Capillary electrophoresis resolution separation technique

Capillary electrophoresis is an exciting, new, high resolution separation technique useful for the determination of drugs and their metabolites in body fluids. The first commercial capillary electrophoresis instruments began to emerge on the market in 1988. Today approximately a dozen companies manufacture electrokinetic capillary instrumentation, with many of these fully automated, that comprise auto samplers with computerized data evaluation.f Capillary electrophoresis involves the electrophoretic separations of minute quantities of molecules in solution according to their different velocities in an applied electrical field. The velocity of these molecules... 

Until the appearance of capillary electrophoresis, electrophoretic separations were not canned out in columns but were performed in a flat stabilized medium such as paper or a porous semisolid gel. Remarkable separations were realized in such media, but the technique was slow, tedious, and required a good deal of operator skill. In the early 1980s,. scientists began to explore the feasibility of performing these same separations on micro amounts of sample in fused silica capillary tubes. Their results proved promising in terms of resolution, speed, and potential for automation. As a consequence, capillary electrophoresis (CE) has become an important tool for a wide variety of analytical separation problems and is the only type of electrophoresis that we will consider. 

Electrophoresis is the migration of ions in an electric field. Anions are attracted to the anode and cations are attracted to the cathode. Different ions migrate at different speeds. Capillary electrophoresis is a high-resolution separation technique conducted with solutions of ions in a narrow capillary tube. A clever modification of the technique allows us to separate neutral analytes also. Capillary electrophoresis applies with equal ease to the separation of macromolecules, such as proteins and DNA, and small species, such as Na" and benzene. Capillary electrophoresis can analyze the contents of a single cell. 

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). 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Capillary Electrophoresis. Capillary electrophoresis (ce) is an analytical technique that can achieve rapid high resolution separation of water-soluble components present in small sample volumes. The separations are generally based on the principle of electrically driven ions in solution. Selectivity can be varied by the alteration of pH, ionic strength, electrolyte composition, or by incorporation of additives. Typical examples of additives include organic solvents, surfactants (qv), and complexation agents (see Chelating agents). 

The heating effect is the limiting factor for all electrophoretic separations. When heat is dissipated rapidly, as in capillary electrophoresis, rapid, high resolution separations are possible. For electrophoretic separations the higher the separating driving force, ie, the electric field strength, the better the resolution. This means that if a way to separate faster can be found, it should also be a more effective separation. This is the opposite of most other separation techniques. 

This chapter will first cover the nature of electrophoretic separations, especially those concerning capillary electrophoresis. Comprehensive multidimensional separations will then be defined, specifically in terms of orthogonality and resolution. The history of planar and non-comprehensive electrodriven separations will then be discussed. True comprehensive multidimensional separations involving chromatography and capillary electrophoresis will be described next. Finally, the future directions of these multidimensional techniques will be outlined. 

In the last decade, capillary electrophoresis (CE) has become one of the most powerful and conceptually simple separation techniques for the analysis of complex mixtures. The main reasons are its high resolution, relatively short analysis times, and low operational cost when compared to high-performance liquid chromatography (HPLC). The ability to analyze ultrasmall volume samples in the picoliter-to-nanoliter ranges makes it an ideal analytical method for extremely volume-limited biological microenvironments. 

Capillary zone electrophoresis is a separation technique that benefits from very high efficiency, not selectivity. This is in contrast to chromatography, for which the converse is true. Differences in mobility in the range of 0.01% can be enough for complete resolution of neighboring peaks. The resolution R is defined as... 

Capillary electrophoresis offers a set of important advantages that make it a premier technique for the investigation of enantioselective effects in the affinity interactions between chiral drugs and cyclodextrins. The most important advantage of CE is the inherently high separation efficiency offered by this technique. As already known, the most important contributors to peak resolution (R) are a separation selectivity (a) and an efficiency (N). A relationship between these parameters in CE is described by the following equation (2) ... 

Electrophoresis has been used for a long time as one of the most important separation principles in analytical biochemistry. In particular, it has been applied to the separation of DNA and DNA components. Electrophoretical techniques have predominated in this field for decades and will continue to do so in the future. The advent of capillary electrophoresis (CE) has boosted the development of electrophoretic techniques, because it opened access to higher sensitivity, better resolution, and greater speed of separation (1-3). 

Operational procedure of capillary electrophoresis (CE) is similar to TEF. Proteins are separated in an electrical field, migrating until they reach the point where they carry zero charge. Analysis is carried out in a microcapillary tube which provides high resolution. CE can be coupled directly to a MS instrument. The superiority of CE to other analytical techniques is its high resolution. However, it is not widely used for proteomic analysis as there is no commercially available and reliable -MS instrument. 

Emerging separation technologies such as capillary electrophoresis may lead to the improved and cost-effective resolution of complex mixtures of drug residues. This is a valuable tool that is finding its place as a workhorse technique in the analytical laboratory. Capillary electrophoresis is expected to provide a wide variety of new methods that may well prove superior to currently available technology. 

Hall et al. (127) compared free solution capillary electrophoresis (FSCE) and micellar elec-trokinetic capillary chromatography (MEKC) techniques with HPLC analysis. Four major food-grade antioxidants, propyl gallate (PG), BHA, BHT, and TBHQ, were separated. Resolution of the 4 antioxidants was not successful with FSCE, but was with MEKC. Separation was completed with excellent resolution and efficiency within 6 min and picomole amounts of the antioxidants were detectable using UV absorption. In contrast, reversed-phase HPLC separation was not as efficient and required larger sample amounts and longer separation time. 

Among the electrophoretic methods of chiral resolution, various forms of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary isotachophoresis (CIF), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), affinity capillary electrophoresis (ACE), and separation on microchips have been used. However, in contrast to others, the CZE model has been used frequently for this purpose [44]. On the other hand, drawbacks associated with the electrophoretic technique due to lack of development of modem chiral phases have limited the application of these methods. Moreover, the electrophoretic techniques cannot be used at the preparative scale, which represents an urgent need of chiral separation science. 

Many groups have used electrophoresis to enhance a primary chromatographic separation. These techniques can be considered to be two-dimensional, but they are not comprehensive, usually due to the loss of resolution in the interface between the two methods. For instance, capillary electrophoresis was used in 1989 by Grossman and co-workers to analyze fractions from an HPLC separation of peptide fragments. In this study, CE was employed for the separation of protein fragments that were not resolved by HPLC. These two techniques proved to be truly orthogonal, since there was no correlation between the retention time in HPLC and the elution order in CE. The analysis time for CE was found to be four times faster than for HPLC (12), which demonstrated that CE is a good candidate for the second dimension in a two-dimensional separation system, as will be discussed in more detail later. 

Because of polydisperse nature of HS, the importance of separation methods increased as the science evolved. Various separation methods were widely used for conventional fractionation and characterization of components based on differences in component solubility, charge, molecular weight, and/or size, polarity, hydropho-bicity, and so on (Janos, 2003). More recent research focused on advanced molecular-level analyses of humic mixtures (Hertkorn and Schmitt-Kopplin, 2007), in which a combination of separation techniques, mostly, chromatography, or capillary electrophoresis) were coupled with high-resolution instrumental analysis [e.g., mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy]. Several examples appeared in the literature, including those that used size exclusion chro-... 

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