Electrophoretic injection

Sample injection is an event common to all modes of CE except CIEF, and the most relevant aspects of this step are described here. Because the total volume of the capillary is very low, sample injection in CE requires the precise introduction of very small amounts of analyte at the capillary inlet. There are two basic procedures for sample injection electromigration and hydraulic displacement. All commercial instruments offer electromigration, or electrophoretic, injection and at least one type of displacement injection. 

In electrophoretic injection, the capillary inlet is immersed in the sample solution and a voltage is applied for a determined period of time. The amount of sample introduced into the capillary depends on the voltage and the time it was applied. Sample injection is a compromise between detection and resolution, and its parameters are often best determined experimentally. If detection is not a problem, resolution can be greatly improved by maintaining the sample plug as narrow as possible. If EOF is present, sample ions will be introduced by a combination of electrophoretic mobility and EOF under these conditions, this injection mode is generally termed electrokinetic injection. 

Electrophoretic injection can be used as a means for zone sharpening or sample concentration if the amount of ions, particularly salt or buffer ions, is lower in the sample than the running buffer. Because sample ions enter the capillary based on mobility, low-mobility ions will be loaded to a lesser extent than high-mobility ions. For this reason, the presence of nonsample ions will reduce injection efficiency, so electrophoretic injection is very sensitive to the presence of salts or buffers in the sample matrix. The disadvantages of electrophoretic injection argue against its use in routine analysis except in cases where displacement injection is not possible, e.g., in capillary gel electrophoresis (CGE) or when sample concentration by stacking is necessary. 

For quantitative purposes, the peak area must be corrected for mobility when using displacement injection. This correction is necessary because peak area is a result of sample response and time. Analytes with lower mobility spend more time in front of the detector, thus generating larger relative area counts. Such correction is not necessary for electrophoretic injections. 

In some cases, sample preparation for CZE requires only the dilution of the sample, mostly to accommodate detection (for signal and linearity of response). However, as was previously mentioned, sample characteristics such as viscosity, buffer composition (pH and excipients), and salt content can especially affect electrophoretic injection and performance. 

Alarie, J.P., Jacobson, S.C., Ramsey, J.M., Electrophoretic injection bias in a microchip valving scheme. Electrophoresis 2001, 22, 312-317. 

Van Harreveld, A. and Fifkova, E., Light- and electron-microscopic changes in central nervous tissue after electrophoretic injection of glutamate, Exp. Mol. Pathol., 15, 61, 1971. 

Figure 6. Rate of the glucose-6-phosphate dehydrogenase reaction as a function of the concentration of the substrate glucose-6-phosphate expressed in log-log coordinates. An isolated ascites cell (EL2) was impaled with a microelectrode, which then was used to electrophoretically inject known concentrations of the substrate into a localized region of the cell. The oxidatiorVreduction state of the cofactor to which the reaction is coupled was monitored by microspectrophotometric methods to determine the rate of the corresponding reaction in situ. (Replotted from the data of Kohen et al., 1973.)...

Electromigration (also known as electrophoretic) injection. The capillary inlet is inunersed in the sample vial and a high voltage applied (usually three to five times lower than used for separation) for a controlled... 

McKillop and associates have examined the electrophoretic separation of alkylpyridines by CZE. Separations were carried out using either 50-pm or 75-pm inner diameter capillaries, with a total length of 57 cm and a length of 50 cm from the point of injection to the detector. The run buffer was a pH 2.5 lithium phosphate buffer. Separations were achieved using an applied voltage of 15 kV. The electroosmotic flow velocity, as measured using a neutral marker, was found to be 6.398 X 10 cm s k The diffusion coefficient,... 

Electroosmotic flow in a capillary also makes it possible to analyze both cations and anions in the same sample. The only requirement is that the electroosmotic flow downstream is of a greater magnitude than electrophoresis of the oppositely charged ions upstream. Electro osmosis is the preferred method of generating flow in the capillary, because the variation in the flow profile occurs within a fraction of Kr from the wall (49). When electro osmosis is used for sample injection, differing amounts of analyte can be found between the sample in the capillary and the uninjected sample, because of different electrophoretic mobilities of analytes (50). Two other methods of generating flow are with gravity or with a pump. 

A number of developments have increased the importance of capillary electrophoretic methods relative to pumped column methods in analysis. Interactions of analytes with the capillary wall are better understood, inspiring the development of means to minimize wall effects. Capillary electrophoresis (CE) has been standardized to the point of being useful as a routine technique. Incremental improvements in column coating techniques, buffer preparation, and injection techniques, combined with substantive advances in miniaturization and detection have potentiated rugged operation and high capacity massive parallelism in analysis. 

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