Revival of Capillary Electrophoretic Techniques in the Pharmaceutical Industry - - Chromatography Online
Revival of Capillary Electrophoretic Techniques in the Pharmaceutical Industry


LCGC North America
Volume 30, Issue 11, pp. 954-971

The first observations of electrophoresis were made in 1807 by Ferdinand Frederic Reuss. Arne Tiselius (1902–1971) introduced electrophoresis as an analytical technique in 1937 (1) and was rewarded with a Nobel Prize for it in 1948 (2). One of his PhD students was Stellan Hjertén (1928–), who further developed the technique in open tube electrophoresis. His thesis was published in English in 1967 in Chromatographic Reviews (3) and he has been rewarded many times over the years for his pioneering work, most recently he was given the 2011 CASSS Award for Separation Science (4). Other pioneers in the field include Virtanen (5) and Mikkers and Everaerts (6) who performed electrophoresis in 200-μm PTFE and glass tubing. In 1981, Jorgenson and Lukacs made the important step to apply 75-μm fused-silica capillaries (7); afterward capillary electrophoresis (CE) really took off.

What Is Capillary Electrophoresis?

CE can be described as an automated, analytical version of the conventional electrophoresis techniques. The two sidebars in this article explain the separation principles of CE. The main advantages of performing electrophoresis in a capillary are the magnificent efficiencies and the automation possibilities. Because of the small diameters of the capillary, typically in the 20–100 μm inner diameter range, the Joule heat dissipation is very efficient. Consequently, high voltages, usually up to 30,000 V, can be applied before excessive Joule heating becomes an issue. Applying high voltages results in fast separations with very little band broadening.

Usually, the capillary is made from fused silica, so on-column UV detection is feasible. Therefore, time-consuming staining and destaining procedures as seen with slab-gel electrophoresis are no longer needed. The combination of the small-inner-diameter capillary and on-column UV detection means that automated equipment has been developed with peaks resulting in electropherograms, simplifying quantitative analysis. Small bands show as efficient peaks with high plate numbers.


Table I: The different modes of capillary electrophoresis
Electrophoresis separates analytes that differ in charge-to-size ratio, so charge is a prerequisite for separation by CE. Several solutions have been created to induce charge and size differences in a capillary, as well as to add extra separation mechanisms. Table I lists some of the most frequently used modes of CE.

CE is applicable over a wide range of analytes. Anything from small anions and cations to chiral separations, large proteins, DNA, cell organelles, and even complete cells and viruses have been analyzed with CE. The small scale of CE makes it a very green technique. Typically, a 50-cm long, 50-μm i.d. capillary has a capillary volume of 1 μL. Most often the separation medium is aqueous. So, the consumption and cost of chemicals are very low.

The small scale of the techniques also means that only a few nanoliters of sample are injected. This is advantageous for application areas in which sample size is an issue, such as bioanalysis or the discovery phase of drug development.

Orthogonality

Electrophoresis is a fundamentally different separation technique from chromatography. Chromatographic separations are based on partition differences of the analytes between the stationary and mobile phase. Electrophoresis is based on differences in migration of charged particles in an electric field. Consequently, chromatography and electrophoresis are complementary tools in the analytical chemist's toolbox. Of course, there are separation problems that can be solved with both techniques. But, even so, there are problems for which one technique proves superior. The art of good analytical science is to select the tool that uses the strength and thus robustness of a technique rather than apply it at the limit of its capabilities and, therefore, have it be intrinsically less robust.


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