Separation techniques zone electrophoresis

One of the major advantages of CE as a separation technique is the wide variety of separation modes available. Analytes can be separated on the basis of charge, molecular size or shape, pi, or hydrophobicity. The same CE instrument can be used for zone electrophoresis, IEF, sieving separations, isotachophoresis, and chromatographic techniques such as MEKC and capillary electrokinetic chromatography. This section provides a brief description of each separation mode. Zone electrophoresis, IEF, and sieving are the primary modes used for protein separations, and these will be discussed in detail in the following sections. 

Isoenzymes have usually been separated by zone electrophoresis on various carriers and identified by subsequent specific staining. Starch, agar, agarose, and polyacrylamide gels, cellulose acetate foils, and dex-trans such as Sephadex are the most commonly used carrier media. Markert and Mpller (1959) were the first to apply the technique of starch gel electrophoresis in separating dehydrogenases. Apart from the fact that such electrophoreses are easily carried out, they require only... 

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). 

Electrodriven Separation Techniques encompass a wide range of analytical procedures based on several distinct physical and chemical principles, usually acting together to perform the requh ed separation. Example of electrophoretic-based techniques includes capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP), and capillary gel electrophoresis (CGE) (45-47). Some other electrodriven separation techniques are based not only on electrophoretic principles but rather on chromatographic principles as well. Examples of the latter are micellar... 

Zone electrophoresis is defined as the differential migration of a molecule having a net charge through a medium under the influence of an electric field (1). This technique was first used in the 1930s, when it was discovered that moving boundary electrophoresis yielded incomplete separations of analytes (2). The separations were incomplete due to Joule heating within the system, which caused convection which was detrimental to the separation. 

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. 

Very recently, HPLC with fluorescence detection was recommended for improving detection sensitivities of betalains. " While this technique may be worthwhile for betaxanthin analyses, its use for betacyanins cannot be recommended. Although this technique represents a worthwhile approach requiring low amounts of solvent and sample and generally characterized by a high separation efhciency, only one study dealt with the use of capillary zone electrophoresis for betalain analyses. ... 

Table 1 summarizes several of the experimental methods discussed in this chapter. A need exists for new or revised methods for transport experimentation, particularly for therapeutic proteins or peptides in polymeric systems. An important criterion for the new or revised methods includes in situ sampling using micro techniques which simultaneously sample, separate, and analyze the sample. For example, capillary zone electrophoresis provides a micro technique with high separation resolution and the potential to measure the mobilities and diffusion coefficients of the diffusant in the presence of a polymer. Combining the separation and analytical components adds considerable power and versatility to the method. In addition, up-to-date separation instrumentation is computer-driven, so that methods development is optimized, data are acquired according to a predetermined program, and data analysis is facilitated. 

Neomycin is a readily ionisable molecule and should thus be separable from other antibiotics by application of an electric field (zone electrophoresis). Various workers have successfully applied this technique to neomycin and Table 10 summarises the conditions reported in the literature. A number of authors described the qualitative separation of neomycin from other chemical-types of antibiotics using paper-electro-phoresis181,185,187 while Ochabl89 described systems specifically designed to separate compounds within the aminoglycoside group of antibiotics. 

Another separation technique utilizes an electric field. An electric held is an electrically charged region of space, such as between a pair of electrodes connected to a power supply. The technique utilizes the varied rates and direction with which different organic ions (or large molecules with charged sites) migrate while under the influence of the electric held. This technique is called electrophoresis. Zone electrophoresis refers to the common case in which a medium such as cellulose or gel is used to contain the solution. A schematic diagram of the electrophoresis apparatus resembles an electrochemical apparatus in many... 

Zonal techniques are the most frequently used form of electrophoresis and involve the application of a sample as a small zone to a relatively large area of inert supporting medium which enables the subsequent detection of the separated sample zones. A wide range of supporting media have been developed either to eliminate difficulties caused by some media (e.g. the adsorptive effects of paper) or to offer additional features (e.g. the molecular sieving effects of polyacrylamide gel). 

Amino acids derivatized with 9-fluorenylmethyl chloroformate (90) were separated by CE and determined by LIF with a pulsed laser LOD 0.5 nM (SNR 2)322. A sensitive technique for amino acids is capillary zone electrophoresis (CZE) combined with LIF of their fluorescein isothiocyanate (133) derivatives. Not all amino acids give good resolution. LOD for proline and arginine were 0.3 and 0.5 nM, respectively323. 

CE is a family of techniques similar to those found in conventional electrophoresis zone electrophoresis, displacement electrophoresis, isoelectric focusing (IEF), and sieving separations. Other modes of operation unique to CE include micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC). 

---