Researchers tip their caps to capillary electrophoresis—a pretty powerful method of separating analytes that has found its place in the hearts of old school chromatographiles.
People are always trying to separate one thing from another. They separate whites from colors before washing clothes. They separate their socks from their shirts when putting away their laundry. But these things are, physically, very easy to separate. However, imagine trying to separate one charged molecule from another so that it can be isolated and purified. That is a tougher task, requiring a much more complex separation technology than the human hand. It requires electrophoresis.
Electrophoresis has been around for ages, predating molecular biology by decades. The basic principle common to all electrophoretic methods, regardless of its application, is that a charged molecule is pulled through a liquid or gel by an electric force, usually generated by an electric power source. But in the 1960s, a variation on this theme was born: capillary electrophoresis. In the case of capillary electrophoresis, the conduit for the analyte’s electrophoretic mobility is, as the name suggests, a capillary. And it is in this capillary that the sample is separated into its different ionic parts, which are also detected within the same capillary … a neat, self-contained system, indeed.
David D.Y. Chen, PhD, professor of chemistry, University of British Columbia, Vancouver, British Columbia, Canada started his career developing fluorescence detectors for capillary electrophoresis about 20 years ago. Then, around 14 years ago—at the beginning of his independent research career—Chen decided to study the fundamental mechanism of capillary electrophoresis. In addition, he is using this technology to determine chemical binding constants such as protein-ligand binding constants.
“It turns out that capillary electrophoresis is probably the most powerful separation techniques because the driving force in capillary electrophoresis is a lot more complex than in chromatography or gel electrophoresis because it uses both the physical field effect and chemical equilibrium for the separation,” says Chen. He adds that it is a little known fact that “the human genome project was largely a success because of the development of the capillary electrophoresis method.”
The chromatography club
General chromatography is based on the partition coefficients of the molecules to be separated. “But in capillary electrophoresis you have the partition coefficient difference and you have what I call a discriminatory field that some molecules feel strongly, others feel weakly, and others don’t feel at all,” says Chen. “The two factors have an additive effect.” Chen adds that although chromatography is a well- established method, scientists in today’s environment should start with the most powerful technique available to them rather than choose an easy way.
Apryll M. Stalcup, PhD, professor, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio, started doing capillary electrophoresis in 1993. The reason: to investigate potential chiral selectors that could be developed into chiral HPLC stationary phases. “In chiral separations at the time, most of the development was done by thin layer chromatography using chiral additives in the mobile phase,” says Stalcup. But development required a lot of work and detection of the chiral analyte in the presence of the chiral additive was not always easy. “In addition, with liquid chromatography, you do generate a lot of waste solvents. So capillary electrophoresis—with very minimal amounts of solvent and that being water—was just very appealing.”
Stalcup performs a specific kind of capillary electrophoresis called electrokinetic chromatography (also known as carrier mode electrophoresis). In this method, the chiral additive that discriminates between the chiral analytes is added to the background buffer. The capillary is filled with the buffer containing the chiral additive and then a voltage is applied. The analytes are detected by an ultraviolet light spectrophotometer.
A lot of Stalcup’s work is related to pharmaceuticals and chiral separation. “Most of the compounds that we look at have aromatic rings in them, and these can be detected at wavelengths you would not ordinarily use in liquid chromatography but you would use them easily in capillary electrophoresis,” says Stalcup. She also adds that there is very little background noise contributing to the signal, so she can get away with measuring absorption at wavelengths down around 210 nanometers.
“One of the things I have noticed is that as wonderful as capillary electrophoresis is, industry has been a little less enthusiastic about it, particularly in the pharmaceutical industry,” says Stalcup. “They need assays in support of drug development or content uniformity and need those assays to be robust and fairly idiot-proof. There is a lot of subtlety to capillary electrophoresis and reproducibility is a challenge.” Stalcup uses a commercially-available capillary electrophoresis system from Beckman Coulter, Fullerton, Calif. Please see www.drugdiscoverytrends.com/capillary-electrophoresis for a list of additional vendors who produce capillary electrophoresis systems.
Lack of manufacturers
A number of years ago, Catherine A. Hammett-Stabler, PhD, DABCC, FACB, Department of Pathology and Laboratory Medicine, University of North Carolina Hospital, Chapel Hill, N.C., faced the challenge of finding a manufacturer of capillary electrophoresis systems that were designed for use in a clinical laboratory. Hammett-Stabler uses capillary electrophoresis instead of the traditional protein electrophoresis method to analyze human serum for atypical proteins, to detect, for example, monoclonal gammopathies.
“UNC has a fairly active hematology/oncology program that takes care of patients who have multiple myeloma. And so, we often run anywhere from 20 to 30 patient samples for serum and urine protein electrophoresis everyday. Capillary electrophoresis lets us get our work done faster and a little more cost-effectively,” says Hammett-Stabler. In fact, capillary electrophoresis allows the laboratory to run unlimited samples throughout the day, in contrast to traditional protein electrophoresis using gels, which requires batching and is restrictive in terms of the number of samples that can be loaded onto a gel.
The biggest limitation for Hammett-Stabler is that there are not many manufacturers that develop capillary electrophoresis systems for clinical labs, leaving very little to choose from. They use a capillary electrophoresis system developed and sold by Sebia Electrophoresis, Norcross, Ga., because it was specifically designed for use in clinical labs and because the company provides technical support should the system go down. “We cannot wait two to three days for service. We have patients who are waiting for results to have their next phase of treatment. So we wanted a company that was able to meet our needs,” says Hammett-Stabler.
In summary, the collection of separation techniques on the market today is surely overwhelming. And often times, the researcher must sift through the method literature to find the best method for their problem. There is no doubting a gold standard, though. And chromatography and gel electrophoresis surely fall into this category. Capillary electrophoresis has been so widely adopted by basic researchers and industry scientists that it soon might join the ranks of this elite class of methods. Welcome to the club!
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 5, May, 2008, pp. 42-44.
Filed Under: Genomics/Proteomics