Equilibrium capillary electrophoresis

Two methods have been used so far for aptamer-related applications nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM). An equilibrium mixture is defined as a mixture of the target and the ligand (or a mixture of ligands) which is incubated until equilibrium is reached in reaction (9.1), so that the concentrations of molecules and complexes are related as [L][T]/[L T] = K. ... 

Drabovich, A., Berezovski, M., Krylov, S. N. (2005). Selection of smart aptamers by equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM). J Am Chem Soc 127, 11224-11225. 

Several variants of separation methods based on dialysis, ultrafiltration, and size exclusion chromatography have been developed that work under equilibrium conditions. Size exclusion chromatography especially has become the method of choice for binding measurements. The Hummel-Dreyer method, the vacancy peak method, and frontal analysis are variants that also apply to capillary electrophoresis. In comparison to chromatographic methods, capillary electrophoresis is faster, needs only minimal amounts of substances, and contains no stationary phase that may absorb parts of the equilibrium mixture or must be pre-equilibrated. 

As the other methods, the Hummel-Dreyer method was first developed for chromatography and then adapted to capillary electrophoresis (21). A small sample of the ligand, dissolved in buffer, is injected into the capillary. The capillary as well as source and destination vial are filled with buffer containing the substrate. In the ligand-containing sample, the initial concentration of the free substrate is depleted to the equilibrium concentration (see Fig. 7a). The depletion peak moves with the velocity of the substrate, and its area corresponds to the bound substrate. As demonstrated in Fig. 7b, the... 

LZ Avila, Y-H Chu, EC Blossey, GM Whitesides. Use of affinity capillary electrophoresis to determine kinetic and equilibrium constants for binding of arylsulfonamides to bovine carbonic anhydrase. J Med Chem 36 126-133,... 

MA Schwarz, K Raith, G Dongowski, R Neubert. Effect on the partition equilibrium of various drugs by the formation of mixed bile salt/phosphatidylcho-line/fatty acid micelles. A characterization by micellar affinity capillary electrophoresis. Part IV. J Chromatogr A 809 219-229, 1998. 

Fig. 1 Schematic representation of the experimental setups of the mobility-shift method and the Hummel-Dreyer method (A) the vacancy peak method and the vacancy affinity capillary electrophoresis method (B) the equilibrium-mixture method and the frontal analysis method (C) for drug-protein binding analysis. drug protein gg drug-protein complex Q buffer. (Reprinted with permission from Ref. 38. Copyright 1992 Elsevier Science.)...

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. 

In the fiber mode, the sorbent coated fiber is housed in a microsyringelike protective holder. With the fiber retracted inside the syringe needle, the needle is used to pierce the septum of the sample vial. The plunger is depressed to expose the sorbent-coated fiber to the sample. After equilibrium is reached or at a specified time prior to reaching equilibrium, the fiber is retracted into the protection of the microsyringe needle and the needle is withdrawn from the sample. The sorbent is then interfaced with an analytical instrument where the analyte is desorbed thermally for GC or by solvents for HPLC or capillary electrophoresis. For the in-tube mode, a sample aliquot is repeatedly aspirated and dispensed into an internally coated capillary. An organic solvent desorbs the analyte and sweeps it into the injector [68,130,133]. An SPME autosampler has been introduced by Varian, Inc., that automates the entire process for GC analyses. 

From the great variety of methods for the determination of protein binding three separation methods, equilibrium dialysis (ED), ultrafiltration (UF), and ultracentrifugation (UC) and a non-conventional method with the binding to immobilized proteins has been chosen. The first methods are undoubtedly the most widely used because of their simplicity and general applicability to many different systems. Other methods e.g. size exclusion chromatography, capillary electrophoresis, or spectroscopic methods have been not described. Oravcova et al. (1996) gives a comprehensive review and comparison for these applications. 

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. 

A scale that would enable us to compare the acidity in all solvents could be based on the transfer activity coefficient on the proton (see the entry Capillary Electrophoresis in Nonaqueous Media). The effect of the solvent of any species can be expressed in the same way as for the proton by this concept and applied to all particles involved in the thermodynamic equilibrium. 

Taking silica (bulk composition silicium oxide, Si02), from which capillary material in capillary electrophoresis (CE) is manufactured, as an example, one can readily understand this when taking into account that on the silica surface, there are silicium hydroxide groups that are subject to an acid-base equilibrium according to... 

Suzuki, T., Yamada, M., Ide, H., Kanaori, K., Tajima, K., Morii, T., and Makino, K. (2000) Influence of ring opening-closure equilibrium of oxanine, a novel damaged nucleobase, on migration behavior in capillary electrophoresis. J. Chromatogr. A, 877, 225-232. 

At equilibrium, the effective mobility of an enantiomer in the presence of a chiral selector in capillary electrophoresis is the sum of the electrophoretic mobility of each species containing the enantiomer, weighted by the mole fraction of each species. Assuming for a given separation the enantiomers exist as either the free enantiomer with a mobility, ixr, or the enantiomer-chiral selector complex, p,c,R, then the effective mobility for the enantiomer is given by... 

Whereas oligonucleotide libraries were traditionally partitioned by filtration or capture on beads or on affinity columns, capillary electrophoresis has been validated as a promising alternative. Aptamers displaying a high affinity were obtained in a very limited number of selection rounds. Nonequilibrium capillary electrophoresis of equilibrium mixtures led to the identification of DNA aptamers... 

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