Capillary electrophoresis migration rate

Capillary electrophoresis offers several useful methods for (i) fast, highly efficient separations of ionic species (ii) fast separations of macromolecules (biopolymers) and (iii) development of small volume separations-based sensors. The very low-solvent flow (l-10nL min-1) CE technique, which is capable of providing exceptional separation efficiencies, places great demands on injection, detection and the other processes involved. The total volume of the capillaries typically used in CE is a few microlitres. CE instrumentation must deliver nL volumes reproducibly every time. The peak width of an analyte obtained from an electropherogram depends not only on the bandwidth of the analyte in the capillary but also on the migration rate of the analyte. 

Capillary electrophoresis systems are also likely to play an increasingly prominent analytical role in the QC laboratory (Figure 7.2). As with other forms of electrophoresis, separation is based upon different rates of protein migration upon application of an electric field. 

In some cases the equilibration rate is very slow compared to the time scale of the analytical separation. The pre-equilibrated reaction mixture behaves indeed as a mixture of inert components and can be separated by capillary electrophoresis. The concentrations are directly derived from the peak areas or peak heights after calibration. This method is suitable if ligand and substrate are separable and the migration time does not exceed 1% of the half-life of complex decomposition. 

Affinity capillary electrophoresis can be used for the detection of Ag-Ab interactions, because the complexation is likely to change the migration properties. Therefore, it is possible to separate free and complexed Ag or Ab in the case of a high-affinity interaction and slow dissociation rate constants of the complex. These experiments are executed in the equilibrium-mixture mode and called CEIA. Additionally, ACE investigations covering weak Ag-Ab interactions can be carried out using the migration-shift approach. 

A method of separating charged molecules through their different rates of migration under an electric field. Usually a medium that minimizes diffusion is used, e.g., gel, paper, or capillary electrophoresis. 

A unique feature of capillary electrophoresis is electroosmotic flow. When a high voltage is applied across a fused-silica capillary tube containing a buffer solution, electroosmotic flow usually occurs in which the solvent migrates toward the cathode. The rate of migration can be substantial. For example, a 50-mM pH 8 buffer has been found to flow through a 50-cm capillary toward the cathode at approximately 5 cm/min with an applied potential of 25 kV. ... 

Capillary electrophoresis (CE) MS is another technique used to separate and measure the m/z ratios of a mixture of peptides and proteins. In this method, the peptide mixture is separated by different migration rates through the electrophoresis media and the effluent is again directly sprayed into the mass spectrometer using a micro ESI device. This method is capable of femtomole or lower detection limits and is further discussed in a review. In 1996, a group at the University of Washington described a solid phase extraction (SPE) capillary electrophoresis MS-MS approach for the analysis of peptides and showed limits of detection at the 100 s of attomole level. " ... 

Capillary zone electrophoresis (CZE) is a relatively recent separation technique based on the differential migration rates of ionic species in an electrical... 

Capillary Zone Electrophoresis (CZE). CZE is a widely used CE technique and separates peptides and proteins based on differences in their charge-to-mass ratios. Separations occur in a capillary filled with a buffer of constant composition. For CZE, the run buffer choice is extremely important because it determines the charge on the analyte molecule and its migration rate. Thus, the type of buffer, its ionic strength, and its pH are optimized for particular separation problems. Buffers based on sodium phosphate, citrate, acetate, or combinations thereof with concentrations ranging from 10 to 200 mM are frequently used [14]. 

If weak complexes are rapidly formed, on-capillary partial complexation can be used. A ligand is added to the running electrolyte and a rapid equilibrium between the free metal ions and their complexes is established, with most of the ions present in the free form. Owing to different complexation degrees with various charges on the complexes, the ions have different migration rates. The capillary zone electrophoresis (CZE) mode and an indirect UV photometric detection method are usually employed in this case, as only a small fraction of the cations is complexed. 

The ionization and net charge of the sample components are affected significantly by changes in the pH of the sample. As a result, the migration rate, the solubility, the theoretical plate number, and the peak height could aU get affected. Proper dilution of the sample with an appropriate buffer is the first step in separation. Usually, the sample is diluted with the same solvent used as the electrophoresis buffer however, in some instances, a pH of the sample different from that of the buffer is selected to concentrate the sample on the capillary (stacking). 

I he liquid chromatography discussed in Chapter 22 separates solutes by adsorption or partition mechanisms. In this chapter, we consider separations by ion exchange, molecular exclusion, and affinity chromatography, which were illustrated in Figure 21-2. We also consider capillary electrophoresis, which separates species on the basis of their different rates of migration in an electric field. 

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