Separation methods capillary electrophoresis

Extraction 7.3 Separation methods GC, HPLC, thin layer, column, capillary, electrophoresis Identification methods UV, visible, IR, NMR, X-ray, fluorescence, AAS, ICP, MS... 

The hyphenation of CE and NMR combines a powerful separation technique with an information-rich detection method. Although compared with LC-NMR, CE-NMR is still in its infancy it has the potential to impact a variety of applications in pharmaceutical, food chemistry, forensics, environmental, and natural products analysis because of the high information content and low sample requirements of this method [82-84]. In addition to standard capillary electrophoresis separations, two CE variants have become increasingly important in CE-NMR, capillary electrochromatography and capillary isotachophoresis, both of which will be described later in this section. 

Miller, J. H. McB., and Rose, U. (2001). Comparison of chiral liquid chromatographic methods and capillary electrophoresis, separation of the enantiomers of ephedrine hydrochloride. Pharmeuropa 13(1), 3-7. 

Persson Stubberud, K., and Astrom, O. (1998). Separation of ibuprofen, codeine phosphate, their degradation products and impurities by capillary electrophoresis I. Method development and optimization with fractional factorial design. ]. Chromatogr. A 798, 307—314. 

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. 

The attraction of using capillary electrophoresis (CE) methods mainly lies in the short analysis time, high separation efficiencies, and reduced consumption of... 

In the outside-out model, the pipette is attached to the entire cell as in the whole cell model, followed by a sharp pull that causes the cell membrane to break and reseal with the pipette tip (Fig. 3b). With the extracellular region exposed, channel activity as a response to different external stimuli can be probed. This configuration is less common than the inside-out method. Using an outside-out method, single-channel opening activity has been recorded while various neurotransmitters were released. For example, this patch clamp method was used as a detector for capillary electrophoresis separations of GABA, glutamate, and NMDA (7). 

Electrophoretic methods are widely used alternatives for the analytical determination of the enantiomeric purity of chiral compounds [194]. Due to the high elTi-ciency of capillary electrophoresis, separations can be achieved even when very low selectivities are observed. At a preparative scale, these methods are well established for the purification of proteins and cells [195] but there is very little published on enantioselective separations. Only recently, some interest in chiral preparative applications has been manifested. Separation of the enantiomers ofterbu-taline [196] and piperoxan [197] have been reported by classical gel electrophoresis using sulfated cyclodextrin as a chiral additive, while the separation of the enantiomers of methadone could be successfully achieved by using free-fluid isotachophoresis [198] and by applying a process called interval-flow electrophoresis [199]. 

Taking this into account, more sensitive methods of sample detection have been developed, for example, special detection cell constructions for ultraviolet (UV) adsorption and laser-induced fluorescence (LIE), and particularly the introduction of mass spectrometry (MS) brought tremendous progress in online and offline characterization not only of modified peptides but also of modified proteins. In this chapter, we try to provide information about capillary electrophoresis (CE) methods developed for the separation of proteins and peptides with various PTMs. 

S.D. Mendonsa and R.J. Hurtubise, Capillary electrophoresis (CE) methods for the separation of carcinogenic heterocyclic aromatic amines, J. Liq. Chrom. Rel. TechnoL, 22, 1027-1040, 1999. 

The direct sampling of solutions is often necessary in a variety of situations such as biological fluids and eluants from liquid chromatography and capillary electrophoresis separation devices. Liquid solutions are difficult to handle by the mass spectrometry vacuum system and require some novel introduction and ionization systems. The last two decades have witnessed the development of some unique ionization methods that are suitable for direct analysis of sample solutions the important ones are discussed below. 

The transposition of capillary electrophoresis (CE) methods from conventional capillaries to channels on planar chip substrates is an emergent separation science that has attracted widespread attention from analysts in many fields. Owing to the miniaturization of the separation format, CE-like separations on a chip typically offer shorter analysis times and lower reagent consumption augmented by the potential for portability of analytical instrumentation. Microchip (p-chip) electrophoresis substrates boast optically flat surfaces, short diffusion distances, low Reynolds numbers, and high surface (or interface)-to-volume ratios. By exploiting these physical advantages of the chip over conventional capillaries, efficient p-chip electrophoresis systems can accomplish multiple complicated tasks that may not be realized by a conventional CE system alone. 

Size exclusion chromatography and gel or capillary electrophoresis are methods that separate protein molecules based on size (or size and charge). In these methods, the protein sample travels down the column, gel slab, or capillary and, for a pure protein, should exit as a single peak traveling past the detector. Denatured proteins should appear to have a larger hydrodynamic radius and should travel more slowly. If the kinetics of interconversion of the native and unfolded species is slower than the time needed to travel through the column (gel or capillary), then it is possible to detect individual peaks for the native and unfolded species. If the interconversion is rapid, a kinetically averaged peak position will be observed. 

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

Capillary Electrophoresis. Capillary electrophoresis (ce) or capillary 2one electrophoresis (c2e), a relatively recent addition to the arsenal of analytical techniques (20,21), has also been demonstrated as a powerful chiral separation method. Its high resolution capabiUty and lower sample loading relative to hplc makes it ideal for the separation of minute amounts of components in complex biological mixtures (22,23). 

We have developed the method for quantitative analysis of urinary albumin with CE. A capillary electrophoresis systems Nanophor 01 (Institute of Analytical Instmmentation, Russian Academy of Sciences, Saint-Petersburg) equipped with a UV-detector was used to determine analyte. Separation was achieved using 45 cmx30 p.m I.D. fused silica capillary column with UV-detection at 214 nm. 

Catechin and epicatechin are two flavanols of the catechin family. They are enantiomers. The capillary zone electrophoresis (CE) methods with UV-detection were developed for quantitative determination of this flavanols in green tea extracts. For this purpose following conditions were varied mnning buffers, pH and concentration of chiral additive (P-cyclodextrin was chosen as a chiral selector). Borate buffers improve selectivity of separation because borate can make complexes with ortho-dihydroxy groups on the flavanoid nucleus. 

At present moment, no generally feasible method exists for the large-scale production of optically pure products. Although for the separation of virtually every racemic mixture an analytical method is available (gas chromatography, liquid chromatography or capillary electrophoresis), this is not the case for the separation of racemic mixtures on an industrial scale. The most widely applied method for the separation of racemic mixtures is diastereomeric salt crystallization [1]. However, this usually requires many steps, making the process complicated and inducing considerable losses of valuable product. In order to avoid the problems associated with diastereomeric salt crystallization, membrane-based processes may be considered as a viable alternative. 

---