Capillary Electrophoresis: Automating A Valuable Technique

Picture a silica tube no thicker than a human hair, and then picture a fluid sample in that tube--a sample so small that the tiniest drop of water seems like a flood in comparison to it. Now imagine zapping the tube with electricity, shining a little ultraviolet or visible light on the migrating particles in the sample, and you have the basics for capillary electrophoresis, an analytical technique that holds great promise for investigators in the life sciences. Robert Palmieri, principal research chemist at Beckman Instruments Inc. in Palo Alto, Calif., says capillary electrophoresis, a method refined in the past decade, is a valuable tool for researchers. "It gives different information about the material you're working with in addition to the information you get from HPLC [high-performance liquid chromatography], and ion exchange and gel electrophoresis," he says. Pharmacologists, molecular biologists, biochemists, and other scientists have used capillary electrophoresis during the last few years to analyze new pharmaceuticals for purity and to separate and detect amino acids, proteins, and oligonucleotides. Scientists have also examined the usefulness of this analytical method in a clinical setting. Possible clinical applications are the analysis of therapeutic doses of administered drugs and the fractionation of hemoglobin. Like its grandfather, the widely used gel electrophoresis (The Scientist, July 23, 1990, page 25), capillary electrophoresis (CE) uses an electric current to separate proteins, polynucleotides, polypeptides, amino acids, and other biomo-lecules. In contrast to gel electrophoresis, however, CE is an instrumental technique, like HPLC, and thus can eliminate a lot of the "grunt work" associated with classic electrophoretic methods. "In gel electrophoresis," says Palmieri, "you have to pour or buy the gel, run the sample, stain the gel, de-stain the gel, read it in a scanning device, or photograph it for qualitative and quantitative information. In CE all [these procedures are] combined in one unit." A standard capillary electrophoresis unit includes a sample injector assembly, the capillary, a power supply, and a detector. Samples are introduced into the capillary through gravity (siphoning), electric current (electromigration), or pressure. The capillaries, which are usually made of fused silica, are inserted by hand or contained in easy-to-load cartridges. Common high-sensitivity detectors include ultraviolet or visible wavelength detectors and fluorescence detectors. The history of capillary electrophoresis is tightly interwoven with that of gel electrophoresis. Capillary electrophoresis came into its own in 1974, when Rauno Virtanen, then of the Helsinki Technical University in Otanieni, Finland, reported on experiments he had conducted using open tubular capillaries with inner diameters of 200 to 500 microns (Acta Polytech. Scand. Chem. Incl. Metall. Ser., 123:67, 1974). Applied Biosystems Inc. 850 Lincoln Center Dr. Foster City, Calif. 94404 (415) 570-6667 Model 270A: $39,500 Beckman Instruments Inc. 1050 Page Mill Rd. Palo Alto, Calif. 94304 (415) 857-1150 (800) 742-2345 P/ACE System 2000: Prices range from $31,190 to $47,750 Bio-Rad Laboratories 1414 Harbour Way South Richmond, Calif. 94804 (415) 232-7000 HPE-100: $17,500 Dionex Corp. 1228 Titan Way P.O. Box 3603 Sunnyvale, Calif. 94088-3603 (408) 737-0700 CES I: $38,000 Isco Inc.P.O. Box 5347 Lincoln, Neb. 68505 (402) 464-0231 (800) 228-4250 Model 3140: $25,000 Model 3850: $14,950 Spectra-Physics 3333 N. First St. San Jose, Calif. 95134 (408) 432-3333 Spectra Phoresis 500: $23,000 Spectra Phoresis 1000: $35,000 SpectroVision Scienta Park 25 Industrial Ave. Chelmsford, Mass. 01824 (508) 250-1911 Modular systems: Prices range from $9,995 to $11,795 Waters-Division of Millipore 34 Maple St. Milford, Mass. 01757 (508) 478-2000 Quanta 4000: $28,000 In the 1980s, James W. Jorgenson and his colleagues at the department of chemistry of the University of North Carolina, Chapel Hill, pioneered the development and applications of this technique. They scaled down the size of the capillaries, thereby reducing the amount of sample needed. In addition, they investigated the effects of various physical parameters (such as heat and the type of buffer used) on the efficiency of the separation. In the latter part of the decade, several companies (see accompanying list) developed CE systems and their applications for the marketplace. In addition to saving labor and time (most separations can be done in minutes), capillary electrophoresis has several other advantages over traditional gel electrophoresis. Since the capillaries are so small (usually 10 to 100 centimeters in length, with an inner diameter of 50 to 100 microns), the Joule heat generated by the electric current is quickly dissipated. This eliminates the potential problem of overheating samples; in addition, it prevents convection currents in the sample that can lead to poor resolution. Faster heat dissipation also allows researchers to use higher voltages than those that are normally used in gel electrophoresis. Higher voltages (as high as 30 kilovolts) can lead to shorter run times. The tiny capillaries also mean that scientists can use very small sample sizes (on the order of