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Bright future for capillary electrophoresis: the efficiency, speed, cost and environmental advantages of new CE instruments are making them strong complementary tools to established HPLC installations.

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.

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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 arc 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 bio-pharmaceutical 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.

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.

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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.

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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.

RELATED ARTICLE: The Family of Capillary Electrophoresis

* Capillary zone electrophoresis (CZE) is the simplest form of CE and the most commonly utilized. CZE is based on differences in the charge-to-mass ratio of the analytes. In CZE, the capillary is filled with a homogeneous buffer, and compounds are separated on the basis of their relative charge and size.

* Micellar electrokinetic chromatography (MEKC) is a modification of CE where the samples are separated by differential partitioning between micelles (pseudo-stationary phase) and a surrounding aqueous buffer (mobile phase). The setup and detection methods used for MEKC are the same as those used in CE. The difference is that the solution contains a surfactant at a concentration greater than the critical micelle concentration (CMC). Above this concentration, surfactant monomers are in equilibrium with micelles. In most applications, MEKC is performed in open capillaries under alkaline conditions to generate a strong electroosmotic flow. Sodium dodecyl sulfate (SDS) is the most commonly used surfactant in MEKC applications. The anionic character of the sulfate groups of SDS cause the surfactant and micelles to have electrophoretic mobility that is counter to the direction of the strong electroosmotic flow.

* Micro-emulsion electrokinetic chromatography (MEECK) is a capillary electrophoretic technique in which a microemuls on is used as carrier electrolyte. Analytes may partition between the aqueous phase of the microemulsion and its oil droplets, which act as a pseudostationary phase. MEECK is well suited for the separation of neutral analytes but can also be employed for charged analytes.

* Capillary gel electrophoresis (CGE) is a form of CE in which a polyacrylamide gel (or other polymeric material) is placed inside the capillary and separation is based on size and charge. CGE is often used to separate oligonucleotides and proteins.

* Capillary isotachophoresis (CITP) is a moving boundary electrophoretic technique in which a combination of two buffers is used to create a state in which separated zones all move at the same velocity. The zones remain sandwiched between leading and terminating electrolytes.

* Capillary electrochromatography (CEC) is a hybrid of HPLC and CE techniques. Here, the CE capillaries are packed with HPLC packing, and a voltage is applied across the packed capillary, which generates an electro-osmotic flow (EOF) that transports solutes along the capillary toward the detector. Both differential partitioning and electrophoretic migration of the solutes occurs during their transportation toward the detector, which leads to CEC separations. It is therefore possible to obtain unique separation selectivities using CEC compared to both HPLC and CE. The beneficial flow profile of EOF reduces flow-related band broadening and separation efficiencies of several hundred thousand piates/m are often obtained in CEC. There is no back pressure when EOF occurs, so small particle sizes such as one to three microns can be used to improve separation efficiencies.

* Capillary isoelectric focusing (CIEF) is an electrophoretic separation that employs a pH gradient within the capillary to separate zwitterons, usually proteins and peptides, based on each solutes pl--the isoelectric points of the protein samples. Differences of only a few hundredths of a pH-unit in isoelectric points are sufficient to resolve proteins from each other.

* Non-aqueous capillary electrophoresis (NACE) can provide a large improvement in the selectivity of conventional CE without the use of aqueous additives, such as surfactants or cyclodextrins.
Capillary Electrophoresis Mode

Analyte           CZE  CITP  MEKC  CGE  CIEF

Small ions        X    X
Small molecules   X    X     X
Peptides          X    X     X     X    X
Proteins          X    X           X    X
Oligonucleotides             X     X
DNA                                     X

Source: Handbook of CE, James Landers


by Tim Studt, Editor In Chief
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Author:Studt, Tim
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Date:Jun 1, 2010
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