Capillary electrophoresis: a product of technological fusion - Technical

Hewlett-Packard Journal, June, 1995 by Robert R. Holloway

Electrophoresis is one of the most powerful, if traditional, analytical methods. Picton and Linder, Hardy, and Ellis were turn of the century pioneers in the analysis of biocolloids (proteins and carbohydrates). By electrophoretic methods they were able to work with these previously intractable materials. Arne Tiselius ushered in the age of instrumentation with a demonstration of the first cell for electrophoretic analysis in the 1940s, and in the 1990s electrophoresis is one of the most visible icons of science, with TV and newspapers daily displaying electrophoretic DNA spot patterns.

The fused silica capillary, a glass tube about the size of a hair, is a spin-off of the optical fiber. It exists more because it could be made (by heat-drawing a large glass tube into a tiny one) than because it was seen as a powerful analytical tool. It is not surprising that one of its grandest applications, capillary electrophoresis (CE), was not foreshadowed.

Various workers in the electrophoretic field (Everaerts, Hjerten, Mikkers, Virtanen), while aware of the benefits of going to small systems, did not employ the fused silica capillary. The growth of the method began when a few workers, including scholars Jorgensen and Lukacs of the University of North Carolina, industrial researchers McManigill and Lauer of HP Laboratories, and others, not necessarily sophisticated in electrophoresis but aware of the power of the capillary in gas chromatography (demonstrated by Ray Dandenau and Ernie Zerenner of Hewlett-Packard in 1979(1)), conducted the first experiments in fused silica.

Separation Science

In analytical chemistry, separation is a fundamental process. A chemical substance is generally intimately mixed with many other substances, and its determination and identification is made considerably easier by its physical separation from the mixture. Thus, analytical chemists have worked hard to understand the separation process and to develop many modes of separation.

The separation principle for a particular separation method is the physical or chemical property that varies in magnitude among the substances in a sample. In chromatography, for example, the separation principle is often chemical affinity for chromatographic materials. A feature of CE is that its separation principle is orthogonal to that of liquid chromatography (LC), or in other words, it has a completely different basis.

Separation implies physical movement or transport. In free solution capillary electrophoresis (FSCE), the simplest form of CE, transport of a particular chemical species is the resultant of several driving forces. The species moves in response to an electric field, which is important if it is charged. It moves in response to the flow in the channel, which can be caused by the electric potential difference across the fluid-silica interface (electroosmotic flow) or by mechanical pumping of the fluid. Finally, its migration is affected by the frictional drag it experiences, which depends on its size and shape and the viscosity of the fluid.

Modes of CE

CE is really a group of several procedures. CGE, or capillary gel electrophoresis, is an enhancement of an older technique. IEF, or isoelectric focusing, has also been done in other formats, and is still in the process of adaptation to the capillary. ITP, or isotachophoresis, has not found wide use but has considerable potential for preparing pure chemicals. MEKC, or micellar electrokinetic chromatography (also called by other acronyms), is a completely new method that combines electrophoresis and partition chromatography. The simplest and most characteristic mode, in terms of which all the others can be described, is FSCE. It is the form most practiced today.

Free Solution CE

In FSCE, a capillary channel, typically a few micrometers to two hundred micrometers in diameter, is filled with a conducting liquid, most often a water solution containing an acid or a base and its salt, termed a buffer, which has a pH that is insensitive to the addition of small amounts of acids or bases. The two ends of the channel are immersed in two reservoirs which are held at different electric potentials; in other words, a voltage is applied to the column. In the most usual situation, this will result in the flow of the liquid from the anodic reservoir to the cathodic reservoir. This is electroosmotic flow (Fig. 1).

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Electroosmotic Flow

The surface of silica in contact with an aqueous medium with a pH no lower than 2.5 or so is loaded with negative charge. This is because silica hydrates and becomes an acid, releasing positive hydrogen ions into the medium (Fig. 2). The excess positive charge is physically localized within a very narrow zone close to the surface (Fig. 3).

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Since capillaries enclose very small channels, and since electrophoretic currents are proportional to the cross-sectional area of the channel, FSCE involves small currents (from a fraction of a microampere to a hundred microamperes or so) and thus small amounts of heat relative to conventional scales of electrophoresis. As a result it is possible to apply much larger voltages, and axial electric fields in the channel are high--hundreds of volts per centimeter.