Capillary electrophoresis electrophoretic injection

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. 

Fan et al. [106] developed a high performance capillary electrophoresis method for the analysis of primaquine and its trifluoroacetyl derivative. The method is based on the mode of capillary-zone electrophoresis in the Bio-Rad HPE-100 capillary electrophoresis system effects of some factors in the electrophoretic conditions on the separation of primaquine and trifluoroacetyl primaquine were studied. Methyl ephedrine was used as the internal standard and the detection was carried out at 210 nm. A linear relationship was obtained between the ratio of peak area of sample and internal standard and corresponding concentration of sample. The relative standard deviations of migration time and the ratio of peak area of within-day and between-day for replicate injections were <0.6% and 5.0%, respectively. 

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. 

Figure 8.22. Capillary electrophoresis on a chip, (a) Schematic of the microchip used for PCR amplification and electrophoresis. The direction of arrows indicate injection (I) and separation (S). (b) Electrophoretic microchip with multiple PCR chambers.

Capillary electrophoresis separations are dependent on the relative mobilities of analytes under the influence of an electric field and do not depend on mobile phase/stationary phase interactions. A fused silica capillary is filled with a buffer and both ends submerged into two reservoirs of the buffer. A platinum electrode is immersed in each reservoir and a potential difference (5-30 kV) is applied across the electrode. An aliquot of sample of a few nanoliters is injected onto the capillary by either hydrostatic or electrokinetic injection, and the components migrate to the negative electrode. Separations of analytes arise from differences in the electrophoretic mobilities, which are dependent on the mass-to-charge ratio of the components, physical size of the analyte, and buffer/analyte interactions. An electro-osmotic flow (EOF) of the buffer occurs in the capillary and arises as a result of interactions of the buffer with dissociated functional groups on the surface of the capillary. Positive ions from the buffer solution are attracted to negative ions... 

Although affinity capillary electrophoresis (ACE) in its classical mode (one of the reagent is dissolved in a BGE, another is injected) is the most widely used technique in the literature, other capillary electrophoretic methods exist which are even more favorable concerning the information about binding parameters obtainable the Hummel-Dreyer (HD) method, frontal analysis (FA), the vacancy peak (VP) method, and vacancy affinity capillary electrophoresis (VACE) (see, e.g., Refs. 49-57). All the methods need as a precondition that the equilibrium between the reactants (say, protein P, drug D, and complex formed PD) is established rapidly compared to the dislocation of the electropho-retically migrating zones. The experimental setup of the HD and the ACE methods is identical, and so is the setup for the VP and the VACE methods. FA differs from all the other techniques. 

Capillary electrophoresis is conducted in capillaries filled with an electrolyte solution. Buffered electrolytes are generally used, since biomolecule mobilities and electroosmotic flow (EOF) are sensitive to pH. The ends of the filled capillaries are placed in electrolyte reservoirs that contain electrodes, and the electrodes are positioned so that electrolysis products do not enter the capillary. A small plug of solution containing the analytes to be separated is pressure- or electrokinetically-injected into one end of the capillary, and a voltage difference is applied to the electrodes such that the analytes of interest migrate toward the other end of the capillary, where they are detected. Analytes with different electrophoretic mobilities migrate at different speeds and become separated as they transverse the capillary. 

Another more recently developed means of separating components in a solution, is known as electrophoresis. This technique is used for the separation of components based upon their ability to travel in an electric field. Many different matrices have been used for electrophoretic separations, including buffered solutions, and gels (e. g. agarose gel). Gel electrophoresis has been used extensively for the separation of biomolecules, however, it is often slow and irreproducible. A faster more reliable form of electrophoretic separation is known as capillary electrophoresis. In this technique, a buffer filed capillary is used to span the distance between two containers of the same buffer solution. A potential of 20—30 kV is typically applied between the two containers, and a small amount of sample is injected into the capillary. The individual components of the sample are then separated, based upon the combination of their overall charge and their friction within the solvent. The individual components can then either be collected or detected upon elution from the column. 

Xu, Z. Q., Hirowaka, T., Nishine, T., and Arai, A., High-sensitivity capillary gel electrophoretic analysis of DNAfragments on an electrophoresis microchip using electrokinetic injection with transient isotachophoretic preconcentration. Journal of Chromatography, 990, 53-61, 2003. 

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