Kinetic capillary electrophoresis methods

A novel capillary electrophoresis method using solutions of non-crosslinked PDADMAC is reported to be effective in the separation of biomolecules [211]. Soil studies conducted with PDADMAC report the minimization of run-off and erosion of selected types of soils [212]. In similar studies, PDADMAC has found to be a good soil conditioner [213]. The use of PDADMAC for the simultaneous determination of inorganic ions and chelates in the kinetic differentiation-mode capillary electrophoresis is reported by Krokhin [214]. Protein multilayer assemblies have been reported with the alternate adsorption of oppositely charged polyions including PDADMAC. Temperature-sensitive flocculants have been prepared based on n-isopropylacrylamide and DADMAC copolymers [215]. A potentiometric titration method for the determination of anionic polyelectrolytes has been developed with the use of PDADMAC, a marker ion and a plastic membrane. The end-point is detected as a sharp potential change due to the rapid decrease in the concentration of the marker due to its association with PDADMAC [216]. 

Drabovich, A. P., Berezovski, M., Okhonin, V., Krylov, S. N. (2006). Selection of smart aptamers by methods of kinetic capillary electrophoresis. Anal Chem 78, 3171-3178. 

Kinetic capillary electrophoresis (KCE) was recently introduced as a conceptual platform for homogeneous kinetic affinity methods (Petrov et al., 2005). KCE is defined as CE separation of species that interact with each other during then-electrophoretic separation in CE. As a brief introduction to CE, the schematic of a CE instrument is depicted in Figure 9.1. 

In general, homogeneous methods are preferable due to their simplicity and kinetic methods are preferable due to their enabling kinetic features. Until recently, the only mixture-based heterogeneous method with comprehensive kinetic capabilities was SPR. In this chapter, kinetic capillary electrophoresis (KCE) is described as a conceptual platform for separation-based homogeneous methods with comprehensive kinetic capabilities, which will find multiple applications in chemistry, biology, medicine, and drug discovery. 

Affinity probe capillary electrophoresis (APCE) uses a fluorescently labeled ligand to bind a receptor. Separation of the bound and free affinity probe has been achieved with CE with LIE detection. One advantage of the rapid CE method is that if the separation is rapid compared to the dissociation time, affinity constants can be determined. Competition experiments can be performed to observe the conditions of complex formation. For example, a fluorescent GTP analog was used as an affinity probe and G protein receptor subunits as the ligand. The complex and free fluorescent probe were separated within 20 s and Ras-like G protein complexes could be separated in less than 15 s. The rapid separations allowed kinetics of binding to be studied. 

Capillary zone electrophoresis (CZE), the most widely used CE mode, is also the mode most frequently used for performing CE immunodetection. The first assay of immunoaffinity capillary electrophoresis described was carried out with CZE in noncompetitive format (Fig. 8a) [79,80]. This format has been used to determine a protein in a matrix as complex as human serum by incubating the sample with specific antibody for 1 hr before the introduction onto the CE column [81]. The size of the immunocomplex peak was seen to increase with incubation time. One drawback of this method is the long incubation time needed because of the slow reaction kinetics of formation of Ab-Ag complex. To prevent noticeable complex dissociation during the analysis, conditions to achieve a separation time shorter than 3 min were chosen. 

Size exclusion chromatography and gel or capillary electrophoresis are methods that separate protein molecules based on size (or size and charge). In these methods, the protein sample travels down the column, gel slab, or capillary and, for a pure protein, should exit as a single peak traveling past the detector. Denatured proteins should appear to have a larger hydrodynamic radius and should travel more slowly. If the kinetics of interconversion of the native and unfolded species is slower than the time needed to travel through the column (gel or capillary), then it is possible to detect individual peaks for the native and unfolded species. If the interconversion is rapid, a kinetically averaged peak position will be observed. 

The most common classification scheme in electrophoresis focuses on the nature of electrolyte system. Using this scheme, electrophoretic modes are classified as continuous or discontinuous systems. Within these groupings the methods may be further divided on the basis of constancy of the electrolyte if the composition of the background electrolyte is constant as in capillary zone electrophoresis, the result is a kinetic process. If the composition of the electrolyte is not constant, as in isoelectric focusing, the result is a steady-state process. 

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