Preparative isotachophoresis

There are three distinct modes of electrophoresis zone electrophoresis, isoelectric focusing, and isotachophoresis. These three methods may be used alone or in combination to separate molecules on both an analytical (p.L of a mixture separated) and preparative (mL of a mixture separated) scale. Separations in these three modes are based on different physical properties of the molecules in the mixture, making at least three different analyses possible on the same mixture. 

In considering the applicability of preparative classical electrophoretic methods to chiral separations, it should be noted that practitioners in the art of classical electrophoresis have been particularly inventive in designing novel separation strategies. For instance, pH, ionic strength and density gradients have all been used. Isoelectric focusing and isotachophoresis are well-established separation modes in classical electrophoresis and are also being implemented in CE separations [7, 8]. These trends are also reflected in the preparative electrophoretic approaches discussed here. 

Klein reported the use of a quantitative and qualitative determination of procaine in pharmaceutical preparations by capillary isotachophoresis [149]. 

Finally, when RPC methods are used in preparative studies with peptides, the opportunity routinely exists for subsequent analysis of the recovered fractions by a variety of analytical methods including high-speed RP-HPLC, HP-IEX, HP-HILIC, or HP-IMAC, zonal or micellar electrokinetic high-performance capillary electrophoresis (HP-CZE and MECK-CZE), capillary electrochromatography (CEC), or capillary isotachophoresis. The combination of the RPC information, drawn from the In k versus i > plots, with the data derived from on-line spectroscopic detection thus readily provides a comprehensive opportunity to assess the purity of an isolated peptide, many of the physicochemical features of the interaction, as well as a means to optimize the resolution in the RPC separation. 

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. 

The development of electrophoretic techniques afforded possibilities for fractionations based on charge density differences. Duxbury (1989) has reviewed applications of different electrophoretic separation methods, including zone electrophoresis, moving boundary electrophoresis, isotachophoresis, and isoelectric focusing (IEF). Preparative column electrophoresis (Clapp, 1957) and continuous flow paper electrophoresis (Hayes, 1960 summarized by Hayes et al., 1985) methods have been used to separate components isolated from sapric histosol soils. These techniques allowed separation of polysaccharides from the colored components the electrophoretograms of the colored components were diffuse, showing a continuum of components of different charge densities. 

Capillary zone electrophoresis (CZE), with direct or indirect photometry and conductivity has become popular in wine analysis. Very little, or sometimes no sample preparation is needed and short analysis times are also apparent advantages of CE and CZE in the analysis of wine. Capillary isotachophoresis (ITP), with conductivity, thermometric, and UV absorption detection, is suitable for the separation of various anionic constituents (organic acids and inorganic anions), currently occurring in wines (Masar et al., 2001). 

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]. 

A15. Arlinger, L., Preparative capillary isotachophoresis—principle and some applications. /. Chromatogr. 119, 9-24 (1976). 

A16. Arlinger, L., The principle of preparative capillary isotachophoresis. /n Electro-focusing and Isotachophoresis (B. J. Radola and D. Graesslin, eds.), pp. 506-514. Walter de Gruyter, Berlin, 1977. 

B8. Bier, M., Cuddeback, R. M., and Kopwillem, A., Preparative plasma protein fractionation by isotachophoresis in Sephadex columns. /. Chromatogr. 132, 437-450 (1977). 

H13. Hjalmarsson, S.-G., Preparative isotachophoresis—the effect of using ampholine of different pH ranges as spacer ions in the fractionation of serum proteins. Sci. Tools 22, 35-38 (1975). 

The most widely used methods by far employ coherent or granular gels. The former are most commonly polyacrylamide or agarose and the latter is usually Sephadex. Gels have been used in the preparative applications of each of the three common electrophoretic modes, isoelectric focusing (lEF), isotachophoresis (ITP) and zone electrophoresis (ZE). Regardless of the mode utilized, the procedure is most often a batch operation and employs either a cylindrical flat bed or annular separation chamber. Isotachophoresis has been applied to preparative scale fractionations in both polyacrylamide (.3) and Sephadex... 

The world of electromigration separations is sharply divided into two areas. Zone electrophoresis on paper and related procedures have (in spite of their wide applicability to diverse organic compounds) already passed their period of favour. The other branch is represented by the more recent techniques some of which have already became widely accepted (such as isoelectric focusing or separations in polyacrylamide gel) and the others that are at the moment in the centre of a rapid development like displacement electrophoresis (isotachophoresis). This chapter is devoted mainly to analytical procedures such as these which are governing the area of electromigration separations at the moment with a single exception flow deviation (curtain) electrophoresis which will be discussed in more detail because it offers several new dimensions in the separation field. The other preparative procedures are summarized only briefly. 

The advantage of isotachophoresis from the point of view of preparative scale operations is the increase in resolution with increasing load. A higher load increases the length of individual zones. Separation is carried out in an adapted LKB prepara-tive electrophoresis column (Uniphor) and the following buffer systems are recommended by Svendsen [293]. 

Preparative isotachophoresis can also be done without supporting gel using a capillary system. The procedure was described by Arlinger [293] the principle is obvious from Fig. 6.32. 

Fig. 6.32. The principle of preparative capillary isotachophoresis. (A) Schematic representation of the analytical equipment. (B) The preparative version with the additional flow of the leading electrolyte. L, Leading electrolyte T, terminating electrolyte D, UV detector S, sample v, migration velocity m,. mj, semipermeable membranes A, flow of L to elute the sample zones.

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