Open-tube capillary electrophoresis

Capillary electrophoresis (CE) is a powerful separation technique. It is especially useful for separation of ionic compounds and chiral mixtures. Mass spectrometry has been coupled with CE to provide a powerful platform for separation and detection of complex mixtures such as combinatorial libraries. However, the full potential of CE in the application of routine analysis of samples has yet to be realized. This is in part due to perceived difficulty in the use of the CE technique compared to the more mature techniques of HPLC and even SFC. Dunayevskiy et al. [136] analyzed a library of 171 theoretically disubstituted xanthene derivatives with a CE/ESI-MS system. The method allowed the purity and makeup of the library to be determined 160 of the expected compounds were found to be present, and 12 side products were also detected in the mixture. Due to the ability of CE to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple CE-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components. Boutin et al. [137] used CE-MS along with NMR and MS/MS to characterize combinatorial peptide libraries that contain 3 variable positions. The CE-MS method was used to provide a rapid and routine method for initial assessment of the construction of the library. Simms et al. [138] developed a micellar electrokinetic chromatography method for the analysis of combinatorial libraries with an open-tube capillary and UV detection. The quick analysis time of the method made it suitable for the analysis of combinatorial library samples. CE-MS was also used in the analysis... 

Cohen, A. S., Paulus, A., and Karger, B. L. High-performance capillary electrophoresis using open tubes and gels, Chromatographia, 24, 15, 1987. 

Electrophoresis in the CZE mode takes place in an open tube and in a free solution without any separation matrix in the capillary. The separation is based on the mass/charge ratio of the analytes. It is appropriate for the separation of nucleosides and nucleotides. It is not well suited for medium to large oligonucleotides, because their mass/charge ratio tend to be smaller. The use of a separation matrix becomes necessary for these species. Various capillary systems, including bare fused silica capillaries and surface-coated capillaries, have been used in CZE. 

Capillary zone electrophoresis (CZE) is the most simple and widely used mode in CE. Separations take place in an open-tube, fused silica capillary under the influence of an electric field. The velocity of the analytes is modified by controlling the pH, viscosity, or concentration of the buffer, or by changing the separation voltage. The electroosmotic flow is often used in this mode to improve resolution or to shorten analysis times. 

Ultramicroelectrodes can also greatly benefit modem microseparation techniques such as open-tube liquid chromatography or capillary-zone electrophoresis (CZE) (73). For example, cylinder-shaped carbon or copper fibers can be inserted into the end of the CE separation capillary (e.g., see Fig. 3.26). Such alignment of the working electrode with the end of the capillary represents a challenge in combining electrochemistry with CZE. 

Higher oligosaccharides or polysaccharides possess unfavorable mass-to-charge ratios, preventing their effective resolution in open tubes. The separation of these carbohydrates is possible with capillary gel electrophoresis (CGE). The analytes are selectively retarded by a sieving network (gel or polymer matrix) due to differences in their sizes and structural conformations. 

Open tube FCCE in narrow capillaries using zone electrophoresis is useful for resolving analytes that can be tagged with fluorescent labels for LIF detection. Culbertson demonstrated resolution of the peptide fragment YAGAVVNDL and YAGAVVNDI (Figure 24.15), which only have an... 

The last original stationary phase used with micellar phases is the wall of capillary open tubes. The forces driving the micellar phase are not mechanical (pressure) but electrical (electric field). This is part of the world of capillary electrophoresis. It deserves special consideration found at the end of this chapter. 

We have already learned about the presence of an electroosmotic flow (EOF) in a silica capillary filled with an electrolytic solution and subjected to a voltage difference at the two ends (Section 6.1.5). This flow is such that the velocity profile, i/eof, is essentially fiat, and the liquid motion is toward the cathode (equation (6.1.22)). If the silica capillary tube is open (and not a packed bed of very small particles), and the surface of the capillary has not been coated in any fashion, capillary electrophoresis occurs in the presence of Veof- Therefore, the net velocity of any ionic species i averaged over the capillary cross section, is given by... 

---