EOF stability

Additional aspects which are relevant to validate CE methods EOF stability Rinsing procedures... 

It is beneficial to develop generic methods, this means methods that are already validated for some aspects, e.g., EOF stability and freedom of matrix interferences. Generic methods that are suitable to separate a number of analytes can be found in CE, especially due to the high separation efficiency. Some aspects, like specificity and repeatability, are still analyte specific, but the entire method validation is substantially speeded up when a generic method already exists. 

Graft polymerization on PDMS channel surface (not channel lumen) in enclosed channels was carried out by first adsorbing the photoinitiator (benzophe-none) on the PDMS surface, followed by UV-mediated photopolymerization. In this manner, the EOF stability of the PDMS channels lasts for 45 h [254]. Besides PDMS chips, PC chips have also been chemically modified by sulfonation (using S03 gas) to produce hydrophilic surfaces [255]. 

Surface modification can be achieved by 02 plasma oxidation. For instance, because of the increase in surface negative charges after oxidation, the oxidized PDMS surface supports EOF. However, because of the instability of the charge created on the polymer surface, EOF was unstable. Better stability can be achieved by immediately filling the PDMS channel with liquids, rather than letting it be exposed to air. The useful lifetime of these devices for quantitative CE analysis, which requires EOF stability, is probably 3 h [1033]. 

Poly(vinyl alcohol). Deposition is achieved by flushing the capillary with a 10% aqueous solution of 50 kDa PVA (99% or more hydrolyzed), pushing the coating solution out of the column with a stream of nitrogen, and then heating the capillary at 140°C under a stream of nitrogen. This produces capillaries with an efficiency of greater than a million plates, essentially no EOF, stability up to pH 10, and the ability to separate basic or acidic proteins (see Fig. 321). A 50 m M sodium phosphate buffer at pH 3.0 is used, and CE is carried out at 30 kV at 20°C. 

Chemical surface modifications The first surface modification for the purpose of eliminating EOF and protein adsorption was recommended by Hjerten.28 The attachment of vinyl silanes allowed the polymerization of a variety of molecules to the surface. Most of the chemical modifications used for preparing capillaries for electrophoresis originated from the experience acquired over the years preparing GC and LC stationary phases. Chemical modification should conform to certain requirements, including the prevention of adsorption, the provision of stable and constant EOF over a wide pH range, chemical stability, ease of preparation, and reproduciblity of preparation. The effects of silanization of the inner surface of capillaries on electrophoretic separations have been extensively studied.26-29... 

The dependence of precision on different parameters has already been discussed. Precision is strongly dependent on the constancy of migration data. Thus, the stability of the EOF is most important. Buffer recipes describe clearly the preparation and avoid errors caused by, e.g., a poorly calibrated pH electrode. 

Capillary coating can also stabilize the migration times and resolutions. This is in particular necessary in the case of peptide and protein analysis, because proteins tend to stick to capillary walls. Often low-concentration polyethylene oxide solutions are recommended as well as dynamic bilayer coating formed by a non-covalent adsorption of polybrene and polyvinylsulfonate (PVS). Due to the stability of the EOF, the variation of intra- and intercapillary migration time was less than 1% relative standard deviation (RSD) with basic analytes and peptides. 

The order of elution of peptides (charged compounds) is governed by a combination of electrophoresis and partitioning, with hydrophobic as well as electrostatic contributions. In this study it was demonstrated that sulfonic acid functionalities in the methacrylate monolith provide high stability and maintain a constant EOF over a wide range of pH (2—12). It was also demonstrated that a better separation of a mixture of therapeutic peptides was obtained at high pH values (Figure 16) due to the suppression of electrostatic attraction. 

In some applications, an alteration in the microchannel surface charge is needed in order to modify EOF, i.e., to increase, suppress, or stabilize EOF. This charge alteration can be achieved by surface modification. 

In the absence of EOF (e.g., in non-cross-linked acrylamide-coated capillaries), the focused zones should be mobilized either chemically or with hydro-dynamic flow. During this mobilization, maintenance of the electric field serves as a stabilizer to prevent zone broadening. A common name for such a setup is two-step capillary isoelectric focusing. 

Separation columns are the heart of any CEC techniques. In order for CEC to become a versatile analytical technique, it is important to have columns with good stability, high EOF velocity generation, and affinity for different analytes. To this end, several different types of capillary columns have been developed for the separation of biomolecules. In this section, a summary of recent advances in column technology, including open tubular columns, duplex columns, and monolithic columns, will be presented. A comparison of different column configurations is shown in Figure 13. 

Another important environmental and forensic application is the identification of explosives and their degradation products. A method based on liquid chromatography was unable to produce baseline resolution of 14 nitroaromatic and nitramine explosive compounds. The problem was solved as Bailey et al. tested the separation of the same mixture in CEC mode (column packed with 1.5-pm C18 silica) [89]. A baseline resolution of all 14 compounds was realized in 7 min in a second run, performed in less than 2 min, only 2 analytes of 14 co-eluted. To stabilize EOF and reduce peak tailing, SDS was added to the mobile phase. As in previous surfactant-mediated studies mentioned, changes in the elution order of the compounds were observed, suggesting a more complex separation mechanism (Figure 18, from Ref. 89). 

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