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Bright Future for Capillary ElectrophoresisThe efficiency, speed, cost and environmental advantages of new CE instruments are making them strong complementary tools to established HPLC installations.by Tim Studt, Editor in Chief
Beckman Coulter’s PA 800 plus is an automated, simplified, and robust CE-based system designed for characterizing therapeutic proteins. | Swedish biochemist Arne Tiselius was awarded the 1948 Nobel Prize in Chemistry for research he did nearly 20 years earlier “on electrophoresis and adsorption analysis, especially for his discoveries concerning the complex nature of serum proteins.” This work has evolved in today’s current capillary electrophoresis (CE) analytical instruments that are increasingly being used in protein-based drug development, genetic analyses, carbohydrate studies, forensics, agrochemical development, and chemical research and manufacturing, among other applications. The basic CE technology has also led to the development of a whole family of CE-based instrumentation technologies (see sidebar on opposite page), some widely used and others still not quite living up to their original expectations.
While a number of commercial CE instruments have been available for more than 20 years, it’s only been within the past five years or so that their acceptance peaked and new instruments were introduced that are competitive with existing analytical instrumentation standards. Since the very first discoveries of DNA more than 50 years ago, electrophoresis techniques have been utilized to identify and characterize genomic entities. Technologies developed before, during and since the completion of the Human Genome Initiative a decade ago, however, have been instrumental in advancing the state-of-the-art in the CE arena.
“Capillary electrophoresis now competes directly with and is complementary to HPLC (high-performance liquid chromatography),” says Hans Dewald, CE marketing manager at Beckman Coulter, Fullerton, Calif. Beckman’s CE-based PA 800 plus Pharmaceutical Analysis System was introduced last year following development collaboration with biopharmaceutical and QC groups. Its automated applications provide reproducible and quantitative results for high-resolution SDS (sodium dodecyl sulfate)-gel separation for protein purity determinations; advanced capillary isoelectric focusing (CIEF) for charge heterogeneity analyses; and carbohydrate profiling by assessing glycoprotein microheterogenity.
“CE’s faster analysis times with much less to no solvent requirements in some applications, along with its software-implemented easy-to-use operation, are proving to be driving factors in its increasing acceptance,” says Dewald. “It used to take a lot of training to operate a CE system—it’s not a constant pressure system like HPLC. But new versions of its software operating system simplifies that to selecting an application, loading the samples and application reagents, and acquiring the data.”
The PA 800 system includes a high-resolution separation module, UV, PDA (photodiode array), and laser-induced fluorescence detection, sample temperature control, a high-speed system controller with integrated applications-based data analysis, validated turn-key method and system application guides, and a starter kit of necessary hardware supplies.
Agilent Technologies also introduced its new Agilent 7100 CE System last year, providing more sensitivity than previous commercial CE systems. “Electrophoresis is one of our core technologies, and we’re seeing strong CE growth in biological drug QA/QC, environmental analysis, food safety, and life sciences,” says Nitin Sood, GM of Agilent’s electrophoresis business. “The 7100 brings unprecedented HPLC-like sensitivity to a wide range of analytical challenges.”
The 7100 offers a wide selection of detectors (from various suppliers) for flexibility and sensitivity. The instrument also performs the full range of CE separation techniques, including CEC for fast separation of closely related compounds. The 7100 also provides plug-and-play connectivity to Agilent’s mass spectrometers, including single- and triple-quadrupole, ion trap, and time-of-flight systems.
The basic operation of capillary electrophoresis is comparatively simple. The speed of movement of an ion in a solution is a reflection of its charge and the effect of the potential difference from an electric field acting on it. The ion experiences motive forces from the electric field and retardant forces from the friction or resistance in the medium (capillary tube) that it is suspended in. The larger the charge on the ion, the faster it moves through the electric field, which allows different ions to be discriminated and identified from each other.
Agilent 7100 Capillary Electrophoresis System offers fast separations with exceptional efficiency and resolution with a broad selection of detectors and plug-and-play compatibility with Agilent mass spectrometers. | CIEF is routinely used for protein pI (isoelectric point) determination, identification, characterization, and stability monitoring of proteins. Compared to conventional slab gel IEF, CIEF offers higher resolution, faster analyses, and quantitation and automation capabilities. In many applications, CIEF has become a completely automated, microprocessor-controlled easy-to-use analytical technique from sample introduction to data readout. A variety of detection methods are available, including fixed wavelength UV, laser-induced fluorescence, and mass spectrometry.
Most CE systems employ UV or UV-Vis absorbance as their primary mode of detection. In these situations, a portion of the capillary tube is optically transparent (window), and the detector is focused on the analyte. For fluorescence detectors, this focusing requirement can become complicated.
CE/MS systems combine the short analysis time and high separation efficiency of CE with the molecular weight and structural information from the mass selective detector.
Applications
The major application areas for CE include life sciences, pharmaceutical, chemical analysis, and food and flavors. The main advantages for life sciences are the higher separation efficacy in comparison to chromatographic methods and the smaller sample volume required. CE is used extensively in the characterization of macromolecules used as biologics, as well as in proteomic or metabolomic studies and the interactions of proteins with other proteins.
The CE advantage for pharmaceutical studies includes the faster and more robust separation methods in order to reduce the time-to-market for new drugs, from drug discovery through quality control.
Lower cost per analysis and reduced cycle times are the advantages CE has in the chemical analysis application area, such as in the analysis of small anions and cations in water and solid waste environmental studies. CE is also used to monitor water quality control in the semiconductor industry.
Typical analysis applications for CE in the food and beverage industry include the characterization of organic acids, inorganic anions, carbohydrates, cations, amino acids, pesticides, herbicides, proteins, and vitamins. CE is ideally suited for these applications due to the compatibility with complex sample matrices, fast run times, easy method development, and the availability of pre-defined methodologies and solution kits.
“The lion’s share of new growth in CE is in the characterization of therapeutic proteins,” says Beckman’s Dewald. This application takes advantage of all of CE’s strengths in analysis speed, cost, waste minimization, strong separation power, software capabilities, and ease-of-use.
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