nanoliters) to do an analysis. Small sample size, says Palmieri, can be a real benefit to those researchers who work with limited amounts of sample to begin with, such as geneticists who perform restriction mapping studies of valuable DNA material. Perhaps the most notable advantage of CE over other electrophoretic methods is its ability to give researchers high-resolution separations. "Because the system is small and can be cooled quickly," says Palmieri, "you don't get band or sample spreading due to heating." The time frame for each run, he says, also plays a role in resolution: "Material will diffuse just on its own [in gel electrophoresis]. The longer you take, the greater the spreading." Better resolution coupled with sensitive detectors increase the wealth of information a researcher can draw from one sample. Even with these advantages, capillary electrophoresis, says Palmieri, will never replace classical gel electrophoresis or HPLC, but will supplement them. However, he notes, CE "definitely has a place in biotechnology. If HPLC could have done it all, it would have. But we [researchers] need additional information." Such "second-dimension" analyses and the extra data they provide are fundamentally important, says Palmeiri, especially to scientists such as pharmacologists, who must ensure the purity of their products. Yet all of the extra information capillary electrophoresis can provide could be a liability, says Palmieri. With the technique's resolving power, researchers now can get more data than they may be capable of analyzing--or than they may be willing to analyze. "A scientist may now be faced with an array of information that he must interpret, but he doesn't have a framework to work against," Palmieri says. He adds that current and future research, particularly in data manipulation and interpretation software, will remove some of the complicating factors involved with quantitation. As with most new forms of analysis, capillary electrophoresis isn't without its glitches. One problem currently under investigation by both industry and university scientists is that of sample interactions with the capillary wall. To circumvent this problem, says Palmieri, researchers are experimenting with various coatings. Another potential setback for capillary electrophoresis in its present state is that the systems cannot be readily scaled up to perform preparative work or to collect fractions. Such processes would require larger capillaries, which, in turn, would raise new challenges in temperature control. "We have yet to see the real final form of this instrument," says Palmieri, who adds that independent scientists and product-development staffs are actively researching cooling systems as well as coatings and packing materials for capillaries, and are looking into ways of varying sample sizes, using multiple detectors, and facilitating interfaces with HPLC systems and mass spectrometers. "Capillary electrophoresis isn't going to do everything," Palmieri says. "It's not a cure-all. But it will definitely be in the analytical chemist's arsenal." Applied Biosystems, based in Foster City, Calif., offers the Model 270A analytical CE system. The complete, compact system occupies 20 inches of bench space. The system, which includes capillaries, buffers, and mobility standards, is controlled by a built-in microprocessor. Users may choose from preprogrammed methods or may set up their own programs. For more data-handling options, the 270A can be hooked up to most liquid chromatography-compatible data acquisition systems. The required sample volume is 1 microliter, but the typical sample injection volume is 5 nanoliters. The remaining sample is available for further analyses or storage. Reproducibility of electrophoretic mobility is said to be less than 1 percent. According to the company, the Model 270A can provide efficiencies of greater than 400,000 theoretical plates per meter. Typical run times are said to be 10 minutes per sample. The Model 270A features an ultraviolet detector. P/ACE System 2000 is an automated CE instrument offered by Beckman Instruments Inc., headquartered in Fullerton, Calif. The unit, which measures 25 inches x 13 inches x 20.25 inches, features interchangeable, temperature-controlled capillary cartridges. Capillaries are continually bathed in liquid coolant to protect thermally labile samples and to improve reproducibility. The injected sample volume ranges from 5 to 50 nanoliters; the required working sample volume is 10 microliters. Samples can be injected either by pressure or by voltage. Samples left in the injection vial can be reused. According to the company, the P/ACE System 2000 has been demonstrated to provide efficiencies of 1 million theoretical plates. Typical run times vary from 1 to 30 minutes. The P/ACE System 2000 is available with or without an IBM PS/2 computer. The system currently features a removable UV selectable-wavelength detector. Bio-Rad Laboratories, located in Richmond, Calif., offers the HPE-100 CE system. This unit, which measures 12.5 inches x 13.5 inches x 16 inches, also uses capillary cartridges, which, according to the company, are rugged and self-aligning. Capillaries are coated with a polymer to minimize sample adsorption and electroendosmosis. Sample loading requires manual injection into the sample reservoir. The minimum sample volume required is 2 to 10 microliters. The HPE-100 system also features a built-in microprocessor and an on-line, variable-wavelength ultraviolet-visible light detector. The Capillary Electrophoresis System I (CES I), offered by the Sunnyvale, Calif.