Capillary Electrophoresis Gains Acceptance As Applications Increase

Through the years, a variety of separation techniques have been used by researchers in their continuing challenge to accurately separate and identify analytes of interest from complex mixtures. Numerous procedures, physical as well as chemical, conducted in an assortment of vessels, are often used to achieve this goal. During the past 25 years, column techniques such as gas chromatography (GC) and high-performance liquid chromatography (HPLC) have led to successful separation and identification of thousands of compounds. DETECTION PROBLEMS: Beckman Instruments P/ACE 5010 CE system with compuetr offers alternatives A relatively new process, capillary electrophoresis (CE), commercialized less than a decade ago, is currently one of the fastest-growing areas in separation technology. This rapid gain in popularity is largely due to the impressive benefits CE offers, including the efficient separation of a wide variety of compounds such as amino acids, peptides, proteins, polymerase chain reaction (PCR) products, oligonucleotides, carbohydrates, vitamins, organic acids, polymers, chiral drugs (see story on page 19), dyes, surfactants, and even whole cells and virus particles. In addition, CE effectively automates most of the separations typically attained by labor-intensive gel electrophoresis, reducing the time required to obtain results from hours to minutes, and often seconds. Separated components are quickly identified by online detectors during the analysis, in contrast to the time-consuming staining steps required for gel separations. Because of the capillary's ability to disperse heat quickly, CE allows for a higher applied voltage, in the range of 10 to 30 kV. Joule heating--the heat generated by the passage of electrical current-- is often a frequent problem with traditional slab gels, but can be overcome by going to a capillary format. With only nanoliter sample volumes required for analysis, CE's speed, sensitivity, and versatility make it an attractive separation technique for many scientists. A typical capillary electrophoresis system consists of a fused-silica capillary, with an inside diameter ranging from 25 to 75 mm, that connects two buffer reservoirs, a power supply, and a detector. Molecules are separated as an electrical force drives them at different rates through the capillary filled with electrolyte. A variety of buffers and additives are used to enhance separations. Capillary zone electrophoresis (CZE) is the simplest form of CE because the open-tube capillary is filled with only buffer solution. Separation occurs as solutes migrate in discrete zones and at different velocities. The separated sample components are monitored by a detector as they travel through a segment of the capillary, and the detector signal is displayed as peaks on an electropherogram, similar to a chromatogram. With several operating modes available, a single instrument, often a single capillary, can be used for the practical separation of many different classes of compounds. "This is unlike any other separation technique," says Milos Novotny, a chemistry professor at Indiana University, "because it spans such a broad range of compounds, from small drug molecules all the way up to particles." Although biochemists are generally conservative in accepting new techniques, the potential for this particular one appears enormous, based on the number of users and their applications, he notes. According to Novotny, the three major types of biomolecules currently being examined with CE are proteins, nucleic acids, and carbohydrates. The last is emerging more slowly as an application area for CE because most glycoconjugate samples still require some form of pretreatment. However, he is quick to point out that major advances have been achieved with proteins, including several recent applications that yield protein separations according to their molec-ular weight, a highly desirable feature for biochemists. ALTERNATIVE METHOD: ATI Unicam's Crystal 310 CE system with conductivity detector CE also holds big potential for clinical determinations, because it's a fast technique and provides quantitative information, explains Novotny. For example, he notes that "the analysis of urinary proteins or lipoproteins could lead to the detection of blood-chemistry defects." Mike Kurokawa, CE product manager at Bio-Rad Laboratories in Hercules, Calif., contends that the next big push in CE could be in the clinical market. "There are a variety of clinically significant protein and DNA assays that can run faster, more economically, and provide excellent analytical results by utilizing capillary electrophoresis technology," he maintains. These predictions for using CE as a clinical analyzer may be realized. John Wheeler, a chemistry professor at Furman University in Greenville, S.C., is applying capillary isoelectric focusing (CIEF), one of CE's modes, to clinical measurements. "We're very excited about using isoelectric focusing to separate hemoglobin isotypes," he says. An improved test could be designed in a capillary format for sickle-cell anemia and other hemoglobin-related disorders that would be much more rapid and convenient than the current three-hour gel assays, claims Wheeler. Equipped with Fremont, Calif.