Eluent capillary electrophoresis

FIGURE 3-23 Schematic of a carbon-fiber amperometric detector for capillary electrophoresis A, fused silica capillary B, eluent drop C, stainless steel plate RE, reference electrode WE, working electrode, AE, auxiliary electrode. (Reproduced with permission from reference 58.)... 

Huber, C.G., Premstaller, A. (1999). Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. I. Liquid chromatography. J. Chromatogr. A 849, 161-173. 

Chromatographic and electrophoretic separations are truly orthogonal, which makes them excellent techniques to couple in a multidimensional system. Capillary electrophoresis separates analytes based on differences in the electrophoretic mobilities of analytes, while chromatographic separations discriminate based on differences in partition function, adsorption, or other properties unrelated to charge (with some clear exceptions). Typically in multidimensional techniques, the more orthogonal two methods are, then the more difficult it is to interface them. Microscale liquid chromatography (p.LC) has been comparatively easy to couple to capillary electrophoresis due to the fact that both techniques involve narrow-bore columns and liquid-phase eluents. 

Additionally, chromatographic techniques such as ion chromatography, gas chromatography and capillary electrophoresis are of increased utility. In part this is because the addition of a mass spectrometric detection system allows for an increased tolerance of nonideal separations. If two eluents are not completely separated at the time of elution, the added dimension of m/z detection often allows the two to be separated on the basis of differences in their mass spectra. The utility of this approach is directly related to the number of mass spectra available during the period of elution. 

Cross-validation should be performed to compare results obtained by methods based on different techniques, e.g. LC-MS and HPLC-UV, or by the same method in different laboratories. Both methods should have been validated independently prior to cross-validation. Capillary electrophoresis (CE) is an alternative for HPLC for a wide range of analytical problems offering shorter analysis times. Both methods are selective and robust. Comparison of robustness implies a variation of different parameters, such as the mobile phase composition, the buffer pH and molarity, temperature, flow-rate and sample solvent [104]. Some concern has been expressed about the reproducibility of CE. Crucial parameters for robustness in CE are the mobile phase composition, which is essential for good separation, the nature of the eluents (volatility), buffer pH and concentration of the additive. Comparison of validated CE and HPLC methods shows that HPLC is about a factor of two better than CE for all quantitative parameters. 

Consider capillary electrophoresis, where there is pressure driven flow of the eluent on top of the electro-osmotic flow. This allows greater control of the flow, since controlling the extent of electroosmotic flow requires either changing the capillary surface or the electrolytic solution or both. 

Most published capillary electrophoresis (CE) methods use buffers containing an organic solvent such as acetonitrile in order to prevent micelle formation. The micelles have different electrophoretic mobility than the isolated surfactant molecules (1). Since individual surfactant molecules are also present, the presence of micelles causes severe tailing of chromatographic peaks (2). Organic eluents also minimize the adsorption of the surfactant to the walls of the capillary. 

Several different analytical and ultra-micropreparative CEC approaches have been described for such peptide separations. For example, open tubular (OT-CEC) methods have been used 290-294 with etched fused silicas to increase the surface area with diols or octadecyl chains then bonded to the surface.1 With such OT-CEC systems, the peptide-ligand interactions of, for example, angiotensin I-III increased with increasing hydrophobicity of the bonded phase on the capillary wall. Porous layer open tubular (PLOT) capillaries coated with anionic polymers 295 or poly(aspartic acid) 296 have also been employed 297 to separate basic peptides on the inner wall of fused silica capillaries of 20 pm i.d. When the same eluent conditions were employed, superior performance was observed for these PLOT capillaries compared to the corresponding capillary zone electrophoresis (HP-CZE) separation. Peptide mixtures can be analyzed 298-300 with OT-CEC systems based on octyl-bonded fused silica capillaries that have been coated with (3-aminopropyl)trimethoxysilane (APS), as well as with pressurized CEC (pCEC) packed with particles of similar surface chemistry, to decrease the electrostatic interactions between the solute and the surface, coupled to a mass spectrometer (MS). In the pressurized flow version of electrochromatography, a pLC pump is also employed (Figure 26) to facilitate liquid flow, reduce bubble formation, and to fine-tune the selectivity of the separation of the peptide mixture. 

The buffer system is a combination of buffer for electrophoresis and eluent for the particular chromatographic mode being employed. Figure 5.14 shows the separation of a group of neutral molecules using a capillary packed with a reversed-phase material.38 The buffer was a mixture of 4 mM sodium tetraborate (pH 9.1) and acetonitrile (20 80, v/v). The separation was compared with a micro-HPLC separation in which the same capillary was used but the eluent was pressure driven. As can be seen in Figure 5.14, sharper peaks were obtained with the EOF-driven system. 

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