-based Dionex Corp., also takes up relatively little bench space (18 inches x 26 inches x 24 inches). The system's 40-position autosampler features three automated injection modes: electro- migration, gravity, and pneumatic. Injection volumes range from 1 to 10 nanoliters. With menu-driven programming, researchers can schedule the unit to screen up to 40 samples in a number of ways, as well as to rinse the capillary and sample and buffer vials. According to the company, sample runs can generate more than 200,000 theoretical plates in 10 minutes. The CES I also features a power supply that offers three control modes and gradients, allowing users to control power, current, or voltage. The unit has two standard detectors (ultraviolet/visible absorbance and fluorescence detection) for both direct and indirect detection. Isco Inc., headquartered in Lincoln, Neb., has made available two CE systems. The first system, the automated Model 3140 Electropherograph, features a 40-position autosampler; computer-based control over separation parameters, including buffer selection; and a 30-kilovolt reversible polarity power supply for flexibility. The Model 3140 has two sample injection modes: vacuum injection and electromigration. In addition, this model features an automated vacuum purge of the capillary with NaOH (sodium hydroxide) to clean adsorbed solutes. Temperature is controlled within ñ0.5øC to maintain reproducibility. The unit has a variable-wavelength ultraviolet detector (190 to 360 nanometers). Isco also offers the Model 3850 Electropherograph, a manual system that features a split flow sample injector that uses an HPLC-style syringe. A syringe also is used to flush the capillary to remove adsorbed solutes. The minimum sample requirement for the Model 3850 is 1 microliter per analysis; the typical sample load is 1 to 30 nanoliters. This unit, which also features a reversible polarity power supply and a variable-wavelength ultraviolet detector, has an optional recorder and integrator/data system available. Spectra-Physics, based in San Jose, Calif., has made available two CE systems: Spectra Phoresis 500 and Spectra Phoresis 1000. Both systems feature capillary cassettes, temperature control (to within ñ0.1øC), voltage regulation (to within 0.1 percent), reversible power supply for gel separations, 80-sample capacity for overnight runs, fiber optic bench for automatic alignment of the capillary, and a dual-lens system to reduce stray light. Both units offer a choice of hydrodynamic or electrokinetic sample injection systems. The autosampler can accommodate both standard 2-milliliter vials and 50-microliter microvials. The Spectra Phoresis 500, which is intended for routine analyses, has a single-wavelength ultraviolet (190 to 365 nanometers) and visible (365 to 800 nanometers) detector. The Spectra Phoresis 1000 system features three detection modes: single wavelength, for detecting parts per million; multiple wavelength, for monitoring several wavelengths simultaneously; and high-speed scanning, for identifying peaks and detecting trace impurities. This unit also offers three separation modes: constant current, constant voltage, and constant resistance. Optional IBM PS/2 computer controller and data station are also available for the Spectra Phoresis 1000. SpectroVision, of Chelmsford, Mass., offers modular CE systems. Customers have the option of purchasing either complete modular systems or separate components. Typical complete modular systems include a detector, a high-voltage power supply, a sample delivery system, and a start-up kit. All systems include the Model DA-30 power supply, which features high voltage selection in steps of 100 volts up to 30 kilovolts, and current selection in steps of 1.0 microamps to 250 microamps. The Model DS-4 sample delivery system is also a standard component in each modular system. The Model DS-4 can hold anode and cathode buffer vials and up to three sample vials. The type of detector used with each modular component system is different: variable-wavelength absorbance detector, fluorescence detector, variable-wavelength fluorescence detector, and dual monochromator fluorescence detector. Another fully automated CE system, the Quanta 4000, is offered by Waters, a division of Millipore. This system from the Milford, Mass.-based company lets researchers preprogram parameters, such as injection time, run time, sample voltage, and run voltage for each sample. The unit features both automatic hydrostatic and electromigration sample injection modes, a low-noise ultraviolet/visible detector, and an interchangeable high-voltage power supply. The sample chamber has a septumless seal to control evaporation and contamination of samples and electrolytes. To improve accuracy, the Quanta 4000 offers an "Autopurge" feature that automatically cleans the capillaries after each run. After each run, the unit can collect fractions for further analyses.

Capillary Electrophoresis: Automating A Valuable Technique

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