-based Thermo Separation Products' SpectraPHORESIS Model 500 and the scanning Model 1000 instruments, Wheeler also has significantly improved CE resolution by using ion-pairing agents--charged surfactant-type molecules --that perform as selective modifiers and change the relative mobility of the analytes of interest, a scheme he adopted from HPLC. "We've been able to provide baseline resolution for several neurotransmitters. Without the modifiers, they would be completely unresolved and elute as a single peak," he explains. Capillary electrophoresis also provides an alternative to traditional slab gel methods for the analysis of DNA restriction fragments, PCR products, and synthetic oligonucleotides. "CE is well suited for high-resolution analysis of DNA in very small sample volumes," says Mike Marino, a section leader at the Armed Forces DNA Identification Laboratory, a Department of Defense facility located in Rockville, Md. Many of the separations typically performed on slab gels or by HPLC can be easily transferred to CE, resulting in enhanced separations with extremely high efficiencies, notes Marino. DNA restriction fragments and PCR products are difficult to separate by conventional open-tube CZE because they all have similar charge-to-mass ratios and tend to migrate through the capillary together. To overcome these difficulties, Marino and his group have implemented two other CE approaches for the analysis of DNA: capillary gel electrophoresis (CGE) and dynamic sieving electrophoresis (DSE). CGE is based on the use of cross-linked linear polyacrylamide gels. By varying the percent concentration of the monomer and the degree of cross-linking, a researcher can create stable gels with a discrete range of pore sizes to separate macromolecules. Covalent bonds between the gel and the capillary wall, and polymer cross-linking, stabilize the gel and create a molecular sieve. Single base-pair resolution, up to 150 base pairs, of synthetic oligonucleotides and PCR fragments can be readily achieved on polyacrylamide gel capillary columns. DSE consists of non-cross-linked entangled or linear polymers, such as hydroxyethyl cellulose, which act as molecular sieves. Unlike CGE, DSE flows freely in the column, and allows samples to be introduced into the sieving system by both electrokinetic and pressure/vacuum injections. DSE can rapidly perform separations in an automated format, and is well suited for high-resolution analysis of compounds present in very small volumes. The use of neutral-coated capillaries eliminates the electroosmotic flow, and ensures that the migration is in order of increasing molecular size. "It's like pouring a new gel for each analysis," Marino observes. "Because gel-filled capillaries can be difficult to make and handle, many users are turning to polymer solutions," says Bio-Rad's Kurokawa. "These hydrophilic polymers create a nonstatic, nongel matrix sieving solution that can be easily pumped in and out of the capillary, so a fresh sieving medium is ready for each separation." Bio-Rad's BioFocus 2000 and BioFocus 3000 CE units offer constant-volume positive-displacement sample injection systems to handle these viscous sieving solutions, he claims. Many areas in the life sciences require reliable analytical techniques for the identification of chiral compounds--materials consisting of three-dimensional isomers that cannot be superimposed on their mirror images. Important applications include pharmaceutical product design and purity control, organic synthetic process development, and medical research investigations, such as metabolism studies. In the pharmaceutical industry, chiral drug enantiomers--mirror-image forms--often have widely different biological properties. Frequently, the undesired enantiomer may take longer to provide the desired effect. It could also lower the efficacy of the beneficial enantiomer, and in the worst cases, it could even have adverse side effects. As a result, regulatory agencies worldwide now recognize the hazards of marketing racemic mixtures of drugs, and have published