy by incorporating them into cartridges that automatically align themselves with the detector window. Application kits, which include the appropriate column, buffers, standards, and operating instructions, are available for common applications, such as proteins, peptides, oligonucleotides, proteins by CGE, DNA fragments, and isoelectric focusing. For those with established
methods, CE manufacturers offer a wide range of consumables, including standards, buffers, and vials.
lysts in different labs can be assured that as long as they are using identical buffers on the same type of capillary, their separations should also be identical. Perhaps the hottest area of activity in CE is that of chiral separations. The high theoretical plate counts (100,00&200,000 plates in a typical capillary), says Jorgenson, make CE particularly amenable to separating chiral molecules. “Many times all you have to do is add some cyclodextrin, and your separation will work the first time.” Some issues still need to be addressed, however, says Jorgenson. “On a concentration basis, W sensitivity in CE is not as good as in LC because of the short pathlength, and most people would like to see a 10-fold increase in sensitivity. This is one of our main goals for the future.” Research is continuing both in this area and in the area of interfacing CE with MS. “CE/MS is fairly clumsy now,” says Jorgenson, “but with better interfacing, it will become a really powerful technique.” Mary Warner
What will the future bring?
CE has enjoyed phenomenal growth in the 13 years since it was first described in the literature. It has gone from a topic of fundamental research on homemade systems to a technique widely used for routine analysis and basic research. What does the future hold? Can CE possibly keep up this extraordinary pace? Jorgenson believes that the popularity of CE should continue to grow because of the advantages it offers analysts. “It provides really great speed without the regeneration times associated with gradient elution LC. And methods development should also be faster than with LC, partially because the runs are shorter, but also because of the mechanism of increased separation efficiency.” CE also has the potential for better reproducibility than LC because there is no stationary phase to change over time. Ana-
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Analytical Chemistry, Vol. 66, No. 22, November 15, 1994 1141 A
