Unit Allows Molecule Measurement Via Capillary Electrophoresis

Applied Biosystems of Foster City, Calif. has introduced the first commercially available instrument that allows scientists—biochemists, for instance—studying biomolecules to take advantage of the innovative technique of capillary electrophoresis (CE). Basically, the Model 270A CE system is an analytical tool that can separate and identify molecules by differences in charge density. CE promises to complement and, in some cases, improve upon the three standard separation techniques: liquid chromatography, ultracentrifugation, and electrophoretic techniques. Liquid chromatography is primarily a preparative separation technique based on molecular weight or net charge; ultracentrifugation is also a preparative technique, which separates molecules by differences in sedimentation; and electrophoresis techniques provide analytical separations through isoelectric point or molecular weight. It has long been recognized that many types of molecules can be identified by differences in electrophoretic mobility, and these three techniques have provided the experimental foundation for the isolation and analysis of biomolecules. For example, polyacrylamide gel electrophoresis (PAGE) has been one of the most widely used methods in protein analysis, resolving proteins on the basis of their molecular weight or isoelectric point. But gel electrophoresis is time-consuming. It requires gel casting, sample preparation, lengthy running time, and gel staining and destaining. None of these procedures is readily automated, and all suffer from problems with quantitative accuracy and reproducibility. In addition, gel electrophoresis generally requires denaturation of the sample proteins and thus limits the amount of structural information that can be obtained. Capillary electrophoresis is a form of free-zone electrophoresis in which molecules are resolved via differential electrophoretic mobility while free in solution. Unlike PAGE, which achieves separation of molecules by either molecular weight or isoelectric point, CE resolves molecules by differences in charge density, thus offering an analytical technique which complements existing electrophoretic and chromatographic techniques. While traditional methods of native gel electrophoresis yield information on the native molecular weight of proteins, capillary electrophoresis offers advantages over such methodology in four ways: a minimum volume of sample is required separation time is greatly reduced concomitant with non-compromised resolution; staining and destaining of genes is not required and multiple gels of different percentage acrylamide are not necessary. The new electrophoresis system from Applied Biosystems couples the separation capabiities of electrophoresis with the more advanced analytical sophistication of the high-performance liquid chromatography (HPLC). The Model 270A uses a small bore (50 pm) capillary tube to achieve the separation of molecules under non-denaturing conditions. The small capillary diameter provides an efficient heat dissipation mechanism so that resolution is not compromised by convective heating, thus permitting the use of higher voltages for faster, more efficient separations. Fast Separation The Model 270A routinely provides efficiencies of separation greater than 400,000 theoretical plates per meter. In addition, the sample volume required is only 1 ul and only 5 ul is actually injected. The remaining sample is available for subsequent analysis. Separation is extremely fast with run times between six and 12 minutes with less than four minutes waiting time between injections. Sample injection can be achieved either by aspiration or electrokinetically (the former method is preferred). Sample detection is achieved with a UV/visible detector. The detector’s window is on the capillary itself, thus eliminating column dead space prior to sample detection. As little as 10 to 20 picograms of a compound can be detected and quantitated. The new CE system offers single-point control (with a built-in microprocessor) that includes sample and internal standard injection and high voltage control and detection. The Model 270A interfaces easily with most liquid chromatography-compatible data acquisition systems, from chart recorders to PC-based systems for increased data handing capacity. Multiple samples can be loaded onto the instrument allowing automatic, unattended separation. Potential applications for the CE system include many areas in chemistry, biochemistry, and pharmacology. The Model 270A has successfully resolved peptides differing only by one amino acid in D-versus L-isomeric forms. Large proteins, identical in primary structure, and differing only slightly in glycosylation, can be resolved easily via CE analysis. Apparent homogeneous peaks obtained via HPLC analysis have, in certain cases, been resolved as heterogeneous peaks following subsequent CE analysis. Even so, CE should not necessarily be considered a replacement for these older techniques. Instead, capillary electrophoresis methodology is, in many ways, a complementary technique to PAGE and HPLC. Molecules purified via PAGE or HPLC can be analyzed further with CE to detect more subtle differences. “Unlike traditional gel methods, the Model 270A CE uses a different fundamental property of molecules to achieve separation. CE offers scientists an orthogonal insight into sample compositional analysis,” says David Ransom, product manager for Applied Biosystems. Capillary electrophoresis, in addition to bridging the gap between gel electrophoresis and HPLC analysis, offers a new approach to the separation and analysis of biomolecules. The system is available for $39,000 and can be tailored to suit the individual needs of the investigator. Wendy Wilson Sheridan is associate director of foundation and corporate relations, and V. Richard Sheridan is a postdoctoral associate in pharmacology both at Fox Chase Cancer Center, Philadelphia.

Unit Allows Molecule Measurement Via Capillary Electrophoresis

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