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Chapter: Modern Analytical Chemistry: Chromatographic and Electrophoretic Methods

Electrophoresis

Thus far all the separations we have considered involve a mobile phase and a stationary phase.

Electrophoresis

Thus far all the separations we have considered involve a mobile phase and a stationary phase. Separation of a com- plex mixture of analytes occurs because each analyte has a different ability to partition between the two phases. An analyte whose distribution ratio favors the stationary phase is retained on the column for a longer time, thereby eluting with a longer retention time. Although the meth- ods described in the preceding sections involve different types of stationary and mobile phases, all are forms of chromatography.

Electrophoresis is another class of separation tech- niques in which analytes are separated based on their ability to move through a conductive medium, usually an aqueous buffer, in response to an applied electric field. In the absence of other effects, cations migrate toward the electric field’s negatively charged cathode, and anions mi-grate toward the positively charged anode. More highly charged ions and ions of smaller size, which means they have a higher charge-to-size ratio, migrate at a faster rate than larger ions, or ions of lower charge. Neutral species do not experi- ence the electric field and remain stationary. As we will see shortly, under normal conditions even neutral species and anions migrate toward the cathode. In either case, differences in their rate of migration allow for the separation of complex mix- tures of analytes.

There are several forms of electrophoresis. In slab gel electrophoresis the con- ducting buffer is retained within a porous gel of agarose or polyacrylamide. Slabs are formed by pouring the gel between two glass plates separated by spacers. Typical thicknesses are 0.25–1 mm. Gel electrophoresis is an important technique in bio- chemistry, in which it is frequently used for DNA sequencing. Although it is a pow- erful tool for the qualitative analysis of complex mixtures, it is less useful for quanti- tative work.

In capillary electrophoresis the conducting buffer is retained within a capillary tube whose inner diameter is typically 25–75 μm. Samples are injected into one end of the capillary tube. As the sample migrates through the capillary, its components separate and elute from the column at different times. The resulting electrophero- gram looks similar to the chromatograms obtained in GC or HPLC and provides both qualitative and quantitative information. Only capillary electrophoretic meth- ods receive further consideration in this text.

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