Electro-osmotic flow , capillary

Capillary electrophoresis (CE) has several unique advantages compared to HPLC, snch as higher efficiency dne to non-parabolic fronting, shorter analytical time, prodnction of no or much smaller amounts of organic solvents, and lower cost for capillary zone electrophoresis (CZE) and fused-silica capillary techniques. However, in CZE, the most popular separation mode for CE, the analytes are separated on the basis of differences in charge and molecular sizes, and therefore neutral compounds snch as carotenoids do not migrate and all co-elute with the electro-osmotic flow. 

Tsuda, T., Modification of electro-osmotic flow with cetyltrimethylammonium bromide in capillary zone electrophoresis, /. High Resolut. Chromatogr., 10,622, 1987. 

Landers, J. P., Oda, R. P., Madden, B. J., and Spelsberg, T. C., High-performance capillary electrophoresis of glycoproteins the use of modifiers of electro-osmotic flow for analysis of microheterogeneity, Anal. Biochem., 205, 115, 1992. 

The efficacy of CE separation depends considerably on the type of capillary. Fused-silica capillaries without pretreatment are used most frequently. Its outside is coated with a polymer layer to make it flexible and to lessen the occurrence of breakage. The polymer coating has to be dissolved with acid or burned away at the detection point. Capillaries with an optically transparent outer coating have also found application in CE. The objectives of the development of chemically modified capillary walls were the elimination of electro-osmotic flow and the prevention of adsorption on the inner wall of the capillary. Another method to prevent the adsorption of cationic analyses and proteins is the use of mobile phase additives. The modification of the pH of the buffer, the addition of salts, amines and polymers have all been successfully employed for the improvement of separation. 

In MEKC, mainly anionic surface-active compounds, in particular SDS, are used. SDS and all other anionic surfactants have a net negative charge over a wide range of pH values, and therefore the micelles have a corresponding electrophoretic mobility toward the anode (opposite the direction of electro-osmotic flow). Anionic species do not interact with the negatively charged surface of the capillary, which is favorable in common CZE but especially in ACE. Therefore, SDS is the best-studied tenside in MEKC. Long-chain cationic ammonium species have also been employed for mainly anionic and neutral solutes (16). Bile salts as representatives of anionic surfactants have been used for the analysis of ionic and nonionic compounds and also for the separation of optical isomers (17-19). 

Separation is carried out by applying a high potential (10-30 kV) to a narrow (25-75 pm) fused silica capillary filled with a mobile phase. The mobile phase generally contains an aqueous component and must contain an electrolyte. Analytes migrate in the applied electric field at a rate dependent on their charge and ionic radius. Even neutral analytes migrate through the column due to electro-osmotic flow, which usually occurs towards the cathode. 

Variation of ion mobility with pH is only part of the story with regard to separation by capillary electrophoresis - the other major factor is electro-osmotic flow (EOF). 

CITP employs two buffers in which the analyte zone is enclosed between. Either anions or cations can be analyzed in sharply separated zones. In addition, the analyte concentrations are the same in each zone thus, the length of each zone is proportional to the amount of the particular analyte. And finally, CGE, which is analogous to gel filtration, uses gel-tilled capillaries to separate molecules on the basis of relative differences in their respective molecular weight or molecular size. It was first used for the separation of proteins, peptides, and oligomers. Advantages of the gel include decreasing the electro-osmotic flow and also reducing the adsorption of protein onto the inner wall of the capillary (von Brocke et al. 2001). 

In capillary electrophoresis, components of a mixture are separated according to two main factors electrophoretic mobility and electro-osmotic flow. These terms apply to ions, molecules or micelles. 

Another factor that controls the migration of the solute is the electro-osmotic mobility //EOS, which results in movement of the electrolyte or electro-osmotic flow. This flow is present in gel electrophoresis to a small extent and to a greater extent in capillary electrophoresis because of the internal wall of the capillary. 

Figure 8.5—Effect of the nature of the capillary inner wall on migration velocities If the inner wall has not been treated (glass or silica naturally have a negative polyanionic layer) the liquid is pumped from the anodic towards the cathodic reservoir. This is called the electro-osmotic flow. Thus an anion can move towards the cathode. Between pH 7 and 8. vE0S can increase by 35%. However, if the wall is coated with a nonpolar film (e.g. octadecyl) this flow does not exist.

The electro-osmotic flow vEOS must first be determined in order to calculate eos-vEos can be calculated by measuring the migration time fnm for a neutral marker to migrate over the distance / of the capillary. An organic molecule that is nonpolar at the pH of the electrolyte used and easily detected in the UV can be used as the neutral marker (e.g. mesityl oxide or benzyl alcohol). vE0S = //fnm. 

In capillary electrophoresis instruments, the electro-osmotic flow is used to impose, on all charged species in the sample, a direction of migration that is oriented from the anode towards the cathode. An increase in the electro-osmotic flow vEOS decreases, at the detector, the gap in migration times of ions travelling in the same direction. The use of fused silica capillaries partially deactivated by coating the inner wall allows modulation of the electro-osmotic flow. A voltage gradient can also be used to this end. 

This mode of electrophoresis, in which the electrolyte migrates through the capillary, is the most widely used. The electrolyte can be an acidic buffer (phosphate, citrate, etc.) or basic buffer (borate) or an amphoteric substance (a molecule that possesses both an acidic and an alkaline function). The electro-osmotic flow increases with the pH of the liquid phase, or can be rendered non-existent. 

This technique represents the transposition of classical polyacrylamide or agarose gel electrophoresis into a capillary. Under these conditions, the electro-osmotic flow is relatively weak. In this approach, the capillary is filled with an electrolyte impregnated into a gel that minimises diffusion and convection phenomena. In contrast to its use for proteins that are fragile and thermally unstable, CGE is ideal for separating the more rugged oligonucleotides. 

Electro-osmotic flow depends largely on the zeta potential arising at the interface of the stationary phase with the electrolyte. For this potential to develop, the stationary phase has to carry some amount of ionic or ionizable groups at its surface. When MIPs are used as stationary phase in CEC their composition has to be designed with this point in mind besides the usual criteria for an MIP. Some of the more usual MIP compositions, like MIPs made with the functional monomer methacrylic acid, satisfy this criterion quite well. It is also important that the stationary phase is well retained in the capillary. Frits at the ends of the packing are difficult to make in capillaries, and therefore monoliths covalently bound to the (prederivatized) surface of the capillary are advantageous. The preparation of MIP monoliths has been mostly promoted in the CEC field. 

Figure 5.13 Electro-osmotic flow profile in a capillary.

Sinibaldi et al. [95] resolved the enantiomers of 2-arylpropionic acids (flobufens) and dansyl amino acids using a coated capillary of polyterguride using capillary electrochromatography (CEC). It was found that the analytes, in the range of the buffer pH between 2.5 and 4.0, were driven by anodic electro-osmotic flow... 

Electrodriven separations, such as capillary electrophoresis (CE) and capillary electrochromatography (CEC), are based on the different electrophoretic mobilities in an electric field of the molecules to be separated. They provide a higher separation efficiency then conventional HPLC since the electrophoretic flow (EOF) has a plug-flow profile. Whereas the mobile phase in CE is driven only by the electro-osmotic flow, it is generated in CEC by a combination of EOF and pressure. CEC has a high sample capacity which favours its hyphenation with NMR. 

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