guidelines that provide for the development and availability of chiral compounds as pure enantiomers. Although separating enantiomers has been one of the most challenging assignments in analytical chemistry, capillary electrophoresis is emerging as an attractive system because of its high efficiencies and minimal consumption of solvents, buffers, and additives. In optimizing enantiomeric separations, a significant effort is devoted to searching for an appropriate chiral selector to perform the separation. Since universally applicable chiral selectors do not currently exist, a variety of materials such as cyclodextrins, detergents, and crown ethers_have been used with CE, in combination with other buffers and additives, to achieve these chiral separations. "We are attempting to really simplify the chiral-separation process for CE users," says Jeffrey Mazzeo, a senior research chemist at the Waters Corp., located in Milford, Mass. Mazzeo is developing a comprehensive series of chiral surfactants that will efficiently separate nearly all enantiomers by micellar electrokinetic capillary chromatography (MECC), a CE mode that separates molecules based on their hydrophobicity. "As analytes partition in and out of these micelles and separate by hydrophobic differences, chiral discrimination can be achieved, as well." Some of the chiral compounds Mazzeo and his colleagues have successfully separated are beta blockers, pseudoephedrine (the ingredient in Sudafed), atenolol, and nicotine, the active ingredient in the transdermal patch designed to cure tobacco addiction. Also, the sweetener aspartame is a dipeptide with two chiral centers, and Mazzeo is able to readily separate all four stereoisomers, a difficult task with other techniques. In addition to Waters, other manufacturers recognize the power of CE for the separation of optical isomers, and are developing their own methodologies. For example, Beckman Instruments recently introduced the eCAP Chiral Methods Development Kit, and Thermo Separation Products offers the Chiral Chemistry Kit to assist researchers in optimizing methods for chiral separations. Both products are cyclodextrin-based systems. A variety of gel-filled and other specialty capillaries are commercially available from several suppliers, including Folsom, Calif.-based J&W Scientific in its PAGE product line; Supelco Inc., located in Bellefonte, Pa.; and Scientific Resources Inc. of Eatontown, N.J. "Developing an optimized, quantitative CE method requires a great deal of experimentation and skill," says Syd Horn, CE marketing specialist at Perkin-Elmer Corp.'s Applied Biosystems division, located in Foster City, Calif. "We focus on trying to make these techniques more reliable and easier to use by developing specialty-application kits." These kits contain specific buffers, chemistries, and very precise protocols so that a user can become successful very rapidly, explains Horn. Among the recently introduced CE Specialty Application Kits from Perkin-Elmer are the MicroCoat for protein analysis, ProSort for protein molecular weight determinations, ProFocus for protein isoelectric focusing, and the Anitron Analysis Kit for inorganic anions and organic acids. The process of detection in CE presents a major challenge due to the small dimensions of the capillary. Although there are several detection methods available, greater sensitivity is still high on many users' wish lists. "I would like to see sensitivity increased by an order of magnitude," comments Steve Edelmuth, a senior research analytical chemist at Abbott Laboratories in North Chicago, Ill. Edelmuth has been using his system mostly for proteins and other large analytes that are difficult to separate by HPLC. UV-Visible (UV-Vis) absorption is the most frequently used detection method, and is achieved by monitoring absorbance directly online through a window in the capillary. With fused-silica capillaries, wavelengths below 200 nm and up through the visible spectrum can be used. Normal fluorescence detection is also available on some instruments. "The Dionex CES I system has the ability to do UV-Vis or fluorescence detection at the touch of a button," says Greg McLaughlin, the CE product manager for Sunnyvale, Calif.-based Dionex Corp. "The overall benefit is to obtain increased sensitivity or detection selectivity." Systems such as the P/ACE 5000 Series from Beckman Instruments, located in Fullerton, Calif., offer other detection options, including laser-induced fluorescence, diode array, and mass spectrometry. "Users are looking for instrumentation with a modular format so they can use different detectors," maintains David Minicuci, a product specialist with Thermo Separation Products. "They often want to use their own lasers and mass spectrometers to improve sensitivity." A SOLUTION FOR SOLUTIONS: Bio-Rad Laboratories' BioFocus 3000 Capillary Electrophoresis System Sensitivity is further enhanced by Extended Light Path capillaries on the HP 3D Capillary Electrophoresis system from Palo Alto, Calif.