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February 2002
Vol. 11, No. 2
pp 59–60, 63–64.
Today's Chemist at Work
Focus: Separation Science


Capillary electrophoresis: Finding a niche

Where HPLC struggles, CE thrives.

Interest in capillary electrophoresis (CE) has waxed and waned in the decade since the first commercial instrument was introduced. Now, CE seems to be establishing itself as the technique of choice for certain applications.

For example, according to company representatives and experts, CE provides chiral analysis at a lower cost and is better than HPLC at separating highly charged and polar species. CE’s facility for small ion analysis has made it a mainstay in the food and beverage industries. And biotech companies have started to incorporate CE in their research as well as into QA and QC. DNA sequencers using CE also played a major role in sequencing the human genome.

When last reviewed in Analytical Chemistry in 1996, the commercial CE field had narrowed to a handful of companies. (Dedicated DNA sequencing instruments were recently reviewed [Anal. Chem. 2002, 73, 327A–331A] and are not included in this discussion.) Since that time, the field has narrowed even further to just a few companies, with two giants, Beckman Coulter and Agilent Technologies, becoming the dominant players.

Table 1 lists representative features of CE instruments from five companies. Readers interested in these products should contact the companies for a complete list of features.

A Short History
As with most new technologies, CE was initially fraught with technical difficulties. Barry Karger, director of the Barnett Institute at Northeastern University, likens it to the early days of LC. In the beginning, he says, “[LC] was horrible. Every column was different. It was many years before the technique became robust.”

However, some of the problem may have been the users who first bought the instruments. “Chromatographers tried to [use CE] and did not fully recognize that it was electrophoresis. And CE was governed by those rules, not chromatographic ones,” says David Heiger of Agilent. “For those who know electrophoresis, you have a much easier entrance.”

Although the chromatography problem still exists, Jeff Chapman of Beckman Coulter says that the adoption of “CE thinking” has been key to the technique’s success. Instead of migration or retention times, which are chromatographic descriptors, CE focuses on mobility. “The use and development of mobility has changed the face of how we operate,” claims Chapman. “It took CE from what was perceived as not reproducible to [a technique] which is highly reproducible.” Chapman adds that coefficients of variation of <0.3% have been demonstrated across CE instruments, capillaries, and buffers using mobility as the “index of identification”.

Another problem was how CE was being applied, says Jim Jorgenson from the University of North Carolina. “In the process of evaluating CE over the past decade, people tried applying it to everything,” recalls Jorgenson. “To those people working on problems that LC is good at handling, CE has often been a disappointment. To those working in fields where LC is inadequate, CE has been a godsend. It is now becoming clearer what its strengths and weaknesses are relative to LC.”

Going with Strengths
Although CE was initially heralded for its speed and low sample volume, the technique is valuable because it is quantitative, can be automated, and will separate compounds that have been traditionally difficult to handle by HPLC, say experts. Jorgenson calls CE the “best-kept secret in chemical analysis,” capable of analyzing complex carbohydrates and protein–protein interactions. He adds that CE is the basis for virtually all microfluidics for lab-on-a-chip devices.

Ed Burton of Procter & Gamble adds that CE can also separate polar substances, which are notoriously difficult to analyze with HPLC. For example, Burton and his colleagues were able to study multicharged and water-soluble bisphosphonates by CE. And they have been able to run analyses in the presence of surfactants, which can be found in practically every product Procter & Gamble produces.

Chiral separations is another area in which CE use has expanded. Chapman reports that he and his colleagues have separated 90% of the compounds that customers have thrown at them with a single capillary and a proprietary line of highly sulfonated cyclodextrins.

What’s New?
The small sample volumes required for CE can be an advantage with limited samples; however, with its short pathlengths, the technique can suffer from low sensitivity. One solution, introduced by Agilent, is the “bubble cell” capillary, which increases the capillary internal diameter at the point of detection, increasing sensitivity by a factor of 3 to 5. Agilent has introduced a newer cell, which the company claims increases sensitivity by a factor of 10 over conventional capillaries. The proprietary cell does this by increasing the pathlength from 75 µm to 1.2 mm while eliminating stray light.

UV is the predominant detection method used with CE; however, laser-induced fluorescence (LIF) and MS have been added to the detection arsenal. Agilent, as well as Beckman Coulter (in conjunction with Thermo Finnigan), offer CE-MS options. According to Heiger, sensitivity with CE-MS is still a problem. But, he adds, CE-MS is now as easy to use as LC-MS. Thus, for problems that can’t be solved by LC-MS, CE-MS is the right choice, says Heiger.

The Goods
In addition to its CE system, Agilent sells the 2100 BioAnalyzers, lab-on-a-chip devices that use electrophoretic separations. The BioAnalyzer is more of an appliance than an analytical instrument, with each chip doing one thing such as RNA, DNA, or protein analysis. With these chips, no method development occurs. If it fits the application and the required sensitivity, you insert your sample and run it.

Beckman Coulter has eight automated, application-based capillary array systems, marketed under the name P/ACE MDQ. The application-based systems—chiral, carbohydrate, molecular characterization, DNA, glycoprotein, education, and QC—differ in the way they are configured and the types of detectors and chemistries coupled to them. The carbohydrate system, for example, comes only with a LIF, whereas the molecular characterization system comes with a UV detector and a two-color LIF unit for detecting nucleic acids and proteins.

CombiSep, a start-up company founded in December 1999, offers the only multiplexed absorbance-based CE instrument. (The other multiplexed instruments are principally for DNA sequencing and are equipped with LIF detectors.) On the basis of technology developed by Edward Yeung’s group at Iowa State University, CombiSep’s MEC 2000 combines massively parallel processing with UV detection and software that can analyze 96 samples in a few minutes.

Prince Technologies offers a completely modular system, which sports a patented dynamic compression injection unit, giving <1% relative standard deviation for migration times and peak areas.

On the Horizon
An area closely related to CE that has yet to reach its stride is capillary electrochromatography (CEC), which combines features of CE with LC by using capillaries packed with chromatographic materials. Although no products have yet hit the CEC market, some of the problems have been solved, so they may start appearing over the next few years.

The term “high throughput” took on a new meaning when Richard Mathies at the University of California–Berkeley built a prototype CE instrument with 1000 capillaries. Mathies has designed a CE–DNA sequencer for Molecular Dynamics; however, he sees chips as the future of CE. “Performing CE on microfabricated channels gives better performance, higher speed, and a more robust and reliable package,” Mathies says. He recently ran a 384-lane genotyping microchip system and found it to be much faster and more reliable than the earlier 384-lane discrete capillary device.

The Niche
Will CE ever overtake HPLC? Some say that’s not the appropriate question anymore. Ian Mutton of GlaxoSmithKline explains, “CE comes into its own for large molecules and when sample size is limited. This tends to give its best applications a biological flavor, and it can powerfully address problems where HPLC has little chance of success—the Human Genome Project being the obvious example.”

Now that CE has been officially recognized by several regulatory agencies—the Food and Drug Administration and the Center for Drug Evaluation and Research among them—it is finding a niche in QA and QC labs as well. And with CE’s hugely successful foray into genomics—it being the undisputed reason the Human Genome Project finished well ahead of schedule—the question is whether it can do the same for proteomics, the next phase of genomic research.

This report is adapted from Analytical Chemistry, Sept. 1, 2001, p 497A.

Laura DeFrancesco is a freelance writer based in Pasadena, CA. Send your comments or questions regarding this article to tcaw@acs.org or the Editorial Office, 1155 16th St N.W., Washington, DC 20036.

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