-based Hewlett-Packard Co. These capillaries contain an expanded-diameter section at the point of detection, reportedly increasing sensitivity three-fold over straight capillaries. The optics have been designed to minimize stray light and noise. Also designed for higher sensitivity is the new immersed-flow cell from Jasco Inc. of Easton, Md., for fluorescence detection. When the unit is used with the Model FP-920 fluorescence detector, the flow cell minimizes the amount of stray light that results from refractive index differences at the air/fused-silica and liquid/fused silica interfaces. The capillary is immersed in a quartz cell containing a liquid with a refractive index identical to that of the fused-silica capillary, significantly reducing stray light and increasing sensitivity, explains Wayne Kott-kamp, Jasco's vice president of marketing. Conductivity--the movement of electrically charged materials--offers another method for CE detection. "We have developed conductivity as a way to detect organic acids and inorganic ions in biological matrices," says Jack Korpi, the marketing manager for CE at ATI Unicam of Boston. It may be possible to measure oxalic acid and some analogous acids in urine samples in order to diagnose metabolic problems involving kidney stones, explains Korpi. "Since there is no column material to become contaminated, the separation is extremely reliable and could be the basis for a future clinical method." Researchers like Rohan Wilmalasena, a scientist at Procter & Gamble Pharmaceuticals in Cincinnati, are currently benefiting from conductivity detection in industrial applications. Wilmalasena is using his ATI Unicam conductivity detection system to measure the concentrations of numerous organic and inorganic ions and cations for a methods-development project. Philip Wyatt, the president of Santa Barbara, Calif.-based Wyatt Technology Corp., proposes using light scattering (LS) measurements to determine the mass of eluting proteins from a CE column by measuring the intensity of the molecules' scattered light from different angles. "Light scattering is an absolute method," notes Wyatt. "If the scattering of molecules can be measured from a solution, their sizes and molecular weights can be determined without calibration or reference to any standard." Although a system is not yet commercially available, Wyatt has successfully demonstrated the feasibility of making online LS measurements from CE-separated samples with his company's multiangle miniDAWN LS detector. Communications is an essential ingredient to the growth of capillary electrophoresis. The number of publications describing CE applications in biochemistry, pharmaceuticals, food, and chemicals is growing rapidly. Scientific conferences also provide excellent opportunities for CE users to meet, exchange ideas, and plan collaborations. Coming up is the 17th International Symposium on Capillary Chromatography and Electrophoresis, to be held from May 7 to 11 at the Wintergreen Conference Center and Resort in Wintergreen, Va. The scientific program features 30 sessions that include more than 150 presentations. Tutorial sessions covering both fundamentals and recent advances are also planned. For more information, contact symposium coordinator Joy Wise, (301) 473-8312. In addition, the Sixth Annual Frederick Conference on Capillary Electrophoresis, sponsored by the Frederick, Md.-based Science Applications International Corp. (SAIC) and the National Cancer Institute's Frederick Cancer Research and Development Center, will be held October 23-25 at Hood College in Frederick. Also, a one-day course will be held there on Sunday, October 22. For more information on the course or the scientific program, contact Margaret L. Fanning, Conference Coordinator, SAIC Frederick, NCI-FCRDC, P.O. Box B, Frederick, Md. 21702-1201; (301) 846-5865. Fax: (301) 846-5866. Early next year, the Eighth International Symposium on High-Performance Capillary Electrophoresis (HPCE '96), attracting prominent researchers from around the world, will be held January 21-25 in Orlando, Fla. For more information, contact Shirley Schlessinger, 400 E. Randolph Dr., Suite 1015, Chicago, Ill. 60601; (312) 527-2011. Fax: (312) 527-3437.

Capillary Electrophoresis Gains Acceptance As Applications